RNAi agents for inhibiting expression of microtubule associated protein tau (MAPT), compositions thereof, and methods of use

Described are RNAi agents, compositions that include RNAi agents, and methods for inhibition of a microtubule associated protein tau (MAPT) gene. The MAPT RNAi agents and RNAi agent conjugates disclosed herein inhibit the expression of a MAPT gene. The MAPT RNAi agents are conjugated to an antigen binding protein that may enable subcutaneous delivery of the RNAi agents by facilitating crossing of the blood brain barrier (BBB). Pharmaceutical compositions that include one or more MAPT RNAi agents, optionally with one or more additional therapeutics, are also described. Delivery of the described MAPT RNAi agents to central nervous system (CNS) tissue, in vivo, provides for inhibition of MAPT gene expression and a reduction in MAPT activity, which can provide a therapeutic benefit to subjects, including human subjects, for the treatment of various diseases including Alzheimer's disease, Frontotemporal lobar degeneration dementia (FTLD), Progressive supranuclear palsy, and other tauopathies.

1. 11. An RNAi agent for inhibiting expression of a microtubule associated protein tau (MAPT) gene, comprising: an antisense strand consisting of the nucleotide sequence cPrpusAfsgucuAfccauGfuCfgAfugcussg (SEQ ID NO: 592), wherein a represents 2′-O-methyl adenosine, c represents 2′-O-methyl cytidine, g represents 2′-O-methyl guanosine, u represents 2′-O-methyl uridine; Af represents 2′-fluoro adenosine, Cf represents 2′-fluoro cytidine, Gf represents 2′-fluoro guanosine, Uf represents 2′-fluoro uridine; cPrpu represents 5′-cyclopropyl phosphonate-2′-O-methyl uridine; s represents a phosphorothioate linkage; and ss represents a phosphorodithioate linkage; and a sense strand comprising a nucleotide sequence that is at least partially complementary to the antisense strand; wherein all of the nucleotides of the sense strand are modified nucleotides.
2. 22. The RNAi agent of claim 1, wherein all or substantially all of the nucleotides of the sense strand are modified with 2′-O-methyl nucleotides, 2′-fluoro nucleotides, or combinations thereof.
3. 33. The RNAi agent of claim 1, wherein the sense strand is 18 to 30 nucleotides in length.
4. 44. The RNAi agent of claim 1, wherein the sense strand is 21 nucleotides in length.
5. 55. The RNAi agent of claim 1, wherein the sense strand comprises one or two terminal caps.
6. 66. The RNAi agent of claim 1, wherein the sense strand comprises one or two inverted abasic residues.
7. 77. The RNAi agent of claim 1, wherein the sense strand comprises a nucleotide sequence of one of the following nucleotide sequences (5′→3′): (SEQ ID NO: 540) GCAUCGACAUGGUAGACUA; or (SEQ ID NO: 777) CAGCAUCGACAUGGUAGACUA.
8. 88. The RNAi agent of claim 1, wherein the sense strand comprises the nucleotide sequence (5′→3′): cagcaucgAfcAfUfgguagacua (SEQ ID NO: 598); wherein a represents 2′-O-methyl adenosine, c represents 2′-O-methyl cytidine, g represents 2′-O-methyl guanosine, u represents 2′-O-methyl uridine; Af represents 2′-fluoro adenosine, Cf represents 2′-fluoro cytidine, Gf represents 2′-fluoro guanosine, and Uf represents 2′-fluoro uridine.

CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 63/572,350, filed on Mar. 31, 2024, U.S. Provisional Patent Application Ser. No. 63/573,135, filed on Apr. 2, 2024, U.S. Provisional Patent Application Ser. No. 63/695,099, filed on Sep. 16, 2024, and U.S. Provisional Patent Application Ser. No. 63/572,349, filed on Mar. 31, 2024, the contents of each of which are incorporated herein by reference in their entirety.


FIELD OF THE INVENTION
The present disclosure relates to RNA interference (RNAi) agents, e.g., double stranded RNAi agents such as chemically modified small interfering RNAs (siRNAs), for inhibition of microtubule associated protein tau (“MAPT”) gene expression, compositions that include MAPT RNAi agents, and methods of use thereof.
SEQUENCE LISTING
This application contains a Sequence Listing (in compliance with Standard ST26), which has been submitted in xml format and is hereby incorporated by reference in its entirety. The xml sequence listing file is named 30743-US1_SeqListing.xml, created Mar. 28, 2025, and is 2660 kb in size.
BACKGROUND
Neurodegenerative diseases, including Alzheimer's disease (AD), frontotemporal dementia (FTD), and other tauopathies, are characterized by the pathological accumulation of microtubule-associated protein tau (MAPT or tau) aggregates. Tau is essential for the stabilization of microtubules within neurons; however, under pathological conditions, it undergoes hyperphosphorylation, leading to its aggregation into neurofibrillary tangles (NFTs). These tau aggregates disrupt cellular function, ultimately resulting in neurotoxicity and neuronal death. The presence of tau pathology strongly correlates with cognitive decline and disease progression, making it a critical therapeutic target for neurodegenerative disorders.
Currently, there are limited therapeutic options for directly targeting pathological tau, and existing treatments for Alzheimer's disease and related tauopathies primarily focus on symptomatic relief rather than disease modification. Various approaches, including small molecules, monoclonal antibodies, antisense oligonucleotides, and gene therapy, have been explored to mitigate tau aggregation, promote tau clearance, or modulate tau phosphorylation. However, challenges such as blood-brain barrier penetration, off-target effects, and limited efficacy have hindered the successful development of tau-targeting therapies.
Given the urgent need for effective disease-modifying treatments, there remains a significant demand for novel therapeutics that can directly and specifically modulate tau pathology. The present disclosure relates to RNA interference (RNAi) agents, e.g., double stranded RNAi agents such as chemically modified small interfering RNAs (siRNAs), for inhibition of microtubule associated protein tau (“MAPT”) gene expression, compositions that include MAPT RNAi agents, and methods of use thereof.
SUMMARY
There exists a need for novel RNA interference (RNAi) agents (termed RNAi agents, RNAi triggers, or triggers), e.g., double stranded RNAi agents such as siRNAs, that are able to selectively and efficiently inhibit the expression of a MAPT gene, including for use as a therapeutic or medicament. Further, there exists a need for compositions of novel MAPT-specific RNAi agents for the treatment of diseases or disorders associated mutant MAPT gene expression and/or disorders that can be mediated at least in part by a reduction in MAPT gene expression.
The nucleotide sequences and chemical modifications of the MAPT RNAi agents disclosed herein, as well as their combination with certain specific antigen binding proteins and/or lipid PK/PD modulators suitable for selectively and efficiently delivering the MAPT RNAi agents to relevant CNS cells in vivo, differ from those previously disclosed or known in the art. The MAPT RNAi agents disclosed herein provide for highly potent and efficient inhibition of the expression of a MAPT gene.
In general, the present disclosure features MAPT gene-specific RNAi agents, compositions that include MAPT RNAi agents, and methods for inhibiting expression of a MAPT gene in vitro and/or in vivo using the MAPT RNAi agents and compositions that include MAPT RNAi agents described herein. The MAPT RNAi agents described herein are able to selectively and efficiently decrease expression of a MAPT gene, and thereby reduce the expression of the MAPT protein.
The described MAPT RNAi agents can be used in methods for therapeutic treatment (including preventative or prophylactic treatment) of symptoms and diseases including, but not limited to, various central nervous system diseases and neurodegenerative diseases (including Alzheimer's disease, Frontotemporal lobar degeneration dementia (FTLD), Progressive supranuclear palsy, and other tauopathies).
In one aspect, the disclosure features RNAi agents for inhibiting expression of a MAPT gene, wherein the RNAi agent includes a sense strand (also referred to as a passenger strand) and an antisense strand (also referred to as a guide strand). The sense strand and the antisense strand can be partially, substantially, or fully complementary to each other. The length of the RNAi agent sense strands described herein each can be 15 to 49 nucleotides in length. The length of the RNAi agent antisense strands described herein each can be 18 to 49 nucleotides in length. In some embodiments, the sense and antisense strands are independently 18 to 26 nucleotides in length. The sense and antisense strands can be either the same length or different lengths. In some embodiments, the sense and antisense strands are independently 21 to 26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 21 to 24 nucleotides in length. In some embodiments, both the sense strand and the antisense strand are 21 nucleotides in length. In some embodiments, the antisense strands are independently 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the sense strands are independently 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides in length. The RNAi agents described herein, upon delivery to a cell expressing MAPT such as endothelial cells, neurons, microglia, and astrocytes, inhibit the expression of one or more MAPT gene variants in vivo and/or in vitro.
The MAPT RNAi agents disclosed herein target a human MAPT gene (see, e.g., SEQ ID NO:1). In some embodiments, the MAPT RNAi agents disclosed herein target a portion of a MAPT gene having the sequence of any of the sequences disclosed in Table 1.
In another aspect, the disclosure features compositions, including pharmaceutical compositions, that include one or more of the disclosed MAPT RNAi agents that are able to selectively and efficiently decrease expression of a MAPT gene. The compositions that include one or more MAPT RNAi agents described herein can be administered to a subject, such as a human or animal subject, for the treatment (including prophylactic treatment or inhibition) of symptoms and diseases associated with MAPT protein levels.
Examples of MAPT RNAi agent sense strands and antisense strands that can be used in a MAPT RNAi agent are provided in Tables 3, 4, 5, and 6. Examples of MAPT RNAi agent duplexes are provided in Tables 7, 8, and 9. Examples of 19-nucleotide core stretch sequences that may consist of or may be included in the sense strands and antisense strands of certain MAPT RNAi agents disclosed herein, are provided in Table 2.
In another aspect, the disclosure features methods for delivering MAPT RNAi agents to neurons, astrocytes, microglia and endothelial cells in a subject, such as a mammal, in vivo. Also described herein are compositions for use in such methods. In some embodiments, disclosed herein are methods for delivering MAPT RNAi agents to central nervous system cells (neurons, astrocytes, microglia and endothelial cells) to a subject in vivo. In some embodiments, the subject is a human subject.
The methods disclosed herein include the administration of one or more MAPT RNAi agents to a subject, e.g., a human or animal subject, by any suitable means known in the art. The pharmaceutical compositions disclosed herein that include one or more MAPT RNAi agents can be administered in a number of ways depending upon whether local or systemic treatment is desired. Administration can be, but is not limited to, for example, intravenous, intraarterial, subcutaneous, intraperitoneal, subdermal (e.g., via an implanted device), and intraparenchymal administration. In some embodiments, the pharmaceutical compositions described herein are administered by intrathecal injection or intracerebroventricular injection. In some embodiments, the pharmaceutical compositions described herein are administered by subcutaneous injection.
In some embodiments, it is desired that the MAPT RNAi agents described herein inhibit the expression of a MAPT gene in central nervous system cells.
The one or more MAPT RNAi agents can be delivered to target cells or tissues using any oligonucleotide delivery technology known in the art. In some embodiments, a MAPT RNAi agent is delivered to cells or tissues by covalently linking the RNAi agent to a targeting group or an antigen binding protein.
The one or more MAPT RNAi agents can be delivered to target cells or tissues using any oligonucleotide delivery technology known in the art. In some embodiments, a MAPT RNAi agent is delivered to cells or tissues by covalently linking the RNAi agent to a targeting group or a lipid moiety.
An antigen binding protein can be linked to the 3′ or 5′ end of a sense strand or an antisense strand of a MAPT RNAi agent. In some embodiments, an antigen binding protein is linked to the 3′ or 5′ end of the sense strand. In some embodiments, an antigen binding protein is linked to the 5′ end of the sense strand. In some embodiments, an antigen binding protein is linked internally to a nucleotide on the sense strand and/or the antisense strand of the RNAi agent. In some embodiments, an antigen binding protein is linked to the RNAi agent via a linker.
A PK/PD modulator can be linked to the 3′ or 5′ end of a sense strand or an antisense strand of a MAPT RNAi agent. In some embodiments, a PK/PD modulator is linked to the 3′ or 5′ end of the sense strand. In some embodiments, a PK/PD modulator is linked to the 5′ end of the sense strand. In some embodiments, a PK/PD modulator is linked internally to a nucleotide on the sense strand and/or the antisense strand of the RNAi agent. In some embodiments, a PK/PD modulator is linked to the RNAi agent via a linker.
In another aspect, the disclosure features compositions that include one or more MAPT RNAi agents that have the duplex structures disclosed in Tables 7, 8, and 9.
The use of MAPT RNAi agents provides methods for therapeutic (including prophylactic) treatment of diseases or disorders for which a reduction in MAPT protein levels can provide a therapeutic benefit. The MAPT RNAi agents disclosed herein can be used to treat various neurodegenerative diseases, including Alzheimer's disease, Frontotemporal lobar degeneration dementia (FTLD), Progressive supranuclear palsy, and other tauopathies. Such methods of treatment include administration of a MAPT RNAi agent to a human being or animal having elevated or MAPT protein or MAPT activity beyond desirable levels.



BRIEF DESCRIPTION OF THE DRAWINGS
These and other features, aspects, and advantages of the present disclosure may be better understood when the following detailed description is read with reference to the accompanying drawings.
FIG. 1 Depicts the knockdown of MAPT protein in hippocampus, frontal cortex, and thoracic spinal cord in cynomolgus monkeys administered MAPT RNAi agents according to the study described in Example 2.
FIG. 2 Depicts the knockdown of MAPT protein in hippocampus, frontal cortex, and thoracic spinal cord in cynomolgus monkeys administered MAPT RNAi agents according to the study described in Example 3.
FIG. 3 Depicts the knockdown of MAPT protein in hippocampus, frontal cortex, temporal cortex, and thoracic spinal cord in cynomolgus monkeys administered MAPT RNAi agents according to the study described in Example 15.



DETAILED DESCRIPTION
Definitions
As used herein, the terms “oligonucleotide” and “polynucleotide” mean a polymer of linked nucleosides each of which can be independently modified or unmodified.
As used herein, an “RNAi agent” (also referred to as an “RNAi trigger”) means a chemical composition of matter that contains an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting (e.g., degrades or inhibits under appropriate conditions) translation of messenger RNA (mRNA) transcripts of a target mRNA in a sequence specific manner. As used herein, RNAi agents may operate through the RNA interference mechanism (i.e., inducing RNA interference through interaction with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) of mammalian cells), or by any alternative mechanism(s) or pathway(s). While it is believed that RNAi agents, as that term is used herein, operate primarily through the RNA interference mechanism, the disclosed RNAi agents are not bound by or limited to any particular pathway or mechanism of action. RNAi agents disclosed herein are comprised of a sense strand and an antisense strand, and include, but are not limited to: small (or short) interfering RNAs (siRNAs), double stranded RNAs (dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), and dicer substrates. The antisense strand of the RNAi agents described herein is at least partially complementary to the mRNA being targeted (i.e. MAPT mRNA). RNAi agents can include one or more modified nucleotides and/or one or more non-phosphodiester linkages.
As used herein, the terms “silence,” “reduce,” “inhibit,” “down-regulate,” or “knockdown” when referring to expression of a given gene, mean that the expression of the gene, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein, or protein subunit translated from the mRNA in a cell, group of cells, tissue, organ, or subject in which the gene is transcribed, is reduced when the cell, group of cells, tissue, organ, or subject is treated with the RNAi agents described herein as compared to a second cell, group of cells, tissue, organ, or subject that has not or have not been so treated.
As used herein, the terms “sequence” and “nucleotide sequence” mean a succession or order of nucleobases or nucleotides, described with a succession of letters using standard nomenclature.
As used herein, a “base,” “nucleotide base,” or “nucleobase,” is a heterocyclic pyrimidine or purine compound that is a component of a nucleotide, and includes the primary purine bases adenine and guanine, and the primary pyrimidine bases cytosine, thymine, and uracil. A nucleobase may further be modified to include, without limitation, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. (See, e.g., Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008). The synthesis of such modified nucleobases (including phosphoramidite compounds that include modified nucleobases) is known in the art.
As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleobase or nucleotide sequence (e.g., RNAi agent sense strand or targeted mRNA) in relation to a second nucleobase or nucleotide sequence (e.g., RNAi agent antisense strand or a single-stranded antisense oligonucleotide), means the ability of an oligonucleotide or polynucleotide including the first nucleotide sequence to hybridize (form base pair hydrogen bonds under mammalian physiological conditions (or otherwise suitable in vivo or in vitro conditions)) and form a duplex or double helical structure under certain standard conditions with an oligonucleotide that includes the second nucleotide sequence. The person of ordinary skill in the art would be able to select the set of conditions most appropriate for a hybridization test. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimics, at least to the extent that the above hybridization requirements are fulfilled. Sequence identity or complementarity is independent of modification. For example, a and Af, as defined herein, are complementary to U (or T) and identical to A for the purposes of determining identity or complementarity.
As used herein, “perfectly complementary” or “fully complementary” means that in a hybridized pair of nucleobase or nucleotide sequence molecules, all (100%) of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence may comprise all or a part of a first or second nucleotide sequence.
As used herein, “partially complementary” means that in a hybridized pair of nucleobase or nucleotide sequence molecules, at least 70%, but not all, of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence may comprise all or a part of a first or second nucleotide sequence.
As used herein, “substantially complementary” means that in a hybridized pair of nucleobase or nucleotide sequence molecules, at least 85%, but not all, of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence may comprise all or a part of a first or second nucleotide sequence.
As used herein, the terms “complementary,” “fully complementary,” “partially complementary,” and “substantially complementary” are used with respect to the nucleobase or nucleotide matching between the sense strand and the antisense strand of an RNAi agent, or between the antisense strand of an RNAi agent and a sequence of a MAPT mRNA.
As used herein, the term “substantially identical” or “substantial identity,” as applied to a nucleic acid sequence means the nucleotide sequence (or a portion of a nucleotide sequence) has at least about 85% sequence identity or more, e.g., at least 90%, at least 95%, or at least 99% identity, compared to a reference sequence. Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window. The percentage is calculated by determining the number of positions at which the same type of nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. The inventions disclosed herein encompass nucleotide sequences substantially identical to those disclosed herein.
As used herein, the terms “treat,” “treatment,” and the like, mean the methods or steps taken to provide relief from or alleviation of the number, severity, and/or frequency of one or more symptoms of a disease in a subject. As used herein, “treat” and “treatment” may include the prevention, management, prophylactic treatment, and/or inhibition or reduction of the number, severity, and/or frequency of one or more symptoms of a disease in a subject.
As used herein, the phrase “introducing into a cell,” when referring to an RNAi agent, means functionally delivering the RNAi agent into a cell. The phrase “functional delivery,” means delivering the RNAi agent to the cell in a manner that enables the RNAi agent to have the expected biological activity, e.g., sequence-specific inhibition of gene expression.
Unless stated otherwise, use of the symbol




as used herein means that any group or groups may be linked thereto that is in accordance with the scope of the inventions described herein.

As used herein, the term “isomers” refers to compounds that have identical molecular formulae, but that differ in the nature or the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.” Stereoisomers that are not mirror images of one another are termed “diastereoisomers,” and stereoisomers that are non-superimposable mirror images are termed “enantiomers,” or sometimes optical isomers. A carbon atom bonded to four non-identical substituents is termed a “chiral center.”
As used herein, unless specifically identified in a structure as having a particular conformation, for each structure in which asymmetric centers are present and thus give rise to enantiomers, diastereomers, or other stereoisomeric configurations, each structure disclosed herein is intended to represent all such possible isomers, including their optically pure and racemic forms. For example, the structures disclosed herein are intended to cover mixtures of diastereomers as well as single stereoisomers.
As used in a claim herein, the phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. When used in a claim herein, the phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.
The person of ordinary skill in the art would readily understand and appreciate that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending upon the environment in which the compound or composition is placed. Accordingly, as used herein, the structures disclosed herein envisage that certain functional groups, such as, for example, OH, SH, or NH, may be protonated or deprotonated. The disclosure herein is intended to cover the disclosed compounds and compositions regardless of their state of protonation based on the environment (such as pH), as would be readily understood by the person of ordinary skill in the art. Correspondingly, compounds described herein with labile protons or basic atoms should also be understood to represent salt forms of the corresponding compound. Compounds described herein may be in a free acid, free base, or salt form. Pharmaceutically acceptable salts of the compounds described herein should be understood to be within the scope of the invention.
As used herein, the term “linked” or “conjugated” when referring to the connection between two compounds or molecules means that two compounds or molecules are joined by a covalent bond. Unless stated, the terms “linked” and “conjugated” as used herein may refer to the connection between a first compound and a second compound either with or without any intervening atoms or groups of atoms.
As used herein, the term “including” is used to herein mean, and is used interchangeably with, the phrase “including but not limited to.” The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless the context clearly indicates otherwise.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other objects, features, aspects, and advantages of the invention will be apparent from the following detailed description, accompanying figures, and from the claims.
RNAi Agents
Described herein are RNAi agents for inhibiting expression of the MAPT (or MAPT) gene (referred to herein as MAPT RNAi agents or MAPT RNAi triggers). Each MAPT RNAi agent disclosed herein comprises a sense strand and an antisense strand. The sense strand can be 15 to 49 nucleotides in length. The antisense strand can be 18 to 30 nucleotides in length. The sense and antisense strands can be either the same length or they can be different lengths. In some embodiments, the sense and antisense strands are each independently 18 to 27 nucleotides in length. In some embodiments, both the sense and antisense strands are each 21-26 nucleotides in length. In some embodiments, the sense and antisense strands are each 21-24 nucleotides in length. In some embodiments, the sense and antisense strands are each independently 19-21 nucleotides in length. In some embodiments, the sense strand is about 19 nucleotides in length while the antisense strand is about 21 nucleotides in length. In some embodiments, the sense strand is about 21 nucleotides in length while the antisense strand is about 23 nucleotides in length. In some embodiments, a sense strand is 23 nucleotides in length and an antisense strand is 21 nucleotides in length. In some embodiments, both the sense and antisense strands are each 21 nucleotides in length. In some embodiments, the RNAi agent sense strands are each independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides in length. In some embodiments, the RNAi agent antisense strands are each independently 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, a double-stranded RNAi agent has a duplex length of about 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides.
Examples of nucleotide sequences used in forming MAPT RNAi agents are provided in Tables 2, 3, 4, 5, 6, and 10. Examples of RNAi agent duplexes, that include the sense strand and antisense strand sequences in Tables 2, 3, 4, 5, 6, are shown in Tables 7A, 7B, 8, 9A, and 10.
In some embodiments, the region of perfect, substantial, or partial complementarity between the sense strand and the antisense strand is 16-26 (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) nucleotides in length and occurs at or near the 5′ end of the antisense strand (e.g., this region may be separated from the 5′ end of the antisense strand by 0, 1, 2, 3, or 4 nucleotides that are not perfectly, substantially, or partially complementary).
A sense strand of the MAPT RNAi agents described herein includes at least 15 consecutive nucleotides that have at least 85% identity to a core stretch sequence (also referred to herein as a “core stretch” or “core sequence”) of the same number of nucleotides in a MAPT mRNA. In some embodiments, a sense strand core stretch sequence is 100% (perfectly) complementary or at least about 85% (substantially) complementary to a core stretch sequence in the antisense strand, and thus the sense strand core stretch sequence is typically perfectly identical or at least about 85% identical to a nucleotide sequence of the same length (sometimes referred to, e.g., as a target sequence) present in the MAPT mRNA target. In some embodiments, this sense strand core stretch is 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length. In some embodiments, this sense strand core stretch is 17 nucleotides in length. In some embodiments, this sense strand core stretch is 19 nucleotides in length.
An antisense strand of a MAPT RNAi agent described herein includes at least 16 consecutive nucleotides that have at least 85% complementarity to a core stretch of the same number of nucleotides in a MAPT mRNA and to a core stretch of the same number of nucleotides in the corresponding sense strand. In some embodiments, an antisense strand core stretch is 100% (perfectly) complementary or at least about 85% (substantially) complementary to a nucleotide sequence (e.g., target sequence) of the same length present in the MAPT mRNA target. In some embodiments, this antisense strand core stretch is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length. In some embodiments, this antisense strand core stretch is 19 nucleotides in length. In some embodiments, this antisense strand core stretch is 17 nucleotides in length. A sense strand core stretch sequence can be the same length as a corresponding antisense core sequence or it can be a different length.
The MAPT RNAi agent sense and antisense strands anneal to form a duplex. A sense strand and an antisense strand of a MAPT RNAi agent can be partially, substantially, or fully complementary to each other. Within the complementary duplex region, the sense strand core stretch sequence is at least 85% complementary or 100% complementary to the antisense core stretch sequence. In some embodiments, the sense strand core stretch sequence contains a sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 nucleotides that is at least 85% or 100% complementary to a corresponding 16, 17, 18, 19, 20, 21, 22, or 23 nucleotide sequence of the antisense strand core stretch sequence (i.e. the sense and antisense core stretch sequences of a MAPT RNAi agent have a region of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 nucleotides that is at least 85% base paired or 100% base paired.)
In some embodiments, the antisense strand of a MAPT RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences in Table 2 or Table 3. In some embodiments, the sense strand of a MAPT RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the sense strand sequences in Table 2, Table 4, Table 5, Table 6, or Table 9.
In some embodiments, the sense strand and/or the antisense strand can optionally and independently contain an additional 1, 2, 3, 4, 5, or 6 nucleotides (extension) at the 3′ end, the 5′ end, or both the 3′ and 5′ ends of the core stretch sequences. The antisense strand additional nucleotides, if present, may or may not be complementary to the corresponding sequence in the MAPT mRNA. The sense strand additional nucleotides, if present, may or may not be identical to the corresponding sequence in the MAPT mRNA. The antisense strand additional nucleotides, if present, may or may not be complementary to the corresponding sense strand's additional nucleotides, if present.
As used herein, an extension comprises 1, 2, 3, 4, 5, or 6 nucleotides at the 5′ and/or 3′ end of the sense strand core stretch sequence and/or antisense strand core stretch sequence. The extension nucleotides on a sense strand may or may not be complementary to nucleotides, either core stretch sequence nucleotides or extension nucleotides, in the corresponding antisense strand. Conversely, the extension nucleotides on an antisense strand may or may not be complementary to nucleotides, either core stretch nucleotides or extension nucleotides, in the corresponding sense strand. In some embodiments, both the sense strand and the antisense strand of an RNAi agent contain 3′ and 5′ extensions. In some embodiments, one or more of the 3′ extension nucleotides of one strand base pairs with one or more 5′ extension nucleotides of the other strand. In other embodiments, one or more of 3′ extension nucleotides of one strand do not base pair with one or more 5′ extension nucleotides of the other strand. In some embodiments, a MAPT RNAi agent has an antisense strand having a 3′ extension and a sense strand having a 5′ extension. In some embodiments, the extension nucleotide(s) are unpaired and form an overhang. As used herein, an “overhang” refers to a stretch of one or more unpaired nucleotides located at a terminal end of either the sense strand or the antisense strand that does not form part of the hybridized or duplexed portion of an RNAi agent disclosed herein.
In some embodiments, a MAPT RNAi agent comprises an antisense strand having a 3′ extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In other embodiments, a MAPT RNAi agent comprises an antisense strand having a 3′ extension of 1, 2, or 3 nucleotides in length. In some embodiments, one or more of the antisense strand extension nucleotides comprise nucleotides that are complementary to the corresponding MAPT mRNA sequence. In some embodiments, one or more of the antisense strand extension nucleotides comprise nucleotides that are not complementary to the corresponding MAPT mRNA sequence.
In some embodiments, a MAPT RNAi agent comprises a sense strand having a 3′ extension of 1, 2, 3, 4, or 5 nucleotides in length. In some embodiments, one or more of the sense strand extension nucleotides comprises adenosine, uracil, or thymidine nucleotides, AT dinucleotide, or nucleotides that correspond to or are the identical to nucleotides in the MAPT mRNA sequence. In some embodiments, the 3′ sense strand extension includes or consists of one of the following sequences, but is not limited to: T, UT, TT, UU, UUT, TTT, or TTTT (each listed 5′ to 3′).
A sense strand can have a 3′ extension and/or a 5′ extension. In some embodiments, a MAPT RNAi agent comprises a sense strand having a 5′ extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In some embodiments, one or more of the sense strand extension nucleotides comprise nucleotides that correspond to or are identical to nucleotides in the MAPT mRNA sequence.
Examples of sequences used in forming MAPT RNAi agents are provided in Tables 2, 3, 4, 5, 6, and 9. In some embodiments, a MAPT RNAi agent antisense strand includes a sequence of any of the sequences in Tables 2, 3, or 10. In certain embodiments, a MAPT RNAi agent antisense strand comprises or consists of any one of the modified sequences in Table 3. In some embodiments, a MAPT RNAi agent antisense strand includes the sequence of nucleotides (from 5′ end→3′ end) 1-17, 2-15, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, or 2-21, of any of the sequences in Tables 2 or 3. In some embodiments, a MAPT RNAi agent sense strand includes the sequence of any of the sequences in Tables 2, 4, 5, or 6. In some embodiments, a MAPT RNAi agent sense strand includes the sequence of nucleotides (from 5′ end→3′ end) 1-18, 1-19, 1-20, 1-21, 2-19, 2-20, 2-21, 3-20, 3-21, or 4-21 of any of the sequences in Tables 2, 4, 5, or 6. In certain embodiments, a MAPT RNAi agent sense strand comprises or consists of a modified sequence of any one of the modified sequences in Table 4, 5, 6, or 9.
In some embodiments, the sense and antisense strands of the RNAi agents described herein contain the same number of nucleotides. In some embodiments, the sense and antisense strands of the RNAi agents described herein contain different numbers of nucleotides. In some embodiments, the sense strand 5′ end and the antisense strand 3′ end of an RNAi agent form a blunt end. In some embodiments, the sense strand 3′ end and the antisense strand 5′ end of an RNAi agent form a blunt end. In some embodiments, both ends of an RNAi agent form blunt ends. In some embodiments, neither end of an RNAi agent is blunt-ended. As used herein a “blunt end” refers to an end of a double stranded RNAi agent in which the terminal nucleotides of the two annealed strands are complementary (form a complementary base-pair).
In some embodiments, the sense strand 5′ end and the antisense strand 3′ end of an RNAi agent form a frayed end. In some embodiments, the sense strand 3′ end and the antisense strand 5′ end of an RNAi agent form a frayed end. In some embodiments, both ends of an RNAi agent form a frayed end. In some embodiments, neither end of an RNAi agent is a frayed end. As used herein a frayed end refers to an end of a double stranded RNAi agent in which the terminal nucleotides of the two annealed strands form a pair (i.e., do not form an overhang) but are not complementary (i.e., form a non-complementary pair). In some embodiments, one or more unpaired nucleotides at the end of one strand of a double stranded RNAi agent form an overhang. The unpaired nucleotides may be on the sense strand or the antisense strand, creating either 3′ or 5′ overhangs. In some embodiments, the RNAi agent contains: a blunt end and a frayed end, a blunt end and 5′ overhang end, a blunt end and a 3′ overhang end, a frayed end and a 5′ overhang end, a frayed end and a 3′ overhang end, two 5′ overhang ends, two 3′ overhang ends, a 5′ overhang end and a 3′ overhang end, two frayed ends, or two blunt ends. Typically, when present, overhangs are located at the 3′ terminal ends of the sense strand, the antisense strand, or both the sense strand and the antisense strand.
The MAPT RNAi agents disclosed herein may also be comprised of one or more modified nucleotides. In some embodiments, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand of the MAPT RNAi agent are modified nucleotides. The MAPT RNAi agents disclosed herein may further be comprised of one or more modified internucleoside linkages, e.g., one or more phosphorothioate linkages. In some embodiments, a MAPT RNAi agent contains one or more modified nucleotides and one or more modified internucleoside linkages. In some embodiments, a 2′-modified nucleotide is combined with modified internucleoside linkage.
In some embodiments, a MAPT RNAi agent is prepared or provided as a salt, mixed salt, or a free-acid. In some embodiments, a MAPT RNAi agent is prepared as a pharmaceutically acceptable salt. In some embodiments, a MAPT RNAi agent is prepared as a pharmaceutically acceptable sodium salt. Such forms that are well known in the art are within the scope of the inventions disclosed herein.
Modified Nucleotides
Modified nucleotides, when used in various oligonucleotide constructs, can preserve activity of the compound in cells while at the same time increasing the serum stability of these compounds, and can also minimize the possibility of activating interferon activity in humans upon administration of the oligonucleotide construct.
In some embodiments, a MAPT RNAi agent contains one or more modified nucleotides. As used herein, a “modified nucleotide” is a nucleotide other than a ribonucleotide (2′-hydroxyl nucleotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides can include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides, 2′-modified nucleotides, inverted nucleotides, modified nucleobase-comprising nucleotides, bridged nucleotides, peptide nucleic acids (PNAs), 2′,3′-seco nucleotide mimics (unlocked nucleobase analogues), locked nucleotides, 3′-O-methoxy (2′ internucleoside linked) nucleotides, 2′-F-Arabino nucleotides, 5′-Me, 2′-fluoro nucleotide, morpholino nucleotides, vinyl phosphonate deoxyribonucleotides, vinyl phosphonate containing nucleotides, and cyclopropyl phosphonate containing nucleotides. 2′-modified nucleotides (i.e., a nucleotide with a group other than a hydroxyl group at the 2′ position of the five-membered sugar ring) include, but are not limited to, 2′-O-methyl nucleotides (also referred to herein or in the art as 2′-methoxy nucleotides), 2′-fluoro nucleotides (also referred to herein or in the art as 2′-deoxy-2′-fluoro nucleotides), 2′-deoxy nucleotides, 2′-methoxyethyl (2′-O-2-methoxylethyl) nucleotides (also referred herein or in the art as 2′-MOE nucleotides), 2′-amino nucleotides, and 2′-alkyl nucleotides. It is not necessary for all positions in a given compound to be uniformly modified. Conversely, more than one modification can be incorporated in a single MAPT RNAi agent or even in a single nucleotide thereof. The MAPT RNAi agent sense strands and antisense strands can be synthesized and/or modified by methods known in the art. Modification at one nucleotide is independent of modification at another nucleotide.
Modified nucleobases include synthetic and natural nucleobases, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.
In some embodiments, the 5′ and/or 3′ end of the antisense strand can include abasic residues (Ab), which can also be referred to as an “abasic site” or “abasic nucleotide.” An abasic residue (Ab) is a nucleotide or nucleoside that lacks a nucleobase at the 1′ position of the sugar moiety. (See, e.g., U.S. Pat. No. 5,998,203). In some embodiments, an abasic residue can be placed internally in a nucleotide sequence. In some embodiments, Ab or AbAb can be added to the 3′ end of the antisense strand. In some embodiments, the 5′ end of the sense strand can include one or more additional abasic residues (e.g., (Ab) or (AbAb)). In some embodiments, UUAb, UAb, or Ab are added to the 3′ end of the sense strand. In some embodiments, an abasic (deoxyribose) residue can be replaced with a ribitol (abasic ribose) residue.
In some embodiments, all or substantially all of the nucleotides of an RNAi agent are modified nucleotides. As used herein, an RNAi agent wherein substantially all of the nucleotides present are modified nucleotides is an RNAi agent having four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides in both the sense strand and the antisense strand being ribonucleotides (i.e., unmodified). As used herein, a sense strand wherein substantially all of the nucleotides present are modified nucleotides is a sense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being unmodified ribonucleotides. As used herein, an antisense strand wherein substantially all of the nucleotides present are modified nucleotides is an antisense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the antisense strand being unmodified ribonucleotides. In some embodiments, one or more nucleotides of an RNAi agent is an unmodified ribonucleotide. Chemical structures for certain modified nucleotides are set forth in Table 10 herein.
Modified Internucleoside Linkages
In some embodiments, one or more nucleotides of a MAPT RNAi agent are linked by non-standard linkages or backbones (i.e., modified internucleoside linkages or modified backbones). Modified internucleoside linkages or backbones include, but are not limited to, phosphorothioate groups (represented herein as a lower case “s”), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g., methyl phosphonates or 3′-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3′-amino phosphoramidate, aminoalkylphosphoramidates, or thionophosphoramidates), thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of boranophosphates, or boranophosphates having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. In some embodiments, a modified internucleoside linkage or backbone lacks a phosphorus atom. Modified internucleoside linkages lacking a phosphorus atom include, but are not limited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixed heteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or more short chain heteroatomic or heterocyclic inter-sugar linkages. In some embodiments, modified internucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methylene formacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S, and CH2 components.
In some embodiments, a sense strand of a MAPT RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, an antisense strand of a MAPT RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages. In some embodiments, a sense strand of a MAPT RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, an antisense strand of a MAPT RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1, 2, 3, or 4 phosphorothioate linkages.
In some embodiments, a MAPT RNAi agent sense strand contains at least two phosphorothioate internucleoside linkages. In some embodiments, the phosphorothioate internucleoside linkages are between the nucleotides at positions 1-3 from the 3′ end of the sense strand. In some embodiments, one phosphorothioate internucleoside linkage is at the 5′ end of the sense strand nucleotide sequence, and another phosphorothioate linkage is at the 3′ end of the sense strand nucleotide sequence. In some embodiments, two phosphorothioate internucleoside linkage are located at the 5′ end of the sense strand, and another phosphorothioate linkage is at the 3′ end of the sense strand. In some embodiments, the sense strand does not include any phosphorothioate internucleoside linkages between the nucleotides, but contains one, two, or three phosphorothioate linkages between the terminal nucleotides on both the 5′ and 3′ ends and the optionally present inverted abasic residue terminal caps. In some embodiments, a targeting ligand is linked to the sense strand via a phosphorothioate linkage.
In some embodiments, a MAPT RNAi agent antisense strand contains four phosphorothioate internucleoside linkages. In some embodiments, the four phosphorothioate internucleoside linkages are between the nucleotides at positions 1-3 from the 5′ end of the antisense strand and between the nucleotides at positions 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5′ end. In some embodiments, three phosphorothioate internucleoside linkages are located between positions 1-4 from the 5′ end of the antisense strand, and a fourth phosphorothioate internucleoside linkage is located between positions 20-21 from the 5′ end of the antisense strand. In some embodiments, a MAPT RNAi agent contains at least three or four phosphorothioate internucleoside linkages in the antisense strand.
Capping Residues or Moieties
In some embodiments, the sense strand may include one or more capping residues or moieties, sometimes referred to in the art as a “cap,” a “terminal cap,” or a “capping residue.” As used herein, a “capping residue” is a non-nucleotide compound or other moiety that can be incorporated at one or more termini of a nucleotide sequence of an RNAi agent disclosed herein. A capping residue can provide the RNAi agent, in some instances, with certain beneficial properties, such as, for example, protection against exonuclease degradation. In some embodiments, inverted abasic residues (invAb) (also referred to in the art as “inverted abasic sites”) are added as capping residues (see Table 10). (See, e.g., F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16). Capping residues are generally known in the art, and include, for example, inverted abasic residues as well as carbon chains such as a terminal C3H7 (propyl), C6H13 (hexyl), or C12H25 (dodecyl) groups. In some embodiments, a capping residue is present at either the 5′ terminal end, the 3′ terminal end, or both the 5′ and 3′ terminal ends of the sense strand. In some embodiments, the 5′ end and/or the 3′ end of the sense strand may include more than one inverted abasic deoxyribose moiety as a capping residue.
In some embodiments, one or more inverted abasic residues (invAb) are added to the 3′ end of the sense strand. In some embodiments, one or more inverted abasic residues (invAb) are added to the 5′ end of the sense strand. In some embodiments, one or more inverted abasic residues or inverted abasic sites are inserted between a targeting ligand and the nucleotide sequence of the sense strand of the RNAi agent. In some embodiments, the inclusion of one or more inverted abasic residues or inverted abasic sites at or near the terminal end or terminal ends of the sense strand of an RNAi agent allows for enhanced activity or other desired properties of an RNAi agent.
In some embodiments, one or more inverted abasic residues (invAb) are added to the 5′ end of the sense strand. In some embodiments, one or more inverted abasic residues can be inserted between a targeting ligand and the nucleotide sequence of the sense strand of the RNAi agent. The inverted abasic residues may be linked via phosphate, phosphorothioate (e.g., shown herein as (invAb)s)), or other internucleoside linkages. In some embodiments, the inclusion of one or more inverted abasic residues at or near the terminal end or terminal ends of the sense strand of an RNAi agent may allow for enhanced activity or other desired properties of an RNAi agent. In some embodiments, an inverted abasic (deoxyribose) residue can be replaced with an inverted ribitol (abasic ribose) residue. In some embodiments, the 3′ end of the antisense strand core stretch sequence, or the 3′ end of the antisense strand sequence, may include an inverted abasic residue. The chemical structures for inverted abasic deoxyribose residues are shown in Table 10 below.
MAPT RNAi Agents
The MAPT RNAi agents disclosed herein are designed to target specific positions on a MAPT gene (e.g., SEQ ID NO: 1 (NM 001123066.4)). As defined herein, an antisense strand sequence is designed to target a MAPT gene at a given position on the gene when the 5′ terminal nucleobase of the antisense strand is aligned with a position that is 21 nucleotides downstream (towards the 3′ end) from the position on the gene when base pairing to the gene. For example, as illustrated in Tables 1 and 2 herein, an antisense strand sequence designed to target a MAPT gene at position 184 requires that when base pairing to the gene, the 5′ terminal nucleobase of the antisense strand is aligned with position 204 of a MAPT gene.
As provided herein, a MAPT RNAi agent does not require that the nucleobase at position 1 (5′→3′) of the antisense strand be complementary to the gene, provided that there is at least 85% complementarity (e.g., at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementarity) of the antisense strand and the gene across a core stretch sequence of at least 16 consecutive nucleotides. For example, for a MAPT RNAi agent disclosed herein that is designed to target position 184 of a MAPT gene, the 5′ terminal nucleobase of the antisense strand of the of the MAPT RNAi agent must be aligned with position 204 of the gene; however, the 5′ terminal nucleobase of the antisense strand may be, but is not required to be, complementary to position 204 of a MAPT gene, provided that there is at least 85% complementarity (e.g., at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementarity) of the antisense strand and the gene transcript across a core stretch sequence of at least 16 consecutive nucleotides. As shown by, among other things, the various examples disclosed herein, the specific site of binding of the gene by the antisense strand of the MAPT RNAi agent (e.g., whether the MAPT RNAi agent is designed to target a MAPT gene at position 184, at position 185, at position 246, or at some other position) is an important factor to the level of inhibition achieved by the MAPT RNAi agent. (See, e.g., Kamola et al., The siRNA Non-seed Region and Its Target Sequences are Auxiliary Determinants of Off-Target Effects, PLOS Computational Biology, 11(12), FIG. 1 (2015)).
In some embodiments, the MAPT RNAi agents disclosed herein target a MAPT gene at or near the positions of the MAPT sequence shown in Table P. In some embodiments, the antisense strand of a MAPT RNAi agent disclosed herein includes a core stretch sequence that is fully, substantially, or at least partially complementary to a target MAPT 19-mer sequence disclosed in Table 1.







TABLE 1







MAPT 19-mer mRNA Target Sequences (taken from homo sapiens


microtubule associated protein tau (MAPT) transcript, GenBank


NM_016834.5 (SEQ ID NO: 1))












Corresponding
Targeted Gene



MAPT 19-mer
Positions of
Position


SEQ ID
Target Sequences
Sequence on
(as referred 


No.
(5′→3′)
SEQ ID NO: 1
to herein)





44
AGAUCACGCUGGGACGUAC
 186-204
 184


 


45
GAUCACGCUGGGACGUACG
 187-205
 185


 


46
AAGACCAAGAGGGUGACAC
 248-266
 246


 


47
AGACGAAGCUGCUGGUCAC
 321-339
 319


 


48
GACCCAAGCUCGCAUGGUC
 342-360
 340


 


49
AAGCUCGCAUGGUCAGUAA
 347-365
 345


 


50
GCUCGCAUGGUCAGUAAAA
 349-367
 347


 


51
UCGCAUGGUCAGUAAAAGC
 351-369
 349


 


52
GCAUGGUCAGUAAAAGCAA
 353-371
 351


 


53
CAUGGUCAGUAAAAGCAAA
 354-372
 352


 


54
UCAGUAAAAGCAAAGACGG
 359-377
 357


 


55
GACUGGAAGCGAUGACAAA
 378-396
 376


 


56
CUGGAAGCGAUGACAAAAA
 380-398
 378


 


57
GAAGCGAUGACAAAAAAGC
 383-401
 381


 


58
CAGGAUUCCAGCAAAAACC
 483-501
 481


 


59
AGGAUUCCAGCAAAAACCC
 484-502
 482


 


60
AAGACACCACCCAGCUCUG
 514-532
 512


 


61
UGGUGAACCUCCAAAAUCA
 531-549
 529


 


62
GGUGAACCUCCAAAAUCAG
 532-550
 530


 


63
UGAACCUCCAAAAUCAGGG
 534-552
 532


 


64
CAAAAUCAGGGGAUCGCAG
 542-560
 540


 


65
GUGCAAAUAGUCUACAAAC
 892-910
 890


 


66
AAUAGUCUACAAACCAGUU
 897-915
 895


 


67
UAGUCUACAAACCAGUUGA
 899-917
 897


 


68
AGUCUACAAACCAGUUGAC
 900-918
 898


 


69
UCUACAAACCAGUUGACCU
 902-920
 900


 


70
CCAGUUGACCUGAGCAAGG
 910-928
 908


 


71
GUUGACCUGAGCAAGGUGA
 913-931
 911


 


72
AAGGUGACCUCCAAGUGUG
 925-943
 923


 


73
CUCCAAGUGUGGCUCAUUA
 933-951
 931


 


74
UCCAAGUGUGGCUCAUUAG
 934-952
 932


 


75
CCAAGUGUGGCUCAUUAGG
 935-953
 933


 


76
CAAGUGUGGCUCAUUAGGC
 936-954
 934


 


77
AAGUGUGGCUCAUUAGGCA
 937-955
 935


 


78
GGCUCAUUAGGCAACAUCC
 943-961
 941


 


79
UAGGCAACAUCCAUCAUAA
 950-968
 948


 


80
AGGCAACAUCCAUCAUAAA
 951-969
 949


 


81
GCAACAUCCAUCAUAAACC
 953-971
 951


 


82
GUGGCCAGGUGGAAGUAAA
 977-995
 975


 


83
CCAGGUGGAAGUAAAAUCU
 981-999
 979


 


84
CAGGUGGAAGUAAAAUCUG
 982-1000
 980


 


85
GGUCCCUGGACAAUAUCAC
1040-1058
1038


 


86
UCCCUGGACAAUAUCACCC
1042-1060
1040


 


87
AGAUUGAAACCCACAAGCU
1085-1103
1083


 


88
AUUGAAACCCACAAGCUGA
1087-1105
1085


 


89
UUGAAACCCACAAGCUGAC
1088-1106
1086


 


90
GCCAAAGCCAAGACAGACC
1120-1138
1118


 


91
CAGCAUCGACAUGGUAGAC
1221-1239
1219


 


92
GCAUCGACAUGGUAGACUC
1223-1241
1221

















Homo sapiens microtubule associated protein tau (MAPT), GenBank NM_016834.5



(SEQ ID NO: 1), gene transcript (5465 bases):









1
gcagtcaccg ccacccacca gctccggcac caacagcagc gccgctgcca ccgcccacct



 


61
tctgccgccg ccaccacagc caccttctcc tcctccgctg tcctctcccg tcctcgcctc


 


121
tgtcgactat caggtgaact ttgaaccagg atggctgagc cccgccagga gttcgaagtg


 


181
atggaagatc acgctgggac gtacgggttg ggggacagga aagatcaggg gggctacacc


 


241
atgcaccaag accaagaggg tgacacggac gctggcctga aagctgaaga agcaggcatt


 


301
ggagacaccc ccagcctgga agacgaagct gctggtcacg tgacccaagc tcgcatggtc


 


361
agtaaaagca aagacgggac tggaagcgat gacaaaaaag ccaagggggc tgatggtaaa


 


421
acgaagatcg ccacaccgcg gggagcagcc cctccaggcc agaagggcca ggccaacgcc


 


481
accaggattc cagcaaaaac cccgcccgct ccaaagacac cacccagctc tggtgaacct


 


541
ccaaaatcag gggatcgcag cggctacagc agccccggct ccccaggcac tcccggcagc


 


601
cgctcccgca ccccgtccct tccaacccca cccacccggg agcccaagaa ggtggcagtg


 


661
gtccgtactc cacccaagtc gccgtcttcc gccaagagcc gcctgcagac agcccccgtg


 


721
cccatgccag acctgaagaa tgtcaagtcc aagatcggct ccactgagaa cctgaagcac


 


781
cagccgggag gcgggaaggt gcagataatt aataagaagc tggatcttag caacgtccag


 


841
tccaagtgtg gctcaaagga taatatcaaa cacgtcccgg gaggcggcag tgtgcaaata


 


901
gtctacaaac cagttgacct gagcaaggtg acctccaagt gtggctcatt aggcaacatc


 


961
catcataaac caggaggtgg ccaggtggaa gtaaaatctg agaagcttga cttcaaggac


 


1021
agagtccagt cgaagattgg gtccctggac aatatcaccc acgtccctgg cggaggaaat


 


1081
aaaaagattg aaacccacaa gctgaccttc cgcgagaacg ccaaagccaa gacagaccac


 


1141
ggggcggaga tcgtgtacaa gtcgccagtg gtgtctgggg acacgtctcc acggcatctc


 


1201
agcaatgtct cctccaccgg cagcatcgac atggtagact cgccccagct cgccacgcta


 


1261
gctgacgagg tgtctgcctc cctggccaag cagggtttgt gatcaggccc ctggggcggt


 


1321
caataattgt ggagaggaga gaatgagaga gtgtggaaaa aaaaagaata atgacccggc


 


1381 
ccccgccctc tgcccccagc tgctcctcgc agttcggtta attggttaat cacttaacct



 


1441
gcttttgtca ctcggctttg gctcgggact tcaaaatcag tgatgggagt aagagcaaat



 


1501
ttcatctttc caaattgatg ggtgggctag taataaaata tttaaaaaaa aacattcaaa


 


1561
aacatggcca catccaacat ttcctcaggc aattcctttt gattcttttt tcttccccct


 


1621
ccatgtagaa gagggagaag gagaggctct gaaagctgct tctgggggat ttcaagggac


 


1681
tgggggtgcc aaccacctct ggccctgttg tgggggtgtc acagaggcag tggcagcaac


 


1741
aaaggatttg aaacttggtg tgttcgtgga gccacaggca gacgatgtca accttgtgtg


 


1801
agtgtgacgg gggttggggt ggggcgggag gccacggggg aggccgaggc aggggctggg


 


1861
cagaggggag aggaagcaca agaagtggga gtgggagagg aagccacgtg ctggagagta


 


1921
gacatccccc tccttgccgc tgggagagcc aaggcctatg ccacctgcag cgtctgagcg


 


1981
gccgcctgtc cttggtggcc gggggtgggg gcctgctgtg ggtcagtgtg ccaccctctg


 


2041
cagggcagcc tgtgggagaa gggacagcgg gtaaaaagag aaggcaagct ggcaggaggg


 


2101
tggcacttcg tggatgacct ccttagaaaa gactgacctt gatgtcttga gagcgctggc


 


2161
ctcttcctcc ctccctgcag ggtagggggc ctgagttgag gggcttccct ctgctccaca


 


2221
gaaaccctgt tttattgagt tctgaaggtt ggaactgctg ccatgatttt ggccactttg


 


2281
cagacctggg actttagggc taaccagttc tctttgtaag gacttgtgcc tcttgggaga


 


2341
cgtccacccg tttccaagcc tgggccactg gcatctctgg agtgtgtggg ggtctgggag


 


2401
gcaggtcccg agccccctgt ccttcccacg gccactgcag tcaccccgtc tgcgccgctg


 


2461
tgctgttgtc tgccgtgaga gcccaatcac tgcctatacc cctcatcaca cgtcacaatg


 


2521
tcccgaattc ccagcctcac caccccttct cagtaatgac cctggttggt tgcaggaggt


 


2581
acctactcca tactgagggt gaaattaagg gaaggcaaag tccaggcaca agagtgggac


 


2641
cccagcctct cactctcagt tccactcatc caactgggac cctcaccacg aatctcatga


 


2701
tctgattcgg ttccctgtct cctcctcccg tcacagatgt gagccagggc actgctcagc


 


2761
tgtgacccta ggtgtttctg ccttgttgac atggagagag ccctttcccc tgagaaggcc


 


2821
tggccccttc ctgtgctgag cccacagcag caggctgggt gtcttggttg tcagtggtgg


 


2881
caccaggatg gaagggcaag gcacccaggg caggcccaca gtcccgctgt cccccacttg


 


2941
caccctagct tgtagctgcc aacctcccag acagcccagc ccgctgctca gctccacatg


 


3001
catagtatca gccctccaca cccgacaaag gggaacacac ccccttggaa atggttcttt


 


3061
tcccccagtc ccagctggaa gccatgctgt ctgttctgct ggagcagctg aacatataca


 


3121
tagatgttgc cctgccctcc ccatctgcac cctgttgagt tgtagttgga tttgtctgtt


 


3181
tatgcttgga ttcaccagag tgactatgat agtgaaaaga aaaaaaaaaa aaaaaaagga


 


3241
cgcatgtatc ttgaaatgct tgtaaagagg tttctaaccc accctcacga ggtgtctctc


 


3301
acccccacac tgggactcgt gtggcctgtg tggtgccacc ctgctggggc ctcccaagtt


 


3361
ttgaaaggct ttcctcagca cctgggaccc aacagagacc agcttctagc agctaaggag


 


3421
gccgttcagc tgtgacgaag gcctgaagca caggattagg actgaagcga tgatgtcccc


 


3481
ttccctactt ccccttgggg ctccctgtgt cagggcacag actaggtctt gtggctggtc


 


3541
tggcttgcgg cgcgaggatg gttctctctg gtcatagccc gaagtctcat ggcagtccca


 


3601
aaggaggctt acaactcctg catcacaaga aaaaggaagc cactgccagc tggggggatc


 


3661
tgcagctccc agaagctccg tgagcctcag ccacccctca gactgggttc ctctccaagc


 


3721
tcgccctctg gaggggcagc gcagcctccc accaagggcc ctgcgaccac agcagggatt


 


3781
gggatgaatt gcctgtcctg gatctgctct agaggcccaa gctgcctgcc tgaggaagga


 


3841
tgacttgaca agtcaggaga cactgttccc aaagccttga ccagagcacc tcagcccgct


 


3901
gaccttgcac aaactccatc tgctgccatg agaaaaggga agccgccttt gcaaaacatt


 


3961
gctgcctaaa gaaactcagc agcctcaggc ccaattctgc cacttctggt ttgggtacag


 


4021
ttaaaggcaa ccctgaggga cttggcagta gaaatccagg gcctcccctg gggctggcag


 


4081
cttcgtgtgc agctagagct ttacctgaaa ggaagtctct gggcccagaa ctctccacca


 


4141
agagcctccc tgccgttcgc tgagtcccag caattctcct aagttgaagg gatctgagaa


 


4201
ggagaaggaa atgtggggta gatttggtgg tggttagaga tatgcccccc tcattactgc


 


4261
caacagtttc ggctgcattt cttcacgcac ctcggttcct cttcctgaag ttcttgtgcc


 


4321
ctgctcttca gcaccatggg ccttcttata cggaaggctc tgggatctcc cccttgtggg


 


4381
gcaggctctt ggggccagcc taagatcatg gtttagggtg atcagtgctg gcagataaat


 


4441
tgaaaaggca cgctggcttg tgatcttaaa tgaggacaat ccccccaggg ctgggcactc


 


4501
ctcccctccc ctcacttctc ccacctgcag agccagtgtc cttgggtggg ctagatagga


 


4561
tatactgtat gccggctcct tcaagctgct gactcacttt atcaatagtt ccatttaaat


 


4621
tgacttcagt ggtgagactg tatcctgttt gctattgctt gttgtgctat ggggggaggg


 


4681
gggaggaatg tgtaagatag ttaacatggg caaagggaga tcttggggtg cagcacttaa


 


4741
actgcctcgt aacccttttc atgatttcaa ccacatttgc tagagggagg gagcagccac


 


4801
ggagttagag gcccttgggg tttctctttt ccactgacag gctttcccag gcagctggct


 


4861
agttcattcc ctccccagcc aggtgcaggc gtaggaatat ggacatctgg ttgctttggc


 


4921
ctgctgccct ctttcagggg tcctaagccc acaatcatgc ctccctaaga ccttggcatc


 


4981
cttccctcta agccgttggc acctctgtgc cacctctcac actggctcca gacacacagc


 


5041
ctgtgctttt ggagctgaga tcactcgctt caccctcctc atctttgttc tccaagtaaa


 


5101
gccacgaggt cggggcgagg gcagaggtga tcacctgcgt gtcccatcta cagacctgca


 


5161
gcttcataaa acttctgatt tctcttcagc tttgaaaagg gttaccctgg gcactggcct


 


5221
agagcctcac ctcctaatag acttagcccc atgagtttgc catgttgagc aggactattt


 


5281
ctggcacttg caagtcccat gatttcttcg gtaattctga gggtgggggg agggacatga


 


5341
aatcatctta gcttagcttt ctgtctgtga atgtctatat agtgtattgt gtgttttaac


 


5401
aaatgattta cactgactgt tgctgtaaaa gtgaatttgg aaataaagtt attactctga


 


5461
ttaaa






In some embodiments, a MAPT RNAi agent includes an antisense strand wherein position 19 of the antisense strand (5′43′) is capable of forming a base pair with position 1 of a 19-mer target sequence disclosed in Table 1. In some embodiments, a MAPT agent includes an antisense strand wherein position 1 of the antisense strand (5′43′) is capable of forming a base pair with position 19 of a 19-mer target sequence disclosed in Table 1.
In some embodiments, a MAPT agent includes an antisense strand wherein position 2 of the antisense strand (5′→3′) is capable of forming a base pair with position 18 of a 19-mer target sequence disclosed in Table 1. In some embodiments, a MAPT agent includes an antisense strand wherein positions 2 through 18 of the antisense strand (5′→3′) are capable of forming base pairs with each of the respective complementary bases located at positions 18 through 2 of the 19-mer target sequence disclosed in Table 1.
For the RNAi agents disclosed herein, the nucleotide at position 1 of the antisense strand (from 5′ end→3′ end) can be perfectly complementary to a MAPT gene, or can be non-complementary to a MAPT gene. In some embodiments, the nucleotide at position 1 of the antisense strand (from 5′ end→3′ end) is a U, A, or dT. In some embodiments, the nucleotide at position 1 of the antisense strand (from 5′ end→3′ end) forms an A:U or U:A base pair with the sense strand.
In some embodiments, a MAPT RNAi agent antisense strand comprises the sequence of nucleotides (from 5′ end→3′ end) 2-18 or 2-19 of any of the antisense strand sequences in Table 2 or Table 3. In some embodiments, a MAPT RNAi sense strand comprises the sequence of nucleotides (from 5′ end→3′ end) 1-17, 1-18, or 2-18 of any of the sense strand sequences in Table 2, Table 4, Table 5, or Table 6.
In some embodiments, a MAPT RNAi agent is comprised of (i) an antisense strand comprising the sequence of nucleotides (from 5′ end→3′ end) 2-18 or 2-19 of any of the antisense strand sequences in Table 2 or Table 3, and (ii) a sense strand comprising the sequence of nucleotides (from 5′ end→3′ end) 1-17 or 1-18 of any of the sense strand sequences in Table 2, Table 4, Table 5, or Table 6.
In some embodiments, the MAPT RNAi agents include core 19-mer nucleotide sequences shown in the following Table 2.







TABLE 2







MAPT RNAi Agent Antisense Strand and Sense Strand Core Stretch Base Sequences


(N = any nucleobase; I = inosine (hypoxanthine nucleobase)













Antisense Strand Base

Sense Strand Base
Corresponding



SEQ
Sequence (5′→3′)
SEQ
Sequence (5′→3′)
Positions of
Targeted


ID
(Shown as an Unmodified
ID
(Shown as an Unmodified
Identified Sequence
Gene


NO:
Nucleotide Sequence)
NO:
Nucleotide Sequence)
on SEQ ID NO: 1
Position















 93
UUACGUCCCAGCGUGAUCU
319
AGAUCACGCUGGGACGUAA
 186-204
184


 


 94
AUACGUCCCAGCGUGAUCU
320
AGAUCACGCUGGGACGUAU
 186-204
184


 


 95
GUACGUCCCAGCGUGAUCU
321
AGAUCACGCUGGGACGUAC
 186-204
184


 


 96
NUACGUCCCAGCGUGAUCU
322
AGAUCACGCUGGGACGUAN
 186-204
184


 


 97
NUACGUCCCAGCGUGAUCN
323
NGAUCACGCUGGGACGUAN
 186-204
184


 


 98
UGUACGUCCCAGCGUGAUC
324
GAUCACGCUGGGACGUACA
 187-205
185


 


 99
AGUACGUCCCAGCGUGAUC
325
GAUCACGCUGGGACGUACU
 187-205
185


 


100
CGUACGUCCCAGCGUGAUC
326
GAUCACGCUGGGACGUACG
 187-205
185


 


101
NGUACGUCCCAGCGUGAUC
327
GAUCACGCUGGGACGUACN
 187-205
185


 


102
NGUACGUCCCAGCGUGAUN
328
NAUCACGCUGGGACGUACN
 187-205
185


 


103
UUGUCACCCUCUUGGUCUU
329
AAGACCAAGAGGGUGACAA
 248-266
246


 


104
AUGUCACCCUCUUGGUCUU
330
AAGACCAAGAGGGUGACAU
 248-266
246


 


105
GUGUCACCCUCUUGGUCUU
331
AAGACCAAGAGGGUGACAC
 248-266
246


 


106
NUGUCACCCUCUUGGUCUU
332
AAGACCAAGAGGGUGACAN
 248-266
246


 


107
NUGUCACCCUCUUGGUCUN
333
NAGACCAAGAGGGUGACAN
 248-266
246


 


108
UUGACCAGCAGCUUCGUCU
334
AGACGAAGCUGCUGGUCAA
 321-339
319


 


109
AUGACCAGCAGCUUCGUCU
335
AGACGAAGCUGCUGGUCAU
 321-339
319


 


110
GUGACCAGCAGCUUCGUCU
336
AGACGAAGCUGCUGGUCAC
 321-339
319


 


111
NUGACCAGCAGCUUCGUCU
337
AGACGAAGCUGCUGGUCAN
 321-339
319


 


112
NUGACCAGCAGCUUCGUCN
338
NGACGAAGCUGCUGGUCAN
 321-339
319


 


113
UACCAUGCGAGCUUGGGUC
339
GACCCAAGCUCGCAUGGUA
 342-360
340


 


114
AACCAUGCGAGCUUGGGUC
340
GACCCAAGCUCGCAUGGUU
 342-360
340


 


115
GACCAUGCGAGCUUGGGUC
341
GACCCAAGCUCGCAUGGUC
 342-360
340


 


116
NACCAUGCGAGCUUGGGUC
342
GACCCAAGCUCGCAUGGUN
 342-360
340


 


117
NACCAUGCGAGCUUGGGUN
343
NACCCAAGCUCGCAUGGUN
 342-360
340


 


118
AUACUGACCAUGCGAGCUU
344
AAGCUCGCAUGGUCAGUAU
 347-365
345


 


119
UUACUGACCAUGCGAGCUU
345
AAGCUCGCAUGGUCAGUAA
 347-365
345


 


120
NUACUGACCAUGCGAGCUU
346
AAGCUCGCAUGGUCAGUAN
 347-365
345


 


121
NUACUGACCAUGCGAGCUN
347
NAGCUCGCAUGGUCAGUAN
 347-365
345


 


122
AUUUACUGACCAUGCGAGC
348
GCUCGCAUGGUCAGUAAAU
 349-367
347


 


123
UUUUACUGACCAUGCGAGC
349
GCUCGCAUGGUCAGUAAAA
 349-367
347


 


124
NUUUACUGACCAUGCGAGC
350
GCUCGCAUGGUCAGUAAAN
 349-367
347


 


125
NUUUACUGACCAUGCGAGN
351
NCUCGCAUGGUCAGUAAAN
 349-367
347


 


126
UCUUUUACUGACCAUGCGA
352
UCGCAUGGUCAGUAAAAGA
 351-369
349


 


127
ACUUUUACUGACCAUGCGA
353
UCGCAUGGUCAGUAAAAGU
 351-369
349


 


128
GCUUUUACUGACCAUGCGA
354
UCGCAUGGUCAGUAAAAGC
 351-369
349


 


129
NCUUUUACUGACCAUGCGA
355
UCGCAUGGUCAGUAAAAGN
 351-369
349


 


130
NCUUUUACUGACCAUGCGN
356
NCGCAUGGUCAGUAAAAGN
 351-369
349


 


131
AUGCUUUUACUGACCAUGC
357
GCAUGGUCAGUAAAAGCAU
 353-371
351


 


132
UUGCUUUUACUGACCAUGC
358
GCAUGGUCAGUAAAAGCAA
 353-371
351


 


133
NUGCUUUUACUGACCAUGC
359
GCAUGGUCAGUAAAAGCAN
 353-371
351


 


134
NUGCUUUUACUGACCAUGN
360
NCAUGGUCAGUAAAAGCAN
 353-371
351


 


135
AUUGCUUUUACUGACCAUG
361
CAUGGUCAGUAAAAGCAAU
 354-372
352


 


136
UUUGCUUUUACUGACCAUG
362
CAUGGUCAGUAAAAGCAAA
 354-372
352


 


137
NUUGCUUUUACUGACCAUG
363
CAUGGUCAGUAAAAGCAAN
 354-372
352


 


138
NUUGCUUUUACUGACCAUN
364
NAUGGUCAGUAAAAGCAAN
 354-372
352


 


139
UCGUCUUUGCUUUUACUGA
365
UCAGUAAAAGCAAAGACGA
 359-377
357


 


140
ACGUCUUUGCUUUUACUGA
366
UCAGUAAAAGCAAAGACGU
 359-377
357


 


141
CCGUCUUUGCUUUUACUGA
367
UCAGUAAAAGCAAAGACGG
 359-377
357


 


142
NCGUCUUUGCUUUUACUGA
368
UCAGUAAAAGCAAAGACGN
 359-377
357


 


143
NCGUCUUUGCUUUUACUGN
369
NCAGUAAAAGCAAAGACGN
 359-377
357


 


144
AUUGUCAUCGCUUCCAGUC
370
GACUGGAAGCGAUGACAAU
 378-396
376


 


145
UUUGUCAUCGCUUCCAGUC
371
GACUGGAAGCGAUGACAAA
 378-396
376


 


146
NUUGUCAUCGCUUCCAGUC
372
GACUGGAAGCGAUGACAAN
 378-396
376


 


147
NUUGUCAUCGCUUCCAGUN
373
NACUGGAAGCGAUGACAAN
 378-396
376


 


148
AUUUUGUCAUCGCUUCCAG
374
CUGGAAGCGAUGACAAAAU
 380-398
378


 


149
UUUUUGUCAUCGCUUCCAG
375
CUGGAAGCGAUGACAAAAA
 380-398
378


 


150
NUUUUGUCAUCGCUUCCAG
376
CUGGAAGCGAUGACAAAAN
 380-398
378


 


151
NUUUUGUCAUCGCUUCCAN
377
NUGGAAGCGAUGACAAAAN
 380-398
378


 


152
UCUUUUUUGUCAUCGCUUC
378
GAAGCGAUGACAAAAAAGA
 383-401
381


 


153
ACUUUUUUGUCAUCGCUUC
379
GAAGCGAUGACAAAAAAGU
 383-401
381


 


154
GCUUUUUUGUCAUCGCUUC
380
GAAGCGAUGACAAAAAAGC
 383-401
381


 


155
NCUUUUUUGUCAUCGCUUC
381
GAAGCGAUGACAAAAAAGN
 383-401
381


 


156
NCUUUUUUGUCAUCGCUUN
382
NAAGCGAUGACAAAAAAGN
 383-401
381


 


157
UGUUUUUGCUGGAAUCCUG
383
CAGGAUUCCAGCAAAAACA
 483-501
481


 


158
AGUUUUUGCUGGAAUCCUG
384
CAGGAUUCCAGCAAAAACU
 483-501
481


 


159
GGUUUUUGCUGGAAUCCUG
385
CAGGAUUCCAGCAAAAACC
 483-501
481


 


160
NGUUUUUGCUGGAAUCCUG
386
CAGGAUUCCAGCAAAAACN
 483-501
481


 


161
NGUUUUUGCUGGAAUCCUN
387
NAGGAUUCCAGCAAAAACN
 483-501
481


 


162
UGGUUUUUGCUGGAAUCCU
388
AGGAUUCCAGCAAAAACCA
 484-502
482


 


163
AGGUUUUUGCUGGAAUCCU
389
AGGAUUCCAGCAAAAACCU
 484-502
482


 


164
GGGUUUUUGCUGGAAUCCU
390
AGGAUUCCAGCAAAAACCC
 484-502
482


 


165
NGGUUUUUGCUGGAAUCCU
391
AGGAUUCCAGCAAAAACCN
 484-502
482


 


166
NGGUUUUUGCUGGAAUCCN
392
NGGAUUCCAGCAAAAACCN
 484-502
482


 


167
UAGAGCUGGGUGGUGUCUU
393
AAGACACCACCCAGCUCUA
 514-532
512


 


168
AAGAGCUGGGUGGUGUCUU
394
AAGACACCACCCAGCUCUU
 514-532
512


 


169
CAGAGCUGGGUGGUGUCUU
395
AAGACACCACCCAGCUCUG
 514-532
512


 


170
NAGAGCUGGGUGGUGUCUU
396
AAGACACCACCCAGCUCUN
 514-532
512


 


171
NAGAGCUGGGUGGUGUCUN
397
NAGACACCACCCAGCUCUN
 514-532
512


 


172
AGAUUUUGGAGGUUCACCA
398
UGGUGAACCUCCAAAAUCU
 531-549
529


 


173
UGAUUUUGGAGGUUCACCA
399
UGGUGAACCUCCAAAAUCA
 531-549
529


 


174
NGAUUUUGGAGGUUCACCA
400
UGGUGAACCUCCAAAAUCN
 531-549
529


 


175
NGAUUUUGGAGGUUCACCN
401
NGGUGAACCUCCAAAAUCN
 531-549
529


 


176
UUGAUUUUGGAGGUUCACC
402
GGUGAACCUCCAAAAUCAA
 532-550
530


 


177
AUGAUUUUGGAGGUUCACC
403
GGUGAACCUCCAAAAUCAU
 532-550
530


 


178
CUGAUUUUGGAGGUUCACC
404
GGUGAACCUCCAAAAUCAG
 532-550
530


 


179
NUGAUUUUGGAGGUUCACC
405
GGUGAACCUCCAAAAUCAN
 532-550
530


 


180
NUGAUUUUGGAGGUUCACN
406
NGUGAACCUCCAAAAUCAN
 532-550
530


 


181
UCCUGAUUUUGGAGGUUCA
407
UGAACCUCCAAAAUCAGGA
 534-552
532


 


182
ACCUGAUUUUGGAGGUUCA
408
UGAACCUCCAAAAUCAGGU
 534-552
532


 


183
CCCUGAUUUUGGAGGUUCA
409
UGAACCUCCAAAAUCAGGG
 534-552
532


 


184
NCCUGAUUUUGGAGGUUCA
410
UGAACCUCCAAAAUCAGGN
 534-552
532


 


185
NCCUGAUUUUGGAGGUUCN
411
NGAACCUCCAAAAUCAGGN
 534-552
532


 


186
UUGCGAUCCCCUGAUUUUG
412
CAAAAUCAGGGGAUCGCAA
 542-560
540


 


187
AUGCGAUCCCCUGAUUUUG
413
CAAAAUCAGGGGAUCGCAU
 542-560
540


 


188
CUGCGAUCCCCUGAUUUUG
414
CAAAAUCAGGGGAUCGCAG
 542-560
540


 


189
NUGCGAUCCCCUGAUUUUG
415
CAAAAUCAGGGGAUCGCAN
 542-560
540


 


190
NUGCGAUCCCCUGAUUUUN
416
NAAAAUCAGGGGAUCGCAN
 542-560
540


 


191
UUUUGUAGACUAUUUGCAC
417
GUGCAAAUAGUCUACAAAA
 892-910
890


 


192
AUUUGUAGACUAUUUGCAC
418
GUGCAAAUAGUCUACAAAU
 892-910
890


 


193
GUUUGUAGACUAUUUGCAC
419
GUGCAAAUAGUCUACAAAC
 892-910
890


 


194
NUUUGUAGACUAUUUGCAC
420
GUGCAAAUAGUCUACAAAN
 892-910
890


 


195
NUUUGUAGACUAUUUGCAN
421
NUGCAAAUAGUCUACAAAN
 892-910
890


 


196
UACUGGUUUGUAGACUAUU
422
AAUAGUCUACAAACCAGUA
 897-915
895


 


197
AACUGGUUUGUAGACUAUU
423
AAUAGUCUACAAACCAGUU
 897-915
895


 


198
NACUGGUUUGUAGACUAUU
424
AAUAGUCUACAAACCAGUN
 897-915
895


 


199
NACUGGUUUGUAGACUAUN
425
NAUAGUCUACAAACCAGUN
 897-915
895


 


200
ACAACUGGUUUGUAGACUA
426
UAGUCUACAAACCAGUUGU
 899-917
897


 


201
UCAACUGGUUUGUAGACUA
427
UAGUCUACAAACCAGUUGA
 899-917
897


 


202
NCAACUGGUUUGUAGACUA
428
UAGUCUACAAACCAGUUGN
 899-917
897


 


203
NCAACUGGUUUGUAGACUN
429
NAGUCUACAAACCAGUUGN
 899-917
897


 


204
UUCAACUGGUUUGUAGACU
430
AGUCUACAAACCAGUUGAA
 900-918
898


 


205
AUCAACUGGUUUGUAGACU
431
AGUCUACAAACCAGUUGAU
 900-918
898


 


206
GUCAACUGGUUUGUAGACU
432
AGUCUACAAACCAGUUGAC
 900-918
898


 


207
NUCAACUGGUUUGUAGACU
433
AGUCUACAAACCAGUUGAN
 900-918
898


 


208
NUCAACUGGUUUGUAGACN
434
NGUCUACAAACCAGUUGAN
 900-918
898


 


209
UGGUCAACUGGUUUGUAGA
435
UCUACAAACCAGUUGACCA
 902-920
900


 


210
AGGUCAACUGGUUUGUAGA
436
UCUACAAACCAGUUGACCU
 902-920
900


 


211
NGGUCAACUGGUUUGUAGA
437
UCUACAAACCAGUUGACCN
 902-920
900


 


212
NGGUCAACUGGUUUGUAGN
438
NCUACAAACCAGUUGACCN
 902-920
900


 


213
UCUUGCUCAGGUCAACUGG
439
CCAGUUGACCUGAGCAAGA
 910-928
908


 


214
ACUUGCUCAGGUCAACUGG
440
CCAGUUGACCUGAGCAAGU
 910-928
908


 


215
CCUUGCUCAGGUCAACUGG
441
CCAGUUGACCUGAGCAAGG
 910-928
908


 


216
NCUUGCUCAGGUCAACUGG
442
CCAGUUGACCUGAGCAAGN
 910-928
908


 


217
NCUUGCUCAGGUCAACUGN
443
NCAGUUGACCUGAGCAAGN
 910-928
908


 


218
ACACCUUGCUCAGGUCAAC
444
GUUGACCUGAGCAAGGUGU
 913-931
911


 


219
UCACCUUGCUCAGGUCAAC
445
GUUGACCUGAGCAAGGUGA
 913-931
911


 


220
NCACCUUGCUCAGGUCAAC
446
GUUGACCUGAGCAAGGUGN
 913-931
911


 


221
NCACCUUGCUCAGGUCAAN
447
NUUGACCUGAGCAAGGUGN
 913-931
911


 


222
UACACUUGGAGGUCACCUU
448
AAGGUGACCUCCAAGUGUA
 925-943
923


 


223
AACACUUGGAGGUCACCUU
449
AAGGUGACCUCCAAGUGUU
 925-943
923


 


224
CACACUUGGAGGUCACCUU
450
AAGGUGACCUCCAAGUGUG
 925-943
923


 


225
NACACUUGGAGGUCACCUU
451
AAGGUGACCUCCAAGUGUN
 925-943
923


 


226
NACACUUGGAGGUCACCUN
452
NAGGUGACCUCCAAGUGUN
 925-943
923


 


227
AAAUGAGCCACACUUGGAG
453
CUCCAAGUGUGGCUCAUUU
 933-951
931


 


228
UAAUGAGCCACACUUGGAG
454
CUCCAAGUGUGGCUCAUUA
 933-951
931


 


229
NAAUGAGCCACACUUGGAG
455
CUCCAAGUGUGGCUCAUUN
 933-951
931


 


230
NAAUGAGCCACACUUGGAN
456
NUCCAAGUGUGGCUCAUUN
 933-951
931


 


231
UUAAUGAGCCACACUUGGA
457
UCCAAGUGUGGCUCAUUAA
 934-952
932


 


232
AUAAUGAGCCACACUUGGA
458
UCCAAGUGUGGCUCAUUAU
 934-952
932


 


233
CUAAUGAGCCACACUUGGA
459
UCCAAGUGUGGCUCAUUAG
 934-952
932


 


234
NUAAUGAGCCACACUUGGA
460
UCCAAGUGUGGCUCAUUAN
 934-952
932


 


235
NUAAUGAGCCACACUUGGN
461
NCCAAGUGUGGCUCAUUAN
 934-952
932


 


236
UCUAAUGAGCCACACUUGG
462
CCAAGUGUGGCUCAUUAGA
 935-953
933


 


237
ACUAAUGAGCCACACUUGG
463
CCAAGUGUGGCUCAUUAGU
 935-953
933


 


238
CCUAAUGAGCCACACUUGG
464
CCAAGUGUGGCUCAUUAGG
 935-953
933


 


239
NCUAAUGAGCCACACUUGG
465
CCAAGUGUGGCUCAUUAGN
 935-953
933


 


240
NCUAAUGAGCCACACUUGN
466
NCAAGUGUGGCUCAUUAGN
 935-953
933


 


241
UCCUAAUGAGCCACACUUG
467
CAAGUGUGGCUCAUUAGGA
 936-954
934


 


242
ACCUAAUGAGCCACACUUG
468
CAAGUGUGGCUCAUUAGGU
 936-954
934


 


243
GCCUAAUGAGCCACACUUG
469
CAAGUGUGGCUCAUUAGGC
 936-954
934


 


244
NCCUAAUGAGCCACACUUG
470
CAAGUGUGGCUCAUUAGGN
 936-954
934


 


245
NCCUAAUGAGCCACACUUN
471
NAAGUGUGGCUCAUUAGGN
 936-954
934


 


246
AGCCUAAUGAGCCACACUU
472
AAGUGUGGCUCAUUAGGCU
 937-955
935


 


247
UGCCUAAUGAGCCACACUU
473
AAGUGUGGCUCAUUAGGCA
 937-955
935


 


248
NGCCUAAUGAGCCACACUU
474
AAGUGUGGCUCAUUAGGCN
 937-955
935


 


249
NGCCUAAUGAGCCACACUN
475
NAGUGUGGCUCAUUAGGCN
 937-955
935


 


250
UGAUGUUGCCUAAUGAGCC
476
GGCUCAUUAGGCAACAUCA
 943-961
941


 


251
AGAUGUUGCCUAAUGAGCC
477
GGCUCAUUAGGCAACAUCU
 943-961
941


 


252
GGAUGUUGCCUAAUGAGCC
478
GGCUCAUUAGGCAACAUCC
 943-961
941


 


253
NGAUGUUGCCUAAUGAGCC
479
GGCUCAUUAGGCAACAUCN
 943-961
941


 


254
NGAUGUUGCCUAAUGAGCN
480
NGCUCAUUAGGCAACAUCN
 943-961
941


 


255
AUAUGAUGGAUGUUGCCUA
481
UAGGCAACAUCCAUCAUAU
 950-968
948


 


256
UUAUGAUGGAUGUUGCCUA
482
UAGGCAACAUCCAUCAUAA
 950-968
948


 


257
NUAUGAUGGAUGUUGCCUA
483
UAGGCAACAUCCAUCAUAN
 950-968
948


 


258
NUAUGAUGGAUGUUGCCUN
484
NAGGCAACAUCCAUCAUAN
 950-968
948


 


259
AUUAUGAUGGAUGUUGCCU
485
AGGCAACAUCCAUCAUAAU
 951-969
949


 


260
UUUAUGAUGGAUGUUGCCU
486
AGGCAACAUCCAUCAUAAA
 951-969
949


 


261
NUUAUGAUGGAUGUUGCCU
487
AGGCAACAUCCAUCAUAAN
 951-969
949


 


262
NUUAUGAUGGAUGUUGCCN
488
NGGCAACAUCCAUCAUAAN
 951-969
949


 


263
UGUUUAUGAUGGAUGUUGC
489
GCAACAUCCAUCAUAAACA
 953-971
951


 


264
AGUUUAUGAUGGAUGUUGC
490
GCAACAUCCAUCAUAAACU
 953-971
951


 


265
GGUUUAUGAUGGAUGUUGC
491
GCAACAUCCAUCAUAAACC
 953-971
951


 


266
NGUUUAUGAUGGAUGUUGC
492
GCAACAUCCAUCAUAAACN
 953-971
951


 


267
NGUUUAUGAUGGAUGUUGN
493
NCAACAUCCAUCAUAAACN
 953-971
951


 


268
AUUACUUCCACCUGGCCAC
494
GUGGCCAGGUGGAAGUAAU
 977-995
975


 


269
UUUACUUCCACCUGGCCAC
495
GUGGCCAGGUGGAAGUAAA
 977-995
975


 


270
NUUACUUCCACCUGGCCAC
496
GUGGCCAGGUGGAAGUAAN
 977-995
975


 


271
NUUACUUCCACCUGGCCAN
497
NUGGCCAGGUGGAAGUAAN
 977-995
975


 


272
UGAUUUUACUUCCACCUGG
498
CCAGGUGGAAGUAAAAUCA
 981-999
979


 


273
AGAUUUUACUUCCACCUGG
499
CCAGGUGGAAGUAAAAUCU
 981-999
979


 


274
NGAUUUUACUUCCACCUGG
500
CCAGGUGGAAGUAAAAUCN
 981-999
979


 


275
NGAUUUUACUUCCACCUGN
501
NCAGGUGGAAGUAAAAUCN
 981-999
979


 


276
UAGAUUUUACUUCCACCUG
502
CAGGUGGAAGUAAAAUCUA
 982-1000
980


 


277
AAGAUUUUACUUCCACCUG
503
CAGGUGGAAGUAAAAUCUU
 982-1000
980


 


278
CAGAUUUUACUUCCACCUG
504
CAGGUGGAAGUAAAAUCUG
 982-1000
980


 


279
NAGAUUUUACUUCCACCUG
505
CAGGUGGAAGUAAAAUCUN
 982-1000
980


 


280
NAGAUUUUACUUCCACCUN
506
NAGGUGGAAGUAAAAUCUN
 982-1000
980


 


281
UUGAUAUUGUCCAGGGACC
507
GGUCCCUGGACAAUAUCAA
1040-1058
1038


 


282
AUGAUAUUGUCCAGGGACC
508
GGUCCCUGGACAAUAUCAU
1040-1058
1038


 


283
GUGAUAUUGUCCAGGGACC
509
GGUCCCUGGACAAUAUCAC
1040-1058
1038


 


284
NUGAUAUUGUCCAGGGACC
510
GGUCCCUGGACAAUAUCAN
1040-1058
1038


 


285
NUGAUAUUGUCCAGGGACN
511
NGUCCCUGGACAAUAUCAN
1040-1058
1038


 


286
UGGUGAUAUUGUCCAGGGA
512
UCCCUGGACAAUAUCACCA
1042-1060
1040


 


287
AGGUGAUAUUGUCCAGGGA
513
UCCCUGGACAAUAUCACCU
1042-1060
1040


 


288
GGGUGAUAUUGUCCAGGGA
514
UCCCUGGACAAUAUCACCC
1042-1060
1040


 


289
NGGUGAUAUUGUCCAGGGA
515
UCCCUGGACAAUAUCACCN
1042-1060
1040


 


290
NGGUGAUAUUGUCCAGGGN
516
NCCCUGGACAAUAUCACCN
1042-1060
1040


 


291
UGCUUGUGGGUUUCAAUCU
517
AGAUUGAAACCCACAAGCA
1085-1103
1083


 


292
AGCUUGUGGGUUUCAAUCU
518
AGAUUGAAACCCACAAGCU
1085-1103
1083


 


293
NGCUUGUGGGUUUCAAUCU
519
AGAUUGAAACCCACAAGCN
1085-1103
1083


 


294
NGCUUGUGGGUUUCAAUCN
520
NGAUUGAAACCCACAAGCN
1085-1103
1083


 


295
ACAGCUUGUGGGUUUCAAU
521
AUUGAAACCCACAAGCUGU
1087-1105
1085


 


296
UCAGCUUGUGGGUUUCAAU
522
AUUGAAACCCACAAGCUGA
1087-1105
1085


 


297
NCAGCUUGUGGGUUUCAAU
523
AUUGAAACCCACAAGCUGN
1087-1105
1085


 


298
NCAGCUUGUGGGUUUCAAN
524
NUUGAAACCCACAAGCUGN
1087-1105
1085


 


299
UUCAGCUUGUGGGUUUCAA
525
UUGAAACCCACAAGCUGAA
1088-1106
1086


 


300
AUCAGCUUGUGGGUUUCAA
526
UUGAAACCCACAAGCUGAU
1088-1106
1086


 


301
GUCAGCUUGUGGGUUUCAA
527
UUGAAACCCACAAGCUGAC
1088-1106
1086


 


302
NUCAGCUUGUGGGUUUCAA
528
UUGAAACCCACAAGCUGAN
1088-1106
1086


 


303
NUCAGCUUGUGGGUUUCAN
529
NUGAAACCCACAAGCUGAN
1088-1106
1086


 


304
UGUCUGUCUUGGCUUUGGC
530
GCCAAAGCCAAGACAGACA
1120-1138
1118


 


305
AGUCUGUCUUGGCUUUGGC
531
GCCAAAGCCAAGACAGACU
1120-1138
1118


 


306
GGUCUGUCUUGGCUUUGGC
532
GCCAAAGCCAAGACAGACC
1120-1138
1118


 


307
NGUCUGUCUUGGCUUUGGC
533
GCCAAAGCCAAGACAGACN
1120-1138
1118


 


308
NGUCUGUCUUGGCUUUGGN
534
NCCAAAGCCAAGACAGACN
1120-1138
1118


 


309
UUCUACCAUGUCGAUGCUG
535
CAGCAUCGACAUGGUAGAA
1221-1239
1219


 


310
AUCUACCAUGUCGAUGCUG
536
CAGCAUCGACAUGGUAGAU
1221-1239
1219


 


311
GUCUACCAUGUCGAUGCUG
537
CAGCAUCGACAUGGUAGAC
1221-1239
1219


 


312
NUCUACCAUGUCGAUGCUG
538
CAGCAUCGACAUGGUAGAN
1221-1239
1219


 


313
NUCUACCAUGUCGAUGCUN
539
NAGCAUCGACAUGGUAGAN
1221-1239
1219


 


314
UAGUCUACCAUGUCGAUGC
540
GCAUCGACAUGGUAGACUA
1223-1241
1221


 


315
AAGUCUACCAUGUCGAUGC
541
GCAUCGACAUGGUAGACUU
1223-1241
1221


 


316
GAGUCUACCAUGUCGAUGC
542
GCAUCGACAUGGUAGACUC
1223-1241
1221


 


317
NAGUCUACCAUGUCGAUGC
543
GCAUCGACAUGGUAGACUN
1223-1241
1221


 


318
NAGUCUACCAUGUCGAUGN
544
NCAUCGACAUGGUAGACUN
1223-1241
1221









The MAPT RNAi agent sense strands and antisense strands that comprise or consist of the nucleotide sequences in Table 2 can be modified nucleotides or unmodified nucleotides. In some embodiments, the MAPT RNAi agents having the sense and antisense strand sequences that comprise or consist of any of the nucleotide sequences in Table 2 are all or substantially all modified nucleotides.
In some embodiments, the antisense strand of a MAPT RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences in Table 2. In some embodiments, the sense strand of a MAPT RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the sense strand sequences in Table 2.
As used herein, each N listed in a sequence disclosed in Table 2 may be independently selected from any and all nucleobases (including those found on both modified and unmodified nucleotides). In some embodiments, an N nucleotide listed in a sequence disclosed in Table 2 has a nucleobase that is complementary to the N nucleotide at the corresponding position on the other strand. In some embodiments, an N nucleotide listed in a sequence disclosed in Table 2 has a nucleobase that is not complementary to the N nucleotide at the corresponding position on the other strand. In some embodiments, an N nucleotide listed in a sequence disclosed in Table 2 has a nucleobase that is the same as the N nucleotide at the corresponding position on the other strand. In some embodiments, an N nucleotide listed in a sequence disclosed in Table 2 has a nucleobase that is different from the N nucleotide at the corresponding position on the other strand.
Certain modified MAPT RNAi agent sense and antisense strands are provided in Table 3, Table 4, Table 5, Table 6, and Table 10. Certain modified MAPT RNAi agent antisense strands, as well as their underlying unmodified nucleobase sequences, are provided in Table 3. Certain modified MAPT RNAi agent sense strands, as well as their underlying unmodified nucleobase sequences, are provided in Tables 4, 5, and 6. In forming MAPT RNAi agents, each of the nucleotides in each of the underlying base sequences listed in Tables 3, 4, 5, and 6, as well as in Table 2, above, can be a modified nucleotide.
The MAPT RNAi agents described herein are formed by annealing an antisense strand with a sense strand. A sense strand containing a sequence listed in Table 2, Table 4, Table 5, or Table 6, can be hybridized to any antisense strand containing a sequence listed in Table 2 or Table 3, provided the two sequences have a region of at least 85% complementarity over a contiguous 16, 17, 18, 19, 20, or 21 nucleotide sequence.
In some embodiments, a MAPT RNAi agent antisense strand comprises a nucleotide sequence of any of the sequences in Table 2 or Table 3.
In some embodiments, a MAPT RNAi agent comprises or consists of a duplex having the nucleobase sequences of the sense strand and the antisense strand of any of the sequences in Table 2, Table 3, Table 4, Table 5, Table 6, or Table 9.
Examples of antisense strands containing modified nucleotides are provided in Table 3. Examples of sense strands containing modified nucleotides are provided in Tables 4, 5 and 6.
As used in Tables 3, 4, 5, 6, and 10, the following notations are used to indicate modified nucleotides, targeting groups, and linking groups:

    
    
        A=adenosine-3′-phosphate
        C=cytidine-3′-phosphate
        G=guanosine-3′-phosphate
        U=uridine-3′-phosphate
        I=inosine-3′-phosphate
        a=2′-O-methyladenosine-3′-phosphate
        as =2′-O-methyladenosine-3′-phosphorothioate
        c=2′-O-methylcytidine-3′-phosphate
        cs=2′-O-methylcytidine-3′-phosphorothioate
        g=2′-O-methylguanosine-3′-phosphate
        gs=2′-O-methylguanosine-3′-phosphorothioate
        i=2′-O-methylinosine-3′-phosphate
        is=2′-O-methylinosine-3′-phosphorothioate
        t=2′-O-methyl-5-methyluridine-3′-phosphate
        ts=2′-O-methyl-5-methyluridine-3′-phosphorothioate
        u=2′-O-methyluridine-3′-phosphate
        us=2′-O-methyluridine-3′-phosphorothioate
        Af=2′-fluoroadenosine-3′-phosphate
        Afs=2′-fluoroadenosine-3′-phosporothioate
        Cf=2′-fluorocytidine-3′-phosphate
        Cfs=2′-fluorocytidine-3′-phosphorothioate
        Gf=2′-fluoroguanosine-3′-phosphate
        Gfs=2′-fluoroguanosine-3′-phosphorothioate
        Tf=2′-fluoro-5′-methyluridine-3′-phosphate
        Tfs=2′-fluoro-5′-methyluridine-3′-phosphorothioate
        Uf=2′-fluorouridine-3′-phosphate
        Ufs=2′-fluorouridine-3′-phosphorothioate
        dT=2′-deoxythymidine-3′-phosphate
        dT=2′-deoxythymidine-3′-phosphorothioate
        dU=2′-deoxyuridine-3′-phosphate
        dUs=2′-deoxyuridine-3′-phosphorothioate
        dA=2′-deoxyadenosine-3′-phosphate
        dAs=2′-deoxyadenosine-3′-phosphorothioate
        dG=2′-deoxyguanosine-3′-phosphate
        dGs=2′-deoxyguanosine-3′-phosphorothioate
        dC=2′-deoxycytidine-3′-phosphate
        dCs=2′-deoxycytidine-3′-phosphorothioate
        AUNA=2′,3′-seco-adenosine-3′-phosphate
        AUNAs=2′,3′-seco-adenosine-3′-phosphorothioate
        CUNA=2′,3′-seco-cytidine-3′-phosphate
        CUNAs=2′,3′-seco-cytidine-3′-phosphorothioate
        GUNA=2′,3′-seco-guanosine-3′-phosphate
        GUNAs=2′,3′-seco-guanosine-3′-phosphorothioate
        UUNA=2′,3′-seco-uridine-3′-phosphate
        UUNAs=2′,3′-seco-uridine-3′-phosphorothioate
        a_2N=2′-O-methyl-2-aminoadenosine-3′-phosphate, see Table 10
        a_2Ns=2′-O-methyl-2-aminoadenosine-3′-phosphorothioate, see Table 10
        (invAb)=inverted abasic deoxyribonucleotide-5′-phosphate, see Table 10
        (invAb)s=inverted abasic deoxyribonucleotide-5′-phosphorothioate, see Table 10
        s=phosphorothioate linkage
        ss=phosphorodithioate linkage
        p=terminal phosphate (as synthesized)
        vpu=vinyl phosphonate 2′-O-methyluridine-3′-phosphate
        cPrpa=5′-cyclopropyl phosphonate-2′-O-methyladenosine-3′-phosphate (see Table 10)
        cPrpas=5′-cyclopropyl phosphonate-2′-O-methyladenosine-3′-phosphorothioate (see Table 10)
        cPrpu=5′-cyclopropyl phosphonate-2′-O-methyluridine-3′-phosphate (see Table 10)
        cPrpus=5′-cyclopropyl phosphonate-2′-O-methyluridine-3′-phosphorothioate (see Table 10)
        (NH2-C6)=see Table 10
        (NH-C6)=see Table 10
        (NH-C6)s=see Table 10
        (L20)=see Table 10
        LP293=see Table 10
        LP310=see Table 10
        LP429=see Table 10
        LP462=see Table 10
        LP183=see Table 10
        L-1026=see Table 10
        uC16=see Table 10
        [CP-1113]=Fabs were capped according to the procedure in Example 1E; (see also Table 10 for structure)
        Fab0070=see Antigen Binding Proteins, infra
    
    


As the person of ordinary skill in the art would readily understand, unless otherwise indicated by the sequence (such as, for example, by a phosphorothioate linkage “s”), when present in an oligonucleotide, the nucleotide monomers are mutually linked by 5′-3′-phosphodiester bonds. As the person of ordinary skill in the art would clearly understand, the inclusion of a phosphorothioate linkage as shown in the modified nucleotide sequences disclosed herein replaces the phosphodiester linkage typically present in oligonucleotides. Further, the person of ordinary skill in the art would readily understand that the terminal nucleotide at the 3′ end of a given oligonucleotide sequence would typically have a hydroxyl (—OH) group at the respective 3′ position of the given monomer instead of a phosphate moiety ex vivo. Additionally, for the embodiments disclosed herein, when viewing the respective strand 5′→3′, the inverted abasic residues are inserted such that the 3′ position of the deoxyribose is linked at the 3′ end of the preceding monomer on the respective strand (see, e.g., Table 10). Moreover, as the person of ordinary skill would readily understand and appreciate, while the phosphorothioate chemical structures depicted herein typically show the anion on the sulfur atom, the inventions disclosed herein encompass all phosphorothioate tautomers (e.g., where the sulfur atom has a double-bond and the anion is on an oxygen atom). Unless expressly indicated otherwise herein, such understandings of the person of ordinary skill in the art are used when describing the MAPT RNAi agents and compositions of MAPT RNAi agents disclosed herein.
Certain examples of antigen binding proteins and linking groups used with the MAPT RNAi agents disclosed herein are included in the chemical structures provided below in Table 10. Each sense strand and/or antisense strand can have any antigen binding protein or linking group listed herein, as well as other targeting groups, antigen binding proteins, linking groups, conjugated to the 5′ and/or 3′ end of the sequence.
Certain examples of PK/PD modulators and linking groups used with the MAPT RNAi agents disclosed herein are included in the chemical structures provided below in Table 10. Each sense strand and/or antisense strand can have any PK/PD modulators or linking groups listed herein, as well as other targeting groups, PK/PD modulators, linking groups, conjugated to the 5′ and/or 3′ end of the sequence.







TABLE 3







MAPT RNAi Agent Antisense Strand Sequences














Underlying Base Sequence





SEQ
(5′ → 3′) (Shown as an
SEQ


AS Strand
Modified Antisense Strand
ID
Unmodified Nucleotide
ID


ID
(5′ → 3′)
NO.
Sequence)
NO.





CA004894
cPrpusAfsgucuAfccauGfuUfgAfugcussg
545
UAGUCUACCAUGUUGAUGCUG
741


 


CA004895
cPrpusAfsgucuAfcuauGfuCfgAfugcussg
546
UAGUCUACUAUGUCGAUGCUG
742


 


CA004897
cPrpusAfsgucuAfCUNAcauGfuCfgAfugcussg
547
UAGUCUACCAUGUCGAUGCUG
743


 


CA004898
cPrpusAfsgucUUNAAfccauGfuCfgAfugcussg
548
UAGUCUACCAUGUCGAUGCUG
743


 


CA004899
cPrpusAfsgucudAccauGfuCfgAfugcussg
549
UAGUCUACCAUGUCGAUGCUG
743


 


CA005104
vpusAfsccdAudAcgagcuUfgGfgucacsgsu
550
UACCAUACGAGCUUGGGUCACGU
744


 


CA005435
cPrpusAfsgucUUNAAfccauGfuUfgAfugcussg
551
UAGUCUACCAUGUUGAUGCUG
741


 


CA005471
cPrpusAfsgucuAfccauGfudTgAfugcussg
552
UAGUCUACCAUGUTGAUGCUG
745


 


CA006181
cPrpusUfsgcuUUNAUfuacuGfaCfcAfugcgssa
553
UUGCUUUUACUGACCAUGCGA
746


 


CA914417
cPrpusUfsasCfgUfcccagCfgUfgAfuCfuusc
554
UUACGUCCCAGCGUGAUCUUC
747


 


CA914420
cPrpusUfsgsAfcCfagcagCfuUfcGfuCfuusc
555
UUGACCAGCAGCUUCGUCUUC
748


 


CA914422
cPrpusUfsasCfuGfaccauGfcGfaGfcUfugsg
556
UUACUGACCAUGCGAGCUUGG
749


 


CA914424
cPrpusUfsusGfcUfuuuacUfgAfcCfaUfgcsg
557
UUUGCUUUUACUGACCAUGCG
750


 


CA914426
cPrpusCfsgsUfcUfuugcuUfuUfaCfuGfacsc
558
UCGUCUUUGCUUUUACUGACC
751


 


CA914428
cPrpusUfsusUfuGfucaucGfcUfuCfcAfgusc
559
UUUUUGUCAUCGCUUCCAGUC
752


 


CA914430
cPrpusCfsusUfuUfuugucAfuCfgCfuUfccsa
560
UCUUUUUUGUCAUCGCUUCCA
753


 


CA914432
cPrpusGfsusUfuUfugcugGfaAfuCfcUfggsu
561
UGUUUUUGCUGGAAUCCUGGU
754


 


CA914434
cPrpusGfsasUfuUfuggagGfuUfcAfcCfagsa
562
UGAUUUUGGAGGUUCACCAGA
755


 


CA914436
cPrpusCfscsUfgAfuuuugGfaGfgUfuCfacsc
563
UCCUGAUUUUGGAGGUUCACC
756


 


CA914438
cPrpusUfsgsCfgAfuccccUfgAfuUfuUfggsa
564
UUGCGAUCCCCUGAUUUUGGA
757


 


CA914440
cPrpasAfscsUfgGfuuuguAfgAfcUfaUfuusg
565
AACUGGUUUGUAGACUAUUUG
758


 


CA914443
cPrpusCfsasAfcUfgguuuGfuAfgAfcUfausu
566
UCAACUGGUUUGUAGACUAUU
759


 


CA914445
cPrpusUfscsAfaCfugguuUfgUfaGfaCfuasu
567
UUCAACUGGUUUGUAGACUAU
760


 


CA914447
cPrpasGfsgsUfcAfacuggUfuUfgUfaGfacsu
568
AGGUCAACUGGUUUGUAGACU
761


 


CA914449
cPrpusCfsusUfgCfucaggUfcAfaCfuGfgusu
569
UCUUGCUCAGGUCAACUGGUU
762


 


CA914451
cPrpusAfsasUfgAfgccacAfcUfuGfgAfggsu
570
UAAUGAGCCACACUUGGAGGU
763


 


CA914453
cPrpusCfsusAfaUfgagccAfcAfcUfuGfgasg
571
UCUAAUGAGCCACACUUGGAG
764


 


CA914455
cPrpusCfscsUfaAfugagcCfaCfaCfuUfggsa
572
UCCUAAUGAGCCACACUUGGA
765


 


CA914457
cPrpusGfscsCfuAfaugagCfcAfcAfcUfugsg
573
UGCCUAAUGAGCCACACUUGG
766


 


CA914459
cPrpusGfsasUfgUfugccuAfaUfgAfgCfcasc
574
UGAUGUUGCCUAAUGAGCCAC
767


 


CA914461
cPrpusUfsasUfgAfuggauGfuUfgCfcUfaasu
575
UUAUGAUGGAUGUUGCCUAAU
768


 


CA914463
cPrpusUfsusAfuGfauggaUfgUfuGfcCfuasc
576
UUUAUGAUGGAUGUUGCCUAC
769


 


CA914465
cPrpusGfsusUfuAfugaugGfaUfgUfuGfccsu
577
UGUUUAUGAUGGAUGUUGCCU
770


 


CA914467
cPrpasGfsasUfuUfuacuuCfcAfcCfuGfgcsc
578
AGAUUUUACUUCCACCUGGCC
771


 


CA914469
cPrpasGfscsUfuGfuggguUfuCfaAfuCfuusc
579
AGCUUGUGGGUUUCAAUCUUC
772


 


CA914471
cPrpusCfsasGfcUfuguggGfuUfuCfaAfucsu
580
UCAGCUUGUGGGUUUCAAUCU
773


 


CA914473
cPrpusUfscsAfgCfuugugGfgUfuUfcAfausc
581
UUCAGCUUGUGGGUUUCAAUC
774


 


CA914475
cPrpusGfsusCfuGfucuugGfcUfuUfgGfcgsu
582
UGUCUGUCUUGGCUUUGGCGU
775


 


CA914477
cPrpusAfsgsUfcUfaccauGfuCfgAfuGfcusg
583
UAGUCUACCAUGUCGAUGCUG
743


 


CA915404
cPrpusGfsccuaAfugagCfcAfcAfcuugsg
584
UGCCUAAUGAGCCACACUUGG
766


 


CA915423
cPrpusAfsgsuCfuaccauGfuCfgAfugcusg
585
UAGUCUACCAUGUCGAUGCUG
743


 


CA915424
cPrpusAfsgsucuAfccauGfuCfgAfugcusg
586
UAGUCUACCAUGUCGAUGCUG
743


 


CA915425
cPrpusAfsgsucuacCfauGfuCfgAfugcusg
587
UAGUCUACCAUGUCGAUGCUG
743


 


CA915426
cPrpusAfsgucuAfccauGfuCfgAfugcusg
588
UAGUCUACCAUGUCGAUGCUG
743


 


CA915430
cPrpusAfsgsucuAfccauGfuCfgaugcusg
589
UAGUCUACCAUGUCGAUGCUG
743


 


CA915431
cPrpusAfsgsucuacCfauguCfgaugcusg
590
UAGUCUACCAUGUCGAUGCUG
743


 


CA915432
cPrpusAfsgsucuAfccauguCfgaugcUfsg
591
UAGUCUACCAUGUCGAUGCUG
743


 


CA915905
cPrpusAfsgucuAfccauGfuCfgAfugcussg
592
UAGUCUACCAUGUCGAUGCUG
743


 


CA916146
cPrpusAfsgucuAfccauGfuCfgAfugcsusg
593
UAGUCUACCAUGUCGAUGCUG
743


 


CA916147
cPrpusAfsgucuAUNAccauGfuCfgAfugcusg
594
UAGUCUACCAUGUCGAUGCUG
743


 


CA916148
cPrpusAfsgucudAccauGfuCfgdAugcusg
595
UAGUCUACCAUGUCGAUGCUG
743


 


CA916149
cPrpusAfsgucudAccaudGuCfgdAugcusg
596
UAGUCUACCAUGUCGAUGCUG
743
















TABLE 4







MAPT RNAi Agent Sense Strand Sequences (Shown Without Linkers,


Conjugates, or Capping Moieties)














Underlying Base






Sequence (5′ → 3′)




Modified Sense Strand
SEQ ID
(Shown as an Unmodified
SEQ ID


Strand ID
(5′ → 3′)
NO.
Nucleotide Sequence)
NO.





CS004896-NL
cagcaucgAfuAfUfgguagacua
597
CAGCAUCGAUAUGGUAGACUA
776


 


CS005096-NL
cagcaucgAfcAfUfgguagacua
598
CAGCAUCGACAUGGUAGACUA
777


 


CS005097-NL
cagcaucgAfcAfUfgguagacua
599
CAGCAUCGACAUGGUAGACUA
777


 


CS005098-NL
cagcaucgAfcAfUfgguagacua
600
CAGCAUCGACAUGGUAGACUA
777


 


CS005099-NL
cagcauC16cgAfcAfUfgguagacua
601
CAGCAUCGACAUGGUAGACUA
777


 


CS005103-NL
gsusgaccC16caAfGfCfucguauggsusa
602
GUGACCCAAGCUCGUAUGGUA
778


 


CS005303-NL
ccaaguGfuGfgCfucauuaggua
603
CCAAGUGUGGCUCAUUAGGUA
779


 


CS005470-NL
cagcaucaAfcAfUfgguagacua
604
CAGCAUCAACAUGGUAGACUA
780


 


CS006178-NL
ucgcauggUfcAfguaaaagcaa
605
UCGCAUGGUCAGUAAAAGCAA
781


 


CS008817-NL
cagcaucgAfcAfUfgguagacua
606
CAGCAUCGACAUGGUAGACUA
777


 


CS011528-NL
cagcaucgAfcAfUfgguagacua
607
CAGCAUCGACAUGGUAGACUA
777


 


CS914416-NL
gaagaucaCfGfCfugggaciuaa
608
GAAGAUCACGCUGGGACIUAA
782


 


CS914418-NL
gaagaucaCfGfCfugggauguaa
609
GAAGAUCACGCUGGGAUGUAA
783


 


CS914419-NL
gaagacgaAfGfCfugcuigucaa
610
GAAGACGAAGCUGCUIGUCAA
784


 


CS914421-NL
ccaagcucGfCfAfuggucaguaa
611
CCAAGCUCGCAUGGUCAGUAA
785


 


CS914423-NL
cgcaugguCfAfGfuaaaagcaaa
612
CGCAUGGUCAGUAAAAGCAAA
786


 


CS914425-NL
ggucaguaAfAfAfgcaaagacia
613
GGUCAGUAAAAGCAAAGACIA
787


 


CS914427-NL
gacuggaaGfCfGfaugacaaaaa
614
GACUGGAAGCGAUGACAAAAA
788


 


CS914429-NL
uggaagcgAfUfGfacaaaaaaga
615
UGGAAGCGAUGACAAAAAAGA
789


 


CS914431-NL
accaggauUfCfCfagcaaaaaca
616
ACCAGGAUUCCAGCAAAAACA
790


 


CS914433-NL
ucuggugaAfCfCfuccaaaauca
617
UCUGGUGAACCUCCAAAAUCA
791


 


CS914435-NL
ggugaaccUfCfCfaaaaucaiga
618
GGUGAACCUCCAAAAUCAIGA
792


 


CS914437-NL
ucca_2NaaauCfAfGfgggaucicaa
619
UCC(A2N)AAAUCAGGGGAUCICAA
793


 


CS914439-NL
caa_2NauaguCfUfAfcaaaccaguu
620
CA(A2N)AUAGUCUACAAACCAGUU
794


 


CS914441-NL
caa_2NauaguCfUfAfcaaacuaguu
621
CA(A2N)AUAGUCUACAAACUAGUU
795


 


CS914442-NL
a_2NauagucuAfCfAfaaccaiuuga
622
(A2N)AUAGUCUACAAACCAIUUGA
796


 


CS914444-NL
a_2NuagucuaCfAfAfaccaguugaa
623
(A2N)UAGUCUACAAACCAGUUGAA
797


 


CS914446-NL
agucuacaAfAfCfcaguuiaccu
624
AGUCUACAAACCAGUUIACCU
798


 


CS914448-NL
aaccaguuGfAfCfcugaicaaga
625
AACCAGUUGACCUGAICAAGA
799


 


CS914450-NL
accuccaaGfUfGfuggcucauua
626
ACCUCCAAGUGUGGCUCAUUA
800


 


CS914452-NL
cuccaaguGfUfGfgcucauuaga
627
CUCCAAGUGUGGCUCAUUAGA
801


 


CS914454-NL
uccaagugUfGfGfcucauuagga
628
UCCAAGUGUGGCUCAUUAGGA
802


 


CS914456-NL
ccaaguguGfGfCfucauuagica
629
CCAAGUGUGGCUCAUUAGICA
803


 


CS914458-NL
guggcucaUfUfAfggcaacauca
630
GUGGCUCAUUAGGCAACAUCA
804


 


CS914460-NL
a_2NuuaggcaAfCfAfuccaucauaa
631
(A2N)UUAGGCAACAUCCAUCAUAA
805


 


CS914462-NL
guaggcaaCfAfUfccaucauaaa
632
GUAGGCAACAUCCAUCAUAAA
806


 


CS914464-NL
aggcaacaUfCfCfaucauaaaca
633
AGGCAACAUCCAUCAUAAACA
807


 


CS914466-NL
ggccagguGfGfAfaguaaaaucu
634
GGCCAGGUGGAAGUAAAAUCU
808


 


CS914468-NL
ga_2NagauugAfAfAfcccacaaicu
635
G(A2N)AGAUUGAAACCCACAAICU
809


 


CS914470-NL
a_2NgauugaaAfCfCfcacaaicuga
636
(A2N)GAUUGAAACCCACAAICUGA
810


 


CS914472-NL
ga_2NuugaaaCfCfCfacaaicugaa
637
G(A2N)UUGAAACCCACAAICUGAA
811


 


CS914474-NL
acgccaaaGfCfCfaagacaiaca
638
ACGCCAAAGCCAAGACAIACA
812


 


CS914476-NL
cagcaucgAfCfAfugguagacua
639
CAGCAUCGACAUGGUAGACUA
777


 


CS915422-NL
cagcaucgAfCfAfugguagacua
640
CAGCAUCGACAUGGUAGACUA
777


 


CS915427-NL
cagcaucgAfcAfugguagacua
641
CAGCAUCGACAUGGUAGACUA
777


 


CS915428-NL
cagcauCfgAfcAfugguagacua
642
CAGCAUCGACAUGGUAGACUA
777


 


CS915429-NL
cagcaucgAfcAfUfgguagacua
643
CAGCAUCGACAUGGUAGACUA
777


 


CS916150-NL
cagcaucgAfcAfdTgguagacua
644
CAGCAUCGACATGGUAGACUA
813





(A2N) = 2-aminoadenine nucleotide; I = hypoxanthine (inosine) nucleotide













TABLE 5







MAPT RNAi Agent Sense Strand Sequences (Shown without antigen binding


protein conjugate and with terminal caps (see Table


10 for structure information.))














Underlying Base





SEQ
Sequence (5′ → 3′)
SEQ




ID
(Shown as an Unmodified
ID


Strand ID
Modified Sense Strand (5′ → 3′)
NO.
Nucleotide Sequence)
NO.





CS004896-C
(invAb)scagcaucgAfuAfUfgguagacuas(invAb)
645
CAGCAUCGAUAUGGUAGACUA
776


 


CS005096-C
(invAb)scagcaucgAfcAfUfgguagacuas(invAb)
646
CAGCAUCGACAUGGUAGACUA
777


 


CS005097-C
(invAb)scagcaucgAfcAfUfgguagacuas(invAb)
647
CAGCAUCGACAUGGUAGACUA
777


 


CS005098-C
(invAb)scagcaucgAfcAfUfgguagacuas(invAb)
648
CAGCAUCGACAUGGUAGACUA
777


 


CS005099-C
(invAb)scagcauC16cgAfcAfUfgguagacuas(invAb)
649
CAGCAUCGACAUGGUAGACUA
777


 


CS005103-C
gsusgaccC16caAfGfCfucguauggsusa
650
GUGACCCAAGCUCGUAUGGUA
778


 


CS005303-C
(invAb)sccaaguGfuGfgCfucauuagguas(invAb)
651
CCAAGUGUGGCUCAUUAGGUA
779


 


CS005470-C
(invAb)scagcaucaAfcAfUfgguagacuas(invAb)
652
CAGCAUCAACAUGGUAGACUA
780


 


CS006178-C
(invAb)sucgcauggUfcAfguaaaagcaas(invAb)
653
UCGCAUGGUCAGUAAAAGCAA
781


 


CS008817-C
(invAb)scagcaucgAfcAfUfgguagacuas(invAb)
654
CAGCAUCGACAUGGUAGACUA
777


 


CS011528-C
(invAb)scagcaucgAfcAfUfgguagacuas(invAb)
655
CAGCAUCGACAUGGUAGACUA
777


 


CS914416-C
(invAb)sgaagaucaCfGfCfugggaciuaas(invAb)
656
GAAGAUCACGCUGGGACIUAA
782


 


CS914418-C
(invAb)sgaagaucaCfGfCfugggauguaas(invAb)
657
GAAGAUCACGCUGGGAUGUAA
783


 


CS914419-C
(invAb)sgaagacgaAfGfCfugcuigucaas(invAb)
658
GAAGACGAAGCUGCUIGUCAA
784


 


CS914421-C
(invAb)sccaagcucGfCfAfuggucaguaas(invAb)
659
CCAAGCUCGCAUGGUCAGUAA
785


 


CS914423-C
(invAb)scgcaugguCfAfGfuaaaagcaaas(invAb)
660
CGCAUGGUCAGUAAAAGCAAA
786


 


CS914425-C
(invAb)sggucaguaAfAfAfgcaaagacias(invAb)
661
GGUCAGUAAAAGCAAAGACIA
787


 


CS914427-C
(invAb)sgacuggaaGfCfGfaugacaaaaas(invAb)
662
GACUGGAAGCGAUGACAAAAA
788


 


CS914429-C
(invAb)suggaagcgAfUfGfacaaaaaagas(invAb)
663
UGGAAGCGAUGACAAAAAAGA
789


 


CS914431-C
(invAb)saccaggauUfCfCfagcaaaaacas(invAb)
664
ACCAGGAUUCCAGCAAAAACA
790


 


CS914433-C
(invAb)sucuggugaAfCfCfuccaaaaucas(invAb)
665
UCUGGUGAACCUCCAAAAUCA
791


 


CS914435-C
(invAb)sggugaaccUfCfCfaaaaucaigas(invAb)
666
GGUGAACCUCCAAAAUCAIGA
792


 


CS914437-C
(invAb)succa_2NaaauCfAfGfgggaucicaas(invAb)
667
UCC(A2N)AAAUCAGGGGAUCICAA
793


 


CS914439-C
(invAb)scaa_2NauaguCfUfAfcaaaccaguus(invAb)
668
CA(A2N)AUAGUCUACAAACCAGUU
794


 


CS914441-C
(invAb)scaa_2NauaguCfUfAfcaaacuaguus(invAb)
669
CA(A2N)AUAGUCUACAAACUAGUU
795


 


CS914442-C
(invAb)sa_2NauagucuAfCfAfaaccaiuugas(invAb)
670
(A2N)AUAGUCUACAAACCAIUUGA
796


 


CS914444-C
(invAb)sa_2NuagucuaCfAfAfaccaguugaas(invAb)
671
(A2N)UAGUCUACAAACCAGUUGAA
797


 


CS914446-C
(invAb)sagucuacaAfAfCfcaguuiaccus(invAb)
672
AGUCUACAAACCAGUUIACCU
798


 


CS914448-C
(invAb)saaccaguuGfAfCfcugaicaagas(invAb)
673
AACCAGUUGACCUGAICAAGA
799


 


CS914450-C
(invAb)saccuccaaGfUfGfuggcucauuas(invAb)
674
ACCUCCAAGUGUGGCUCAUUA
800


 


CS914452-C
(invAb)scuccaaguGfUfGfgcucauuagas(invAb)
675
CUCCAAGUGUGGCUCAUUAGA
801


 


CS914454-C
(invAb)succaagugUfGfGfcucauuaggas(invAb)
676
UCCAAGUGUGGCUCAUUAGGA
802


 


CS914456-C
(invAb)sccaaguguGfGfCfucauuagicas(invAb)
677
CCAAGUGUGGCUCAUUAGICA
803


 


CS914458-C
(invAb)sguggcucaUfUfAfggcaacaucas(invAb)
678
GUGGCUCAUUAGGCAACAUCA
804


 


CS914460-C
(invAb)sa_2NuuaggcaAfCfAfuccaucauaas(invAb)
679
(A2N)UUAGGCAACAUCCAUCAUAA
805


 


CS914462-C
(invAb)sguaggcaaCfAfUfccaucauaaas(invAb)
680
GUAGGCAACAUCCAUCAUAAA
806


 


CS914464-C
(invAb)saggcaacaUfCfCfaucauaaacas(invAb)
681
AGGCAACAUCCAUCAUAAACA
807


 


CS914466-C
(invAb)sggccagguGfGfAfaguaaaaucus(invAb)
682
GGCCAGGUGGAAGUAAAAUCU
808


 


CS914468-C
(invAb)sga_2NagauugAfAfAfcccacaaicus(invAb)
683
G(A2N)AGAUUGAAACCCACAAICU
809


 


CS914470-C
(invAb)sa_2NgauugaaAfCfCfcacaaicugas(invAb)
684
(A2N)GAUUGAAACCCACAAICUGA
810


 


CS914472-C
(invAb)sga_2NuugaaaCfCfCfacaaicugaas(invAb)
685
G(A2N)UUGAAACCCACAAICUGAA
811


 


CS914474-C
(invAb)sacgccaaaGfCfCfaagacaiacas(invAb)
686
ACGCCAAAGCCAAGACAIACA
812


 


CS914476-C
(invAb)scagcaucgAfCfAfugguagacuas(invAb)
687
CAGCAUCGACAUGGUAGACUA
777


 


CS915422-C
(invAb)scagcaucgAfCfAfugguagacuas(invAb)
688
CAGCAUCGACAUGGUAGACUA
777


 


CS915427-C
(invAb)scagcaucgAfcAfugguagacuas(invAb)
689
CAGCAUCGACAUGGUAGACUA
777


 


CS915428-C
(invAb)scagcauCfgAfcAfugguagacuas(invAb)
690
CAGCAUCGACAUGGUAGACUA
777


 


CS915429-C
(invAb)scagcaucgAfcAfUfgguagacuas(invAb)
691
CAGCAUCGACAUGGUAGACUA
777


 


CS916150-C
(invAb)scagcaucgAfcAfdTgguagacuas(invAb)
692
CAGCAUCGACATGGUAGACUA
813





(A2N) = 2-aminoadenine nucleotide; I = hypoxanthine (inosine) nucleotide













TABLE 6







MAPT Agent Sense Strand Sequences (shown with lipid or antigen binding


moiety). The structures of the lipid and antigen binding moieties


are shown in Table 10.














Underlying Base






Sequence (5′ → 3′)





SEQ
(Shown as
SEQ




ID
an Unmodified
ID


Strand ID
Modified Sense Strand (5′ → 3′)
NO.
Nucleotide Sequence)
NO.





CS004896
LP293-(NH-
693
CAGCAUCGAUAUGGUAGACUA
776



C6)s(invAb)scagcaucgAfuAfUfgguagacuas(invAb)





 


CS005096
LP310-(NH-
694
CAGCAUCGACAUGGUAGACUA
777



C6)s(invAb)scagcaucgAfcAfUfgguagacuas(invAb)





 


CS005097
LP429-(NH-
695
CAGCAUCGACAUGGUAGACUA
777



C6)s(invAb)scagcaucgAfcAfUfgguagacuas(invAb)





 


CS005098
LP462-(NH-
696
CAGCAUCGACAUGGUAGACUA
777



C6)s(invAb)scagcaucgAfcAfUfgguagacuas(invAb)





 


CS005099
(invAb)scagcauC16cgAfcAfUfgguagacuas(invAb)
697
CAGCAUCGACAUGGUAGACUA
777


 


CS005103
gsusgaccC16caAfGfCfucguauggsusa
698
GUGACCCAAGCUCGUAUGGUA
778


 


CS005303
LP293-(NH-
699
CCAAGUGUGGCUCAUUAGGUA
779



C6)s(invAb)sccaaguGfuGfgCfucauuagguas(invAb)





 


CS005470
LP293-(NH-
700
CAGCAUCAACAUGGUAGACUA
780



C6)s(invAb)scagcaucaAfcAfUfgguagacuas(invAb)





 


CS006178
LP293-(NH-
701
UCGCAUGGUCAGUAAAAGCAA
781



C6)s(invAb)sucgcauggUfcAfguaaaagcaas(invAb)





 


CS008817
Fab0070-[CP-1113]L20-(NH-
702
CAGCAUCGACAUGGUAGACUA
777



C6)s(invAb)scagcaucgAfcAfUfgguagacuas(invAb)





 


CS011528
Fab0070-L-1026-(NH-
703
CAGCAUCGACAUGGUAGACUA
777



C6)s(invAb)scagcaucgAfcAfUfgguagacuas(invAb)





 


CS914416
LP183-(NH-
704
GAAGAUCACGCUGGGACIUAA
782



C6)s(invAb)sgaagaucaCfGfCfugggaciuaas(invAb)





 


CS914418
LP183-(NH-
705
GAAGAUCACGCUGGGAUGUAA
783



C6)s(invAb)sgaagaucaCfGfCfugggauguaas(invAb)





 


CS914419
LP183-(NH-
706
GAAGACGAAGCUGCUIGUCAA
784



C6)s(invAb)sgaagacgaAfGfCfugcuigucaas(invAb)





 


CS914421
LP183-(NH-
707
CCAAGCUCGCAUGGUCAGUAA
785



C6)s(invAb)sccaagcucGfCfAfuggucaguaas(invAb)





 


CS914423
LP183-(NH-
708
CGCAUGGUCAGUAAAAGCAAA
786



C6)s(invAb)scgcaugguCfAfGfuaaaagcaaas(invAb)





 


CS914425
LP183-(NH-
709
GGUCAGUAAAAGCAAAGACIA
787



C6)s(invAb)sggucaguaAfAfAfgcaaagacias(invAb)





 


CS914427
LP183-(NH-
710
GACUGGAAGCGAUGACAAAAA
788



C6)s(invAb)sgacuggaaGfCfGfaugacaaaaas(invAb)





 


CS914429
LP183-(NH-
711
UGGAAGCGAUGACAAAAAAGA
789



C6)s(invAb)suggaagcgAfUfGfacaaaaaagas(invAb)





 


CS914431
LP183-(NH-
712
ACCAGGAUUCCAGCAAAAACA
790



C6)s(invAb)saccaggauUfCfCfagcaaaaacas(invAb)





 


CS914433
LP183-(NH-
713
UCUGGUGAACCUCCAAAAUCA
791



C6)s(invAb)sucuggugaAfCfCfuccaaaaucas(invAb)





 


CS914435
LP183-(NH-
714
GGUGAACCUCCAAAAUCAIGA
792



C6)s(invAb)sggugaaccUfCfCfaaaaucaigas(invAb)





 


CS914437
LP183-(NH-C6)s
715
UCC(A2N)AAAUCAGGGGAUCICAA
793



(invAb)succa_2NaaauCfAfGfgggaucicaas(invAb)





 


CS914439
LP183-(NH-C6)s
716
CA(A2N)AUAGUCUACAAACCAGUU
794



(invAb)scaa_2NauaguCfUfAfcaaaccaguus(invAb)





 


CS914441
LP183-(NH-C6)s
717
CA(A2N)AUAGUCUACAAACUAGUU
795



(invAb)scaa_2NauaguCfUfAfcaaacuaguus(invAb)





 


CS914442
LP183-(NH-C6)s
718
(A2N)AUAGUCUACAAACCAIUUGA
796



(invAb)sa_2NauagucuAfCfAfaaccaiuugas(invAb)





 


CS914444
LP183-(NH-C6)s
719
(A2N)UAGUCUACAAACCAGUUGAA
797



(invAb)sa_2NuagucuaCfAfAfaccaguugaas(invAb)





 


CS914446
LP183-(NH-C6)s
720
AGUCUACAAACCAGUUIACCU
798



(invAb)sagucuacaAfAfCfcaguuiaccus(invAb)





 


CS914448
LP183-(NH-
721
AACCAGUUGACCUGAICAAGA
799



C6)s(invAb)saaccaguuGfAfCfcugaicaagas(invAb)





 


CS914450
LP183-(NH-
722
ACCUCCAAGUGUGGCUCAUUA
800



C6)s(invAb)saccuccaaGfUfGfuggcucauuas(invAb)





 


CS914452
LP183-(NH-
723
CUCCAAGUGUGGCUCAUUAGA
801



C6)s(invAb)scuccaaguGfUfGfgcucauuagas(invAb)





 


CS914454
LP183-(NH-
724
UCCAAGUGUGGCUCAUUAGGA
802



C6)s(invAb)succaagugUfGfGfcucauuaggas(invAb)





 


CS914456
LP183-(NH-
725
CCAAGUGUGGCUCAUUAGICA
803



C6)s(invAb)sccaaguguGfGfCfucauuagicas(invAb)





 


CS914458
LP183-(NH-
726
GUGGCUCAUUAGGCAACAUCA
804



C6)s(invAb)sguggcucaUfUfAfggcaacaucas(invAb)





 


CS914460
LP183-(NH-C6)s
727
(A2N)UUAGGCAACAUCCAUCAUAA
805



(invAb)sa_2NuuaggcaAfCfAfuccaucauaas(invAb)





 


CS914462
LP183-(NH-
728
GUAGGCAACAUCCAUCAUAAA
806



C6)s(invAb)sguaggcaaCfAfUfccaucauaaas(invAb)





 


CS914464
LP183-(NH-
729
AGGCAACAUCCAUCAUAAACA
807



C6)s(invAb)saggcaacaUfCfCfaucauaaacas(invAb)





 


CS914466
LP183-(NH-
730
GGCCAGGUGGAAGUAAAAUCU
808



C6)s(invAb)sggccagguGfGfAfaguaaaaucus(invAb)





 


CS914468
LP183-(NH-C6)s
731
G(A2N)AGAUUGAAACCCACAAICU
809



(invAb)sga_2NagauugAfAfAfcccacaaicus(invAb)





 


CS914470
LP183-(NH-C6)s
732
(A2N)GAUUGAAACCCACAAICUGA
810



(invAb)sa_2NgauugaaAfCfCfcacaaicugas(invAb)





 


CS914472
LP183-(NH-C6)s
733
GAUUGAAACCCACAAICUGAA
811



(invAb)sga_2NuugaaaCfCfCfacaaicugaas(invAb)





 


CS914474
LP183-(NH-
734
ACGCCAAAGCCAAGACAIACA
812



C6)s(invAb)sacgccaaaGfCfCfaagacaiacas(invAb)





 


CS914476
LP183-(NH-
735
CAGCAUCGACAUGGUAGACUA
777



C6)s(invAb)scagcaucgAfCfAfugguagacuas(invAb)





 


CS915422
LP293-(NH-
736
CAGCAUCGACAUGGUAGACUA
777



C6)s(invAb)scagcaucgAfCfAfugguagacuas(invAb)





 


CS915427
LP293-(NH-
737
CAGCAUCGACAUGGUAGACUA
777



C6)s(invAb)scagcaucgAfcAfugguagacuas(invAb)





 


CS915428
LP293-(NH-
738
CAGCAUCGACAUGGUAGACUA
777



C6)s(invAb)scagcauCfgAfcAfugguagacuas(invAb)





 


CS915429
LP293-(NH-
739
CAGCAUCGACAUGGUAGACUA
777



C6)s(invAb)scagcaucgAfcAfUfgguagacuas(invAb)





 


CS916150
LP293-(NH-
740
CAGCAUCGACATGGUAGACUA
813



C6)s(invAb)scagcaucgAfcAfdTgguagacuas(invAb)





(A2N) = 2-aminoadenine nucleotide; I = hypoxanthine (inosine) nucleotide






The MAPT RNAi agents disclosed herein are formed by annealing an antisense strand with a sense strand. A sense strand containing a sequence listed in Table 2, Table 4, Table 5, or Table 6, can be hybridized to any antisense strand containing a sequence listed in Table 2 or Table 3, provided the two sequences have a region of at least 85% complementarity over a contiguous 16, 17, 18, 19, 20, or 21 nucleotide sequence.
As shown in Table 6 above, certain of the example MAPT RNAi agent nucleotide sequences are shown to further include reactive linking groups at one or both of the 5′ terminal end and the 3′ terminal end of the sense strand. For example, many of the MAPT RNAi agent sense strand sequences shown in Table 6 above have a (NH-C6) linking group at the 5′ end of the nucleotide sequence, which was synthesized first as a (NH2-C6) group and then conjugated to a PK/PD modulator or linker such as L-20 or L-1026 (see Example 1, below, for conjugation details). Other linking groups, such as a (6-SS-6) linking group or a (C6-SS-C6) linking group, may be present as well or alternatively in certain embodiments. Such reactive linking groups are positioned to facilitate the linking of targeting ligands, targeting groups, and/or antigen binding proteins to the MAPT RNAi agents disclosed herein. Linking or conjugation reactions are well known in the art and provide for formation of covalent linkages between two molecules or reactants. Suitable conjugation reactions for use in the scope of the inventions herein include, but are not limited to, amide coupling reaction, Michael addition reaction, hydrazone formation reaction, inverse-demand Diels-Alder cycloaddition reaction, oxime ligation, and Copper (I)-catalyzed or strain-promoted azide-alkyne cycloaddition reaction cycloaddition reaction.
In some embodiments, targeting ligands, can be synthesized as activated esters, such as tetrafluorophenyl (TFP) esters, which can be displaced by a reactive amino group (e.g., NH2-C6) to attach the targeting ligand to the MAPT RNAi agents disclosed herein. In some embodiments, targeting ligands are synthesized as azides, which can be conjugated to a propargyl or DBCO group, for example, via Copper (I)-catalyzed or strain-promoted azide-alkyne cycloaddition reaction.
Additionally, certain of the nucleotide sequences can be synthesized with a dT nucleotide at the 3′ terminal end of the sense strand, followed by (3′→5′) a linker (e.g., C6-SS-C6). The linker can, in some embodiments, facilitate the linkage to additional components, such as, for example, an antigen binding protein or one or more targeting ligands. As described herein, the disulfide bond of C6-SS-C6 is first reduced, removing the dT from the molecule, which can then facilitate the conjugation of the desired component. The terminal dT nucleotide therefore is not a part of the fully conjugated construct.
Additionally, certain of the nucleotide sequences can be synthesized with a dT nucleotide at the 3′ terminal end of the sense strand, followed by (3′ 4 5′) a linker (e.g., C6-SS-C6). The linker can, in some embodiments, facilitate the linkage to additional components, such as, for example, a lipid or one or more targeting ligands. As described herein, the disulfide bond of C6-SS-C6 is first reduced, removing the dT from the molecule, which can then facilitate the conjugation of the desired component. The terminal dT nucleotide therefore is not a part of the fully conjugated construct.
In some embodiments, the antisense strand of a MAPT RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences in Table 3 or Table 10. In some embodiments, the sense strand of a MAPT RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the sense strand sequences in Table 4, Table 5, Table 6, or Table 9.
In some embodiments, a MAPT RNAi agent antisense strand comprises a nucleotide sequence of any of the sequences in Table 2 or Table 3. In some embodiments, a MAPT RNAi agent antisense strand comprises the sequence of nucleotides (from 5′ end→3′ end) 1-17, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, 2-21, 1-22, 2-22, 1-23, 2-23, 1-24, or 2-24 of any of the sequences in Table 2, Table 3, or Table 9. In certain embodiments, a MAPT RNAi agent antisense strand comprises or consists of a modified sequence of any one of the modified sequences in Table 3 or Table 9.
In some embodiments, a MAPT RNAi agent sense strand comprises the nucleotide sequence of any of the sequences in Table 2 or Table 4. In some embodiments, a MAPT RNAi agent sense strand comprises the sequence of nucleotides (from 5′ end→3′ end) 1-17, 2-17, 3-17, 4-17, 1-18, 2-18, 3-18, 4-18, 1-19, 2-19, 3-19, 4-19, 1-20, 2-20, 3-20, 4-20, 1-21, 2-21, 3-21, 4-21, 1-22, 2-22, 3-22, 4-22, 1-23, 2-23, 3-23, 4-23, 1-24, 2-24, 3-24, or 4-24, of any of the sequences in Table 2, Table 4, Table 5, Table 6, or Table 9. In certain embodiments, a MAPT RNAi agent sense strand comprises or consists of a modified sequence of any one of the modified sequences in Table 6 or Table 9.
For the RNAi agents disclosed herein, the nucleotide at position 1 of the antisense strand (from 5′ end→3′ end) can be perfectly complementary to a MAPT gene, or can be non-complementary to a MAPT gene. In some embodiments, the nucleotide at position 1 of the antisense strand (from 5′ end→3′ end) is a U, A, or dT (or a modified version of U, A or dT). In some embodiments, the nucleotide at position 1 of the antisense strand (from 5′ end→3′ end) forms an A:U or U:A base pair with the sense strand.
In some embodiments, a MAPT RNAi agent antisense strand comprises the sequence of nucleotides (from 5′ end→3′ end) 2-18 or 2-19 of any of the antisense strand sequences in Table 2, Table 3, or Table 9. In some embodiments, a MAPT RNAi sense strand comprises the sequence of nucleotides (from 5′ end→3′ end) 1-17 or 1-18 of any of the sense strand sequences in Table 2, Table 4, Table 5, Table 6, or Table 9.
In some embodiments, a MAPT RNAi agent includes (i) an antisense strand comprising the sequence of nucleotides (from 5′ end→3′ end) 2-18 or 2-19 of any of the antisense strand sequences in Table 2, Table 3, or Table 9, and (ii) a sense strand comprising the sequence of nucleotides (from 5′ end→3′ end) 1-17 or 1-18 of any of the sense strand sequences in Table 2, Table 4, Table 5, Table 6, or Table 9.
A sense strand containing a sequence listed in Table 2 or Table 4 can be hybridized to any antisense strand containing a sequence listed in Table 2 or Table 3 provided the two sequences have a region of at least 85% complementarity over a contiguous 16, 17, 18, 19, 20, or 21 nucleotide sequence. In some embodiments, the MAPT RNAi agent has a sense strand consisting of the modified sequence of any of the modified sequences in Table 4, Table 5, Table 6, or Table 9, and an antisense strand consisting of the modified sequence of any of the modified sequences in Table 3 or Table 9. Certain representative sequence pairings are exemplified by the Duplex ID Nos. shown in Tables 7, 8, and 9.
In some embodiments, a MAPT RNAi agent comprises, consists of, or consists essentially of a duplex represented by any one of the Duplex ID Nos. presented herein. In some embodiments, a MAPT RNAi agent consists of any of the Duplex ID Nos. presented herein. In some embodiments, a MAPT RNAi agent comprises the sense strand and antisense strand nucleotide sequences of any of the Duplex ID Nos. presented herein. In some embodiments, a MAPT RNAi agent comprises the sense strand and antisense strand nucleotide sequences of any of the Duplex ID Nos. presented herein and a targeting group, linking group, antigen binding protein and/or other non-nucleotide group wherein the targeting group, linking group, antigen binding protein and/or other non-nucleotide group is covalently linked (i.e., conjugated) to the sense strand or the antisense strand. In some embodiments, a MAPT RNAi agent includes the sense strand and antisense strand modified nucleotide sequences of any of the Duplex ID Nos. presented herein. In some embodiments, a MAPT RNAi agent comprises the sense strand and antisense strand modified nucleotide sequences of any of the Duplex ID Nos. presented herein and a targeting group, linking group, and/or other non-nucleotide group, wherein the targeting group, linking group, antigen binding protein and/or other non-nucleotide group is covalently linked to the sense strand or the antisense strand.
In some embodiments, a MAPT RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand/sense strand duplexes of Tables 2, 7, 8, or 9, and comprises an antigen binding protein. In some embodiments, a MAPT RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand/sense strand duplexes of Tables 2, 7, 8, or 9, and comprises one or more antigen binding proteins.
In some embodiments, a MAPT RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand/sense strand duplexes of Tables 2, 7, 8, or 9, and comprises an antigen binding protein. In some embodiments, a MAPT RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand/sense strand duplexes of Tables 2, 7, 8, or 9, and comprises one or more antigen binding protein.
In some embodiments, a MAPT RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand/sense strand duplexes of Tables 2, 7, 8, or 9, and comprises a PK/PD modulator. In some embodiments, a MAPT RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand/sense strand duplexes of Tables 2, 7, 8, or 9, and comprises one or more lipid moieties.
In some embodiments, a MAPT RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand/sense strand duplexes of Tables 2, 7, 8, or 9, and comprises a lipid moiety. In some embodiments, a MAPT RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand/sense strand duplexes of Tables 2, 7, 8, or 9, and comprises one or more lipid moieties.
In some embodiments, a MAPT RNAi agent comprises an antisense strand and a sense strand having the modified nucleotide sequences of any of the antisense strand/sense strand duplexes of Tables 7, 8, and 9.
In some embodiments, a MAPT RNAi agent comprises an antisense strand and a sense strand having the modified nucleotide sequences of any of the antisense strand/sense strand duplexes of Tables 7, 8, and 9, and comprises an antigen binding protein.
In some embodiments, a MAPT RNAi agent comprises an antisense strand and a sense strand having the modified nucleotide sequences of any of the antisense strand/sense strand duplexes of Tables 7, 8, and 9, and comprises a lipid moiety.
In some embodiments, a MAPT RNAi agent comprises, consists of, or consists essentially of any of the duplexes of Tables 7, 8, and 9.







TABLE 7







MAPT RNAi Agent Duplexes with Corresponding Sense and


Antisense Strand ID Numbers and Sequence ID numbers


for the modified and unmodified nucleotide sequences.















AS
AS

SS
SS




modified
unmodified

modified
unmodified




SEQ ID
SEQ ID

SEQ ID
SEQ ID


Duplex
AS ID
NO:
NO:
SS ID
NO:
NO:
















AC003990
CA004894
545
741
CS915429
739
777


AC003991
CA004895
546
742
CS915429
739
777


AC003992
CA004894
545
741
CS004896
693
776


AC003993
CA004897
547
743
CS915429
739
777


AC003994
CA004898
548
743
CS915429
739
777


AC003995
CA004899
549
743
CS915429
739
777


AC004123
CA915905
592
743
CS005096
694
777


AC004124
CA915905
592
743
CS005097
695
777


AC004125
CA915905
592
743
CS005098
696
777


AC004126
CA915905
592
743
CS005099
697
777


AC004130
CA005104
550
744
CS005103
698
778


AC004265
CA915404
584
766
CS005303
699
779


AC004396
CA005435
551
741
CS915429
739
777


AC004434
CA004894
545
741
CS005470
700
780


AC004435
CA005471
552
745
CS915429
739
777


AC005033
CA006181
553
746
CS006178
701
781


AC007414
CA915905
592
743
CS008817
702
777


AC009806
CA915905
592
743
CS011528
703
777


AC911178
CA914417
554
747
CS914416
704
782


AC911179
CA914417
554
747
CS914418
705
783


AC911180
CA914420
555
748
CS914419
706
784


AC911181
CA914422
556
749
CS914421
707
785


AC911182
CA914424
557
750
CS914423
708
786


AC911183
CA914426
558
751
CS914425
709
787


AC911184
CA914428
559
752
CS914427
710
788


AC911185
CA914430
560
753
CS914429
711
789


AC911186
CA914432
561
754
CS914431
712
790


AC911187
CA914434
562
755
CS914433
713
791


AC911188
CA914436
563
756
CS914435
714
792


AC911189
CA914438
564
757
CS914437
715
793


AC911190
CA914440
565
758
CS914439
716
794


AC911191
CA914440
565
758
CS914441
717
795


AC911192
CA914443
566
759
CS914442
718
796


AC911193
CA914445
567
760
CS914444
719
797


AC911194
CA914447
568
761
CS914446
720
798


AC911195
CA914449
569
762
CS914448
721
799


AC911196
CA914451
570
763
CS914450
722
800


AC911197
CA914453
571
764
CS914452
723
801


AC911198
CA914455
572
765
CS914454
724
802


AC911199
CA914457
573
766
CS914456
725
803


AC911200
CA914459
574
767
CS914458
726
804


AC911201
CA914461
575
768
CS914460
727
805


AC911202
CA914463
576
769
CS914462
728
806


AC911203
CA914465
577
770
CS914464
729
807


AC911204
CA914467
578
771
CS914466
730
808


AC911205
CA914469
579
772
CS914468
731
809


AC911206
CA914471
580
773
CS914470
732
810


AC911207
CA914473
581
774
CS914472
733
811


AC911208
CA914475
582
775
CS914474
734
812


AC911209
CA914477
583
743
CS914476
735
777


AC912001
CA914477
583
743
CS915422
736
777


AC912002
CA915423
585
743
CS915422
736
777


AC912003
CA915424
586
743
CS915422
736
777


AC912004
CA915425
587
743
CS915422
736
777


AC912005
CA915426
588
743
CS915422
736
777


AC912006
CA915424
586
743
CS915427
737
777


AC912007
CA915424
586
743
CS915428
738
777


AC912008
CA915424
586
743
CS915429
739
777


AC912009
CA915430
589
743
CS915422
736
777


AC912010
CA915431
590
743
CS915422
736
777


AC912011
CA915432
591
743
CS915422
736
777


AC912669
CA915426
588
743
CS915429
739
777


AC912670
CA916146
593
743
CS915429
739
777


AC912671
CA915905
592
743
CS915429
739
777


AC912672
CA916147
594
743
CS915429
739
777


AC912673
CA916148
595
743
CS915429
739
777


AC912674
CA916149
596
743
CS915429
739
777


AC912675
CA916148
595
743
CS916150
740
813
















TABLE 8







Conjugate Duplex ID Numbers Referencing


Position Targeted On MAPT (MAPT) Gene













Targeted MAPT Gene Position


Duplex
AS ID
SS ID
(Of SEQ ID NO: 1)













AC003990
CA004894
CS915429
1221


AC003991
CA004895
CS915429
1221


AC003992
CA004894
CS004896
1221


AC003993
CA004897
CS915429
1221


AC003994
CA004898
CS915429
1221


AC003995
CA004899
CS915429
1221


AC004123
CA915905
CS005096
1221


AC004124
CA915905
CS005097
1221


AC004125
CA915905
CS005098
1221


AC004126
CA915905
CS005099
1221


AC004130
CA005104
CS005103
340


AC004265
CA915404
CS005303
935


AC004396
CA005435
CS915429
1221


AC004434
CA004894
CS005470
1221


AC004435
CA005471
CS915429
1221


AC005033
CA006181
CS006178
351


AC007414
CA915905
CS008817
1221


AC009806
CA915905
CS011528
1221


AC911178
CA914417
CS914416
184


AC911179
CA914417
CS914418
184


AC911180
CA914420
CS914419
319


AC911181
CA914422
CS914421
345


AC911182
CA914424
CS914423
352


AC911183
CA914426
CS914425
357


AC911184
CA914428
CS914427
378


AC911185
CA914430
CS914429
381


AC911186
CA914432
CS914431
481


AC911187
CA914434
CS914433
529


AC911188
CA914436
CS914435
532


AC911189
CA914438
CS914437
540


AC911190
CA914440
CS914439
895


AC911191
CA914440
CS914441
895


AC911192
CA914443
CS914442
897


AC911193
CA914445
CS914444
898


AC911194
CA914447
CS914446
900


AC911195
CA914449
CS914448
908


AC911196
CA914451
CS914450
931


AC911197
CA914453
CS914452
933


AC911198
CA914455
CS914454
934


AC911199
CA914457
CS914456
935


AC911200
CA914459
CS914458
941


AC911201
CA914461
CS914460
948


AC911202
CA914463
CS914462
949


AC911203
CA914465
CS914464
951


AC911204
CA914467
CS914466
979


AC911205
CA914469
CS914468
1083


AC911206
CA914471
CS914470
1085


AC911207
CA914473
CS914472
1086


AC911208
CA914475
CS914474
1118


AC911209
CA914477
CS914476
1221


AC912001
CA914477
CS915422
1221


AC912002
CA915423
CS915422
1221


AC912003
CA915424
CS915422
1221


AC912004
CA915425
CS915422
1221


AC912005
CA915426
CS915422
1221


AC912006
CA915424
CS915427
1221


AC912007
CA915424
CS915428
1221


AC912008
CA915424
CS915429
1221


AC912009
CA915430
CS915422
1221


AC912010
CA915431
CS915422
1221


AC912011
CA915432
CS915422
1221


AC912669
CA915426
CS915429
1221


AC912670
CA916146
CS915429
1221


AC912671
CA915905
CS915429
1221


AC912672
CA916147
CS915429
1221


AC912673
CA916148
CS915429
1221


AC912674
CA916149
CS915429
1221


AC912675
CA916148
CS916150
1221
















TABLE 9







Conjugate ID Numbers With Chemically Modified Antisense and


Sense Strands (including Linkers and Conjugates)












Sense Strand (fully modified






with conjugated antigen






binding protein or
SEQ

SEQ


ACID
conjugated PK/PD modulator)
ID

ID


Number
(5′ → 3′)
NO:
Antisense Strand (5′ → 3′)
NO:





AC003990
LP293-(NH-C6)s(invAb)scagcaucg
739
cPrpusAfsgucuAfccauGfuUfgAfugc
545



AfcAfUfgguagacuas(invAb)

ussg



 


AC003991
LP293-(NH-C6)s(invAb)scagcaucg
739
cPrpusAfsgucuAfcuauGfuCfgAfugc
546



AfcAfUfgguagacuas(invAb)

ussg



 


AC003992
LP293-(NH-C6)s(invAb)scagcaucg
693
cPrpusAfsgucuAfccauGfuUfgAfugc
545



AfuAfUfgguagacuas(invAb)

ussg



 


AC003993
LP293-(NH-C6)s(invAb)scagcaucg
739
cPrpusAfsgucuAfCUNAcauGfuCfgAf
547



AfcAfUfgguagacuas(invAb)

ugcussg



 


AC003994
LP293-(NH-C6)s(invAb)scagcaucg
739
cPrpusAfsgucUUNAAfccauGfuCfgAf
548



AfcAfUfgguagacuas(invAb)

ugcussg



 


AC003995
LP293-(NH-C6)s(invAb)scagcaucg
739
cPrpusAfsgucudAccauGfuCfgAfugc
549



AfcAfUfgguagacuas(invAb)

ussg



 


AC004123
LP310-(NH-C6)s(invAb)scagcaucg
694
cPrpusAfsgucuAfccauGfuCfgAfugc
592



AfcAfUfgguagacuas(invAb)

ussg



 


AC004124
LP429-(NH-C6)s(invAb)scagcaucg
695
cPrpusAfsgucuAfccauGfuCfgAfugc
592



AfcAfUfgguagacuas(invAb)

ussg



 


AC004125
LP462-(NH-C6)s(invAb)scagcaucg
696
cPrpusAfsgucuAfccauGfuCfgAfugc
592



AfcAfUfgguagacuas(invAb)

ussg



 


AC004126
(invAb)scagcauC16cgAfcAfUfggua
697
cPrpusAfsgucuAfccauGfuCfgAfugc
592



gacuas(invAb)

ussg



 


AC004130
gsusgaccC16caAfGfCfucguauggsus
698
vpusAfsccdAudAcgagcuUfgGfgucac
550



a

sgsu



 


AC004265
LP293-(NH-C6)s(invAb)sccaaguGf
699
cPrpusGfsccuaAfugagCfcAfcAfcuu
584



uGfgCfucauuagguas(invAb)

gsg



 


AC004396
LP293-(NH-C6)s(invAb)scagcaucg
739
cPrpusAfsgucUUNAAfccauGfuUfgAf
551



AfcAfUfgguagacuas(invAb)

ugcussg



 


AC004434
LP293-(NH-C6)s(invAb)scagcauca
700
cPrpusAfsgucuAfccauGfuUfgAfugc
545



AfcAfUfgguagacuas(invAb)

ussg



 


AC004435
LP293-(NH-C6)s(invAb)scagcaucg
739
cPrpusAfsgucuAfccauGfudTgAfugc
552



AfcAfUfgguagacuas(invAb)

ussg



 


AC005033
LP293-(NH-C6)s(invAb)sucgcaugg
701
cPrpusUfsgcuUUNAUfuacuGfaCfcAf
553



UfcAfguaaaagcaas(invAb)

ugcgssa



 


AC007414
Fab0070-[CP-1113]-L20-(NH-C6)s
702
cPrpusAfsgucuAfccauGfuCfgAfugc
592



(invAb)scagcaucgAfcAfUfgguagac

ussg




uas(invAb)





 


AC009806
Fab0070-L-1026-(NH-C6)s(invAb)
703
cPrpusAfsgucuAfccauGfuCfgAfugc
592



scagcaucgAfcAfUfgguagacuas(inv

ussg




Ab)





 


AC911178
LP183-(NH-C6)s(invAb)sgaagauca
704
cPrpusUfsasCfgUfcccagCfgUfgAfu
554



CfGfCfugggaciuaas(invAb)

Cfuusc



 


AC911179
LP183-(NH-C6)s(invAb)sgaagauca
705
cPrpusUfsasCfgUfcccagCfgUfgAfu
554



CfGfCfugggauguaas(invAb)

Cfuusc



 


AC911180
LP183-(NH-C6)s(invAb)sgaagacga
706
cPrpusUfsgsAfcCfagcagCfuUfcGfu
555



AfGfCfugcuigucaas(invAb)

Cfuusc



 


AC911181
LP183-(NH-C6)s(invAb)sccaagcuc
707
cPrpusUfsasCfuGfaccauGfcGfaGfc
556



GfCfAfuggucaguaas(invAb)

Ufugsg



 


AC911182
LP183-(NH-C6)s(invAb)scgcauggu
708
cPrpusUfsusGfcUfuuuacUfgAfcCfa
557



CfAfGfuaaaagcaaas(invAb)

Ufgcsg



 


AC911183
LP183-(NH-C6)s(invAb)sggucagua
709
cPrpusCfsgsUfcUfuugcuUfuUfaCfu
558



AfAfAfgcaaagacias(invAb)

Gfacsc



 


AC911184
LP183-(NH-C6)s(invAb)sgacuggaa
710
cPrpusUfsusUfuGfucaucGfcUfuCfc
559



GfCfGfaugacaaaaas(invAb)

Afgusc



 


AC911185
LP183-(NH-C6)s(invAb)suggaagcg
711
cPrpusCfsusUfuUfuugucAfuCfgCfu
560



AfUfGfacaaaaaagas(invAb)

Ufccsa



 


AC911186
LP183-(NH-C6)s(invAb)saccaggau
712
cPrpusGfsusUfuUfugcugGfaAfuCfc
561



UfCfCfagcaaaaacas(invAb)

Ufggsu



 


AC911187
LP183-(NH-C6)s(invAb)sucugguga
713
cPrpusGfsasUfuUfuggagGfuUfcAfc
562



AfCfCfuccaaaaucas(invAb)

Cfagsa



 


AC911188
LP183-(NH-C6)s(invAb)sggugaacc
714
cPrpusCfscsUfgAfuuuugGfaGfgUfu
563



UfCfCfaaaaucaigas(invAb)

Cfacsc



 


AC911189
LP183-(NH-C6)s(invAb)succa_2Na
715
cPrpusUfsgsCfgAfuccccUfgAfuUfu
564



aauCfAfGfgggaucicaas(invAb)

Ufggsa



 


AC911190
LP183-(NH-C6)s(invAb)scaa_2Nau
716
cPrpasAfscsUfgGfuuuguAfgAfcUfa
565



aguCfUfAfcaaaccaguus(invAb)

Ufuusg



 


AC911191
LP183-(NH-C6)s(invAb)scaa_2Nau
717
cPrpasAfscsUfgGfuuuguAfgAfcUfa
565



aguCfUfAfcaaacuaguus(invAb)

Ufuusg



 


AC911192
LP183-(NH-C6)s(invAb)sa_2Nauag
718
cPrpusCfsasAfcUfgguuuGfuAfgAfc
566



ucuAfCfAfaaccaiuugas(invAb)

Ufausu



 


AC911193
LP183-(NH-C6)s(invAb)sa_2Nuagu
719
cPrpusUfscsAfaCfugguuUfgUfaGfa
567



cuaCfAfAfaccaguugaas(invAb)

Cfuasu



 


AC911194
LP183-(NH-C6)s(invAb)sagucuaca
720
cPrpasGfsgsUfcAfacuggUfuUfgUfa
568



AfAfCfcaguuiaccus(invAb)

Gfacsu



 


AC911195
LP183-(NH-C6)s(invAb)saaccaguu
721
cPrpusCfsusUfgCfucaggUfcAfaCfu
569



GfAfCfcugaicaagas(invAb)

Gfgusu



 


AC911196
LP183-(NH-C6)s(invAb)saccuccaa
722
cPrpusAfsasUfgAfgccacAfcUfuGfg
570



GfUfGfuggcucauuas(invAb)

Afggsu



 


AC911197
LP183-(NH-C6)s(invAb)scuccaagu
723
cPrpusCfsusAfaUfgagccAfcAfcUfu
571



GfUfGfgcucauuagas(invAb)

Gfgasg



 


AC911198
LP183-(NH-C6)s(invAb)succaagug
724
cPrpusCfscsUfaAfugagcCfaCfaCfu
572



UfGfGfcucauuaggas(invAb)

Ufggsa



 


AC911199
LP183-(NH-C6)s(invAb)sccaagugu
725
cPrpusGfscsCfuAfaugagCfcAfcAfc
573



GfGfCfucauuagicas(invAb)

Ufugsg



 


AC911200
LP183-(NH-C6)s(invAb)sguggcuca
726
cPrpusGfsasUfgUfugccuAfaUfgAfg
574



UfUfAfggcaacaucas(invAb)

Cfcasc



 


AC911201
LP183-(NH-C6)s(invAb)sa_2Nuuag
727
cPrpusUfsasUfgAfuggauGfuUfgCfc
575



gcaAfCfAfuccaucauaas(invAb)

Ufaasu



 


AC911202
LP183-(NH-C6)s(invAb)sguaggcaa
728
cPrpusUfsusAfuGfauggaUfgUfuGfc
576



CfAfUfccaucauaaas(invAb)

Cfuasc



 


AC911203
LP183-(NH-C6)s(invAb)saggcaaca
729
cPrpusGfsusUfuAfugaugGfaUfgUfu
577



UfCfCfaucauaaacas(invAb)

Gfccsu



 


AC911204
LP183-(NH-C6)s(invAb)sggccaggu
730
cPrpasGfsasUfuUfuacuuCfcAfcCfu
578



GfGfAfaguaaaaucus(invAb)

Gfgcsc



 


AC911205
LP183-(NH-C6)s(invAb)sga_2Naga
731
cPrpasGfscsUfuGfuggguUfuCfaAfu
579



uugAfAfAfcccacaaicus(invAb)

Cfuusc



 


AC911206
LP183-(NH-C6)s(invAb)sa_2Ngauu
732
cPrpusCfsasGfcUfuguggGfuUfuCfa
580



gaaAfCfCfcacaaicugas(invAb)

Afucsu



 


AC911207
LP183-(NH-C6)s(invAb)sga_2Nuug
733
cPrpusUfscsAfgCfuugugGfgUfuUfc
581



aaaCfCfCfacaaicugaas(invAb)

Afausc



 


AC911208
LP183-(NH-C6)s(invAb)sacgccaaa
734
cPrpusGfsusCfuGfucuugGfcUfuUfg
582



GfCfCfaagacaiacas(invAb)

Gfcgsu



 


AC911209
LP183-(NH-C6)s(invAb)scagcaucg
735
cPrpusAfsgsUfcUfaccauGfuCfgAfu
583



AfCfAfugguagacuas(invAb)

Gfcusg



 


AC912001
LP293-(NH-C6)s(invAb)scagcaucg
736
cPrpusAfsgsUfcUfaccauGfuCfgAfu
583



AfCfAfugguagacuas(invAb)

Gfcusg



 


AC912002
LP293-(NH-C6)s(invAb)scagcaucg
736
cPrpusAfsgsuCfuaccauGfuCfgAfug
585



AfCfAfugguagacuas(invAb)

cusg



 


AC912003
LP293-(NH-C6)s(invAb)scagcaucg
736
cPrpusAfsgsucuAfccauGfuCfgAfug
586



AfCfAfugguagacuas(invAb)

cusg



 


AC912004
LP293-(NH-C6)s(invAb)scagcaucg
736
cPrpusAfsgsucuacCfauGfuCfgAfug
587



AfCfAfugguagacuas(invAb)

cusg



 


AC912005
LP293-(NH-C6)s(invAb)scagcaucg
736
cPrpusAfsgucuAfccauGfuCfgAfugc
588



AfCfAfugguagacuas(invAb)

usg



 


AC912006
LP293-(NH-C6)s(invAb)scagcaucg
737
cPrpusAfsgsucuAfccauGfuCfgAfug
586



AfcAfugguagacuas(invAb)

cusg



 


AC912007
LP293-(NH-C6)s(invAb)scagcauCf
738
cPrpusAfsgsucuAfccauGfuCfgAfug
586



gAfcAfugguagacuas(invAb)

cusg



 


AC912008
LP293-(NH-C6)s(invAb)scagcaucg
739
cPrpusAfsgsucuAfccauGfuCfgAfug
586



AfcAfUfgguagacuas(invAb)

cusg



 


AC912009
LP293-(NH-C6)s(invAb)scagcaucg
736
cPrpusAfsgsucuAfccauGfuCfgaugc
589



AfCfAfugguagacuas(invAb)

usg



 


AC912010
LP293-(NH-C6)s(invAb)scagcaucg
736
cPrpusAfsgsucuacCfauguCfgaugcu
590



AfCfAfugguagacuas(invAb)

sg



 


AC912011
LP293-(NH-C6)s(invAb)scagcaucg
736
cPrpusAfsgsucuAfccauguCfgaugcU
591



AfCfAfugguagacuas(invAb)

fsg



 


AC912669
LP293-(NH-C6)s(invAb)scagcaucg
739
cPrpusAfsgucuAfccauGfuCfgAfugc
588



AfcAfUfgguagacuas(invAb)

usg



 


AC912670
LP293-(NH-C6)s(invAb)scagcaucg
739
cPrpusAfsgucuAfccauGfuCfgAfugc
593



AfcAfUfgguagacuas(invAb)

susg



 


AC912671
LP293-(NH-C6)s(invAb)scagcaucg
739
cPrpusAfsgucuAfccauGfuCfgAfugc
592



AfcAfUfgguagacuas(invAb)

ussg



 


AC912672
LP293-(NH-C6)s(invAb)scagcaucg
739
cPrpusAfsgucuAUNAccauGfuCfgAfu
594



AfcAfUfgguagacuas(invAb)

gcusg



 


AC912673
LP293-(NH-C6)s(invAb)scagcaucg
739
cPrpusAfsgucudAccauGfuCfgdAugc
595



AfcAfUfgguagacuas(invAb)

usg



 


AC912674
LP293-(NH-C6)s(invAb)scagcaucg
739
cPrpusAfsgucudAccaudGuCfgdAugc
596



AfcAfUfgguagacuas(invAb)

usg



 


AC912675
LP293-(NH-C6)s(invAb)scagcaucg
740
cPrpusAfsgucudAccauGfuCfgdAugc
595



AfcAfdTgguagacuas(invAb)

usg









In some embodiments, a MAPT RNAi agent is prepared or provided as a salt, mixed salt, or a free-acid. In some embodiments, a MAPT RNAi agent is prepared or provided as a pharmaceutically acceptable salt. In some embodiments, a MAPT RNAi agent is prepared or provided as a pharmaceutically acceptable sodium or potassium salt The RNAi agents described herein, upon delivery to a cell expressing a MAPT gene, inhibit or knockdown expression of one or more MAPT genes in vivo and/or in vitro.
Targeting Groups, Linking Groups, Lipid PK/PD Moieties, and Delivery Vehicles
In some embodiments, a MAPT RNAi agent contains or is conjugated to one or more non-nucleotide groups including, but not limited to, a targeting group, a linking group, a delivery polymer, a pharmacokinetic/pharmacodynamic (PK/PD) modulator, or a delivery vehicle. The non-nucleotide group can enhance targeting, delivery, or attachment of the RNAi agent. The non-nucleotide group can be covalently linked to the 3′ and/or 5′ end of either the sense strand and/or the antisense strand. In some embodiments, a MAPT RNAi agent contains a non-nucleotide group linked to the 3′ and/or 5′ end of the sense strand. In some embodiments, a non-nucleotide group is linked to the 5′ end of a MAPT RNAi agent sense strand. A non-nucleotide group can be linked directly or indirectly to the RNAi agent via a linker/linking group. In some embodiments, a non-nucleotide group is linked to the RNAi agent via a labile, cleavable, or reversible bond or linker.
In some embodiments, a non-nucleotide group enhances the pharmacokinetic or biodistribution properties of an RNAi agent or conjugate to which it is attached to improve cell- or tissue-specific distribution and cell-specific uptake of the conjugate. In some embodiments, a non-nucleotide group enhances endocytosis of the RNAi agent.
Targeting groups or targeting moieties enhance the pharmacokinetic or biodistribution properties of a conjugate or RNAi agent to which they are attached to improve cell-specific (including, in some cases, organ specific) distribution and cell-specific (or organ specific) uptake of the conjugate or RNAi agent. A targeting group can be monovalent, divalent, trivalent, tetravalent, or have higher valency for the target to which it is directed. Representative targeting groups include, without limitation, compounds with affinity to cell surface molecule, cell receptor ligands, hapten, antibodies, monoclonal antibodies, antibody fragments, and antibody mimics with affinity to cell surface molecules. In some embodiments, a targeting group is linked to an RNAi agent using a linker, such as a PEG linker or one, two, or three abasic and/or ribitol (abasic ribose) residues, which in some instances can serve as linkers.
A targeting group, with or without a linker, can be attached to the 5′ or 3′ end of any of the sense and/or antisense strands disclosed in Tables 2, 3, 4, 5, 6, and 9. A linker, with or without a targeting group, can be attached to the 5′ or 3′ end of any of the sense and/or antisense strands disclosed in Tables 2, 3, 4, 5, 6, and 9.
The MAPT RNAi agents described herein can be synthesized having a reactive group, such as an amino group (also referred to herein as an amine), at the 5′-terminus and/or the 3′-terminus. The reactive group can be used subsequently to attach a targeting moiety using methods typical in the art.
For example, in some embodiments, the MAPT RNAi agents disclosed herein are synthesized having an NH2-C6 group at the 5′-terminus of the sense strand of the RNAi agent. The terminal amino group subsequently can be reacted to form a conjugate with, for example, a group that includes a lipid moiety or an antigen binding protein. In some embodiments, the MAPT RNAi agents disclosed herein are synthesized having one or more alkyne groups at the 5′-terminus of the sense strand of the RNAi agent.
In some embodiments, targeting groups are linked to the MAPT RNAi agents without the use of an additional linker. In some embodiments, the targeting group is designed having a linker readily present to facilitate the linkage to a MAPT RNAi agent. In some embodiments, when two or more RNAi agents are included in a composition, the two or more RNAi agents can be linked to their respective targeting groups using the same linkers. In some embodiments, when two or more RNAi agents are included in a composition, the two or more RNAi agents are linked to their respective targeting groups using different linkers.
In some embodiments, a linking group is conjugated to the RNAi agent. The linking group facilitates covalent linkage of the agent to a targeting group, pharmacokinetic modulator, delivery polymer, or delivery vehicle. The linking group can be linked to the 3′ and/or the 5′ end of the RNAi agent sense strand or antisense strand. In some embodiments, the linking group is linked to the RNAi agent sense strand. In some embodiments, the linking group is conjugated to the 5′ or 3′ end of an RNAi agent sense strand. In some embodiments, a linking group is conjugated to the 5′ end of an RNAi agent sense strand. Examples of linking groups, include but are not limited to: C6-SS-C6, 6-SS-6, reactive groups such a primary amines (e.g., NH2-C6) and alkynes, alkyl groups, abasic residues/nucleotides, amino acids, tri-alkyne functionalized groups, ribitol, and/or PEG groups. Examples of certain linking groups are provided in Table 10.
A linker or linking group is a connection between two atoms that links one chemical group (such as an RNAi agent) or segment of interest to another chemical group (such as a targeting group, pharmacokinetic modulator, or delivery polymer) or segment of interest via one or more covalent bonds. A labile linkage contains a labile bond. A linkage can optionally include a spacer that increases the distance between the two joined atoms. A spacer may further add flexibility and/or length to the linkage. Spacers include, but are not to be limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups; each of which can contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and saccharides. Spacer groups are well known in the art and the preceding list is not meant to limit the scope of the description. In some embodiments, a MAPT RNAi agent is conjugated to a polyethylene glycol (PEG) moiety, or to a hydrophobic group having 12 or more carbon atoms, such as a cholesterol or palmitoyl group.
In some embodiments, a MAPT RNAi agent is linked to one or more antigen binding proteins. Antigen binding proteins may enhance the bioavailability of the RNAi agent, the delivery of the RNAi agent to a cell of interest, or the facilitation of shuttling the RNAi agent across the blood brain barrier. In some embodiments, the antigen binding protein may be conjugated to a linker at the 3′ or 5′ end of a sense strand or an antisense strand of an RNAi agent described herein. In some embodiments, an antigen binding protein may be linked at both the 3′ or 5′ end of either the sense strand or the antisense strand of an RNAi agent described herein.
In some embodiments, a MAPT RNAi agent is linked to one or more lipid PK/PD moieties (referred to herein as “lipid moieties” or “PK/PD modulators”.) Lipid PK/PD moieties may enhance the pharmacodynamic or pharmacokinetic properties of the RNAi agent. In some embodiments, the lipid moiety may be conjugated to a linker at the 3′ or 5′ end of a sense strand or an antisense strand of an RNAi agent described herein. In some embodiments, a lipid moiety may be linked at both the 3′ or 5′ end of either the sense strand or the antisense strand of an RNAi agent described herein. Examples of PK/PD modulators may be found, for example, in PCT Publication No. WO2023/245061, which is incorporated by reference in its entirety herein.
In some embodiments, an antigen binding protein may be conjugated to a MAPT RNAi agent by reacting a MAPT RNAi agent comprising an amine-comprising linker, for example, (NH2-C6) (see Table 10). In some embodiments, the amine-comprising linker may be located on the 5′ end of the sense strand or the antisense strand of a MAPT RNAi agent. In some embodiments, the amine-comprising linker may be located on the 3′ end of the sense strand or the antisense strand of an RNAi agent.
In some embodiments, a lipid moiety may be conjugated to a MAPT RNAi agent by reacting a MAPT RNAi agent comprising an amine-comprising linker, for example, (NH2-C6) (see Table 10). In some embodiments, the amine-comprising linker may be located on the 5′ end of the sense strand or the antisense strand of a MAPT RNAi agent. In some embodiments, the amine-comprising linker may be located on the 3′ end of the sense strand or the antisense strand of an RNAi agent.
Any of the MAPT RNAi agent nucleotide sequences listed in Tables 2, 3, 4, 5, 6, and 9, whether modified or unmodified, can contain 3′ and/or 5′ targeting group(s), linking group(s), and/or antigen binding fragments. Any of the MAPT RNAi agent duplexes listed in Tables 7, 8, and 9, whether modified or unmodified, can further comprise a targeting group or linking group, but not limited to, those depicted in Table 10, and the targeting group or linking group can be attached to the 3′ or 5′ terminus of either the sense strand or the antisense strand of the MAPT RNAi agent duplex.
In some embodiments, an RNAi agent comprising an amine-comprising linker, such as (NH2-C6) or (NH2-C6)s, may be reacted with a lipid comprising an activated ester moiety.
In some embodiments, a MAPT RNAi agent may be conjugated to a lipid moiety using phosphoramidite synthesis. Synthesizing oligonucleotides using phosphoramidites is well-known in the art. In some embodiments, a lipid moiety may be conjugated to the 5′ end of the sense strand or the antisense strand of a MAPT RNAi agent using a phosphoramidite. In some embodiments, a lipid moiety may be conjugated to the 3′ end of the sense strand or the antisense strand of a MAPT RNAi agent using a phosphoramidite. In some embodiments, a phosphoramidite may be used to conjugate a lipid moiety to a MAPT RNAi agent.
In some embodiments, MAPT RNAi agents may comprise a lipid moiety on an internal nucleotide (i.e., not on the 3′ or 5′ terminal nucleotides.) In some embodiments, an internal nucleotide may be linked to the 2′ position of ribose.
Any of the MAPT RNAi agent nucleotide sequences listed in Tables 2, 3, 4, 5, 6, and 9, whether modified or unmodified, can contain 3′ and/or 5′ targeting group(s), linking group(s), and/or lipid PK/PD moieties. Any of the MAPT RNAi agent sequences listed in Tables 3, 4, 5, 6, and 9, or are otherwise described herein, which contain a 3′ or 5′ targeting group, linking group, and/or lipid PK/PD moiety can alternatively contain no 3′ or 5′ targeting group, linking group, or lipid PK/PD moiety, or can contain a different 3′ or 5′ targeting group, linking group, or lipid PK/PD moiety including, but not limited to, those depicted in Table 10. Any of the MAPT RNAi agent duplexes listed in Tables 7, 8, and 9, whether modified or unmodified, can further comprise a targeting group, linking group, or PK/PD moiety including, but not limited to, those depicted in Table 10, and the targeting group, linking group or PK/PD moiety can be attached to the 3′ or 5′ terminus of either the sense strand or the antisense strand of the MAPT RNAi agent duplex.
Examples of certain modified nucleotides, capping moieties, and linking groups are provided in Table 10.







TABLE 10





Structures Representing Various Modified Nucleotides, Capping Moieties, Lipid


PK/PD Moieties, and Linking Groups (wherein   indicates the point of connection)













 


cPrpu


 








 


cPrpus


 








 


cPrpa


 








 


cPrpas


 








 


a_2N


 








 


a_2Ns


 


When positioned internally:


 








 


(invAb)


 








 


(invAb)s


 








(invAb)


When position at the 3′ terminal end:


 








 


(C6-SS-C6)


When positioned internally:


 








 


(C6-SS-C6)


When position at the 3′ terminal end:


 








 


(6-SS-6)


When positioned internally:


 








 


(6-SS-6)


 








 


(NHC12)s


 








 


(NH2-C6)


 








 


(NH2-C6)s


 








 


(NH-C6)


 








 


(NH-C6)s


 








 


L20


 








 


L20-p


 








 


[CP-1113]


 








 


CP-1113-p


 








 


L-1026


 








 


L-1026-p


 








 


(C6)


 








 


(C6)s


 








 


(C5)


 








 


(C5)s


 








 


LP128


 








 


LP132


 








 


LP183


 








 


LP183-(NH-C6)s


 








 


LP183r


 








 


LP183r-(C5)s


 








 


LP200


 








 


LP232


 








 


LP-233


 








 


LP242


 








 


LP243


 








 


LP245


 








 


LP249


 








 


LP257


 








 


LP259


 








 


LP260


 








 


LP262


 








 


LP269


 








 


LP273


 








 


LP274


 








 


LP276


 








 


LP283


 








 


LP286


 








 


LP287


 








 


LP289


 








 


LP290


 








 


LP293


 








 


LP296


 








 


LP300


 








 


LP303


 








 


LP304


 








 


LP310


 








 


LP383


 








 


LP395


 








 


LP396


 








 


LP409


 








 


LP429


 








 


LP430


 








 


LP431


 








 


LP435


 








 


LP439


 








 


LP440


 








 


LP441


 








 


LP456


 








 


LP462


 








 


LP463


 








 


LP464


 








 


LP465


 








 


LP466


 


When positioned internally at 2′ position:


 








 


LP493 (wherein B is a nucleobase)


 








 


(2C8C12)s


 








 


(2C6C10)s


 








 


HO-C16s


 








 


c16s


 








 


C22s


 








 


aC16


 








 


aC16s


 








 


uC16


 








 


uC16s


 








 


cC16


 








 


cC16s


 








 


gC16


 








 


gC16s









Alternatively, other linking groups known in the art may be used. In many instances, linking groups can be commercially acquired or alternatively, are incorporated into commercially available nucleotide phosphoramidites. (See, e.g., International Patent Application Publication No. WO 2019/161213, which is incorporated herein by reference in its entirety).
In some embodiments, a MAPT RNAi agent is delivered without being conjugated to an antigen binding protein or other targeting group (referred to as being “naked” or a “naked RNAi agent”).
In some embodiments, a MAPT RNAi agent is delivered without being conjugated to a targeting ligand or pharmacokinetic/pharmacodynamic (PK/PD) modulator (referred to as being “naked” or a “naked RNAi agent”).
In some embodiments, a MAPT RNAi agent is conjugated to a targeting group, a linking group, a PK modulator, and/or another non-nucleotide group to facilitate delivery of the MAPT RNAi agent to the cell or tissue of choice, for example, to a CNS cell in vivo. In some embodiments, a MAPT RNAi agent is conjugated to an antigen binding protein. In some embodiments, a MAPT RNAi agent is conjugated to a lipid moiety.
In some embodiments, a delivery vehicle may be used to deliver an RNAi agent to a cell or tissue. A delivery vehicle is a compound that improves delivery of the RNAi agent to a cell or tissue. A delivery vehicle can include, or consist of, but is not limited to: a polymer, such as an amphipathic polymer, a membrane active polymer, a peptide, a melittin peptide, a melittin-like peptide (MLP), a lipid, a reversibly modified polymer or peptide, or a reversibly modified membrane active polyamine.
In some embodiments, the RNAi agents can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs or other delivery systems available in the art for nucleic acid delivery. The RNAi agents can also be chemically conjugated to targeting groups, lipids (including, but not limited to cholesteryl and cholesteryl derivatives), encapsulating in nanoparticles, liposomes, micelles, conjugating to polymers or DPCs (see, for example WO 2000/053722, WO 2008/022309, WO 2011/104169, and WO 2012/083185, WO 2013/032829, WO 2013/158141, each of which is incorporated herein by reference), by iontophoresis, or by incorporation into other delivery vehicles or systems available in the art such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors. In some embodiments the RNAi agents can be conjugated to antibodies having affinity for CNS cells. In some embodiments, the RNAi agents can be linked to targeting ligands that have affinity for CNS cells or receptors present on CNS cells.
Antigen Binding Proteins
In one aspect, MAPT RNAi agents are conjugates to antigen binding proteins. In some embodiments, the antigen binding protein may be selected from the group consisting of: an antibody, an antibody fragment (e.g., an antigen binding fragment, or Fab), scFv, or other functional component or derivative of an antibody encompassing a Fab and/or complementary-determining regions (CDRs) disclosed herein.
In some embodiments, the antigen binding protein may act as a shuttle to facilitate the crossing of the blood brain barrier (BBB) of the RNAi agent, such that the RNAi agent may be administered subcutaneously and reach CNS tissue. In some embodiments, the antigen binding protein is an anti-Transferrin 1 (TfR1) antibody or Fab.
In some embodiments, the antigen binding protein is a Fab. In some embodiments, the Fab comprises (i) 6 complementary determining regions (CDRs), (ii) 3 CDRs on the variable light chain (VL), or (iii) 3 CDRs on the variable heavy chain (VH).
In some embodiments, the Fab comprises a light chain and a heavy chain. In some embodiments, the light chain comprises a variable light chain (VL) and a light constant chain 1 (CL). In some embodiments, the VL comprises three CDRs. In some embodiments, the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3. In some embodiments, the heavy chain comprises a variable heavy chain (VH) and a heavy constant chain 1 (CH). In some embodiments, the VH comprises three CDRs. In some embodiments, the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3.
In some embodiments, the light constant chain 1 (CL) comprises or consists of the sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2). In some embodiments, the light chain comprises or consists of the sequence:









(SEQ ID NO: 3)



DIQLTQSPSSLSASVGDRVTITCRASDKLYSNLAWYQQKPGKAPK



 



LLIYDATLLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQH



 



FWGTPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL



 



LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT



 



LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.






In some embodiments, the heavy constant chain 1 (CH) comprises or consists of the sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH (SEQ ID NO: 4). In some embodiments the heavy chain comprises or consists of the sequence:









(SEQ ID NO: 5)



EVQLVESGGGLVQPGGSLRLSCATSGFTFTSYWMHWVRQAPGKGL



 



EWVAEINPTNGRTNYIEKFKSRITLSVDKSKSTVYLQMNSLRAED



 



TAVYYCARGTRAYHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS



 



GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS



 



LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH.






In some embodiments, the antigen binding protein may have a VL CDR1 sequence selected from the group consisting of: RASDGLYSNLA (SEQ ID NO: 6), RASDNLYRNLA (SEQ ID NO: 7), and RASDKLYSNLA (SEQ ID NO: 8).
In some embodiments, the antigen binding protein may have a VL CDR2 sequence selected from the group consisting of: DATLLAS (SEQ ID NO: 9), DARNLAS (SEQ ID NO: 10), DAFNLAS (SEQ ID NO: 11), DATRLAS (SEQ ID NO: 12), DATKLAS (SEQ ID NO: 13), and DAKNLAS (SEQ ID NO: 14).
In some embodiments, the antigen binding protein may have a VL CDR3 sequence of QHFWGTPLT (SEQ ID NO: 15).
In some embodiments, the antigen binding protein may have a VH CDR1 sequence selected from the group consisting of: GYTFNSYWMH (SEQ ID NO: 16), GYTFKSYWMH (SEQ ID NO: 17), GFTFTSYWMH (SEQ ID NO: 18), GYTFTSYWVH (SEQ ID NO: 19), and GYTFTSYWMH (SEQ ID NO: 20).
In some embodiments, the antigen binding protein may have a VH CDR2 sequence selected from the group consisting of: EINPTNGRVNYIEKFKS (SEQ ID NO: 21), EINPTNGRFNYIEKFKS (SEQ ID NO: 22), EINPTNGRTNYIEKFKS (SEQ ID NO: 23), and EINPTNGRSNYIEKFKS (SEQ ID NO: 24).
In some embodiments, the antigen binding protein may have a VH CDR3 sequence of:









(SEQ ID NO: 25)



GTRAYHY.






In some embodiments, the VL comprises a sequence of any one of the sequences listed in Table A. Each of the Fabs described in Table A may have a light chain constant region that comprises or consists of the sequence:









(SEQ ID NO: 2)



RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN



 



ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT



 



HQGLSSPVTKSFNRGEC













TABLE A







VL chains with CDR mutation combinations in bold.









SEQ




ID




NO.
Fab
VL SEQUENCE





26
Fab0002
DIQMTQSPSSLSASVGDRVTITCRASDNLYSNLAWYQQKPGKSPK




LLVYDATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHF




WGTPLTFGQGTKVEIK


 


27
Fab0060
DIQLTQSPSSLSASVGDRVTITCRASDGLYSNLAWYQQKPGKAPKL




LIYDATKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFW




GTPLTFGQGTKVEIK


 


27
Fab0061
DIQLTQSPSSLSASVGDRVTITCRASDGLYSNLAWYQQKPGKAPKL




LIYDATKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFW




GTPLTFGQGTKVEIK


 


28
Fab0062
DIQLTQSPSSLSASVGDRVTITCRASDGLYSNLAWYQQKPGKAPKL




LIYDAFNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFW




GTPLTFGQGTKVEIK


 


29
Fab0063
DIQLTQSPSSLSASVGDRVTITCRASDGLYSNLAWYQQKPGKAPKL




LIYDATRLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFW




GTPLTFGQGTKVEIK


 


30
Fab0064
DIQLTQSPSSLSASVGDRVTITCRASDGLYSNLAWYQQKPGKAPKL




LIYDAKNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFW




GTPLTFGQGTKVEIK


 


31
Fab0065
DIQLTQSPSSLSASVGDRVTITCRASDNLYRNLAWYQQKPGKAPK




LLIYDATKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHF




WGTPLTFGQGTKVEIK


 


28
Fab0066
DIQLTQSPSSLSASVGDRVTITCRASDGLYSNLAWYQQKPGKAPKL




LIYDAFNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFW




GTPLTFGQGTKVEIK


 


31
Fab0067
DIQLTQSPSSLSASVGDRVTITCRASDNLYRNLAWYQQKPGKAPK




LLIYDATKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHF




WGTPLTFGQGTKVEIK


 


27
Fab0068
DIQLTQSPSSLSASVGDRVTITCRASDGLYSNLAWYQQKPGKAPKL




LIYDATKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFW




GTPLTFGQGTKVEIK


 


27
Fab0069
DIQLTQSPSSLSASVGDRVTITCRASDGLYSNLAWYQQKPGKAPKL




LIYDATKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFW




GTPLTFGQGTKVEIK


 


32
Fab0070
DIQLTQSPSSLSASVGDRVTITCRASDKLYSNLAWYQQKPGKAPKL




LIYDATLLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFW




GTPLTFGQGTKVEIK


 


33
Fab0071
DIQLTQSPSSLSASVGDRVTITCRASDGLYSNLAWYQQKPGKAPKL




LIYDARNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFW




GTPLTFGQGTKVEIK


 


34
Fab0072
DIQLTQSPSSLSASVGDRVTITCRASDNLYRNLAWYQQKPGKAPK




LLIYDARNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHF




WGTPLTFGQGTKVEIK


 


31
Fab0073
DIQLTQSPSSLSASVGDRVTITCRASDNLYRNLAWYQQKPGKAPK




LLIYDATKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHF




WGTPLTFGQGTKVEIK


 


30
Fab0074
DIQLTQSPSSLSASVGDRVTITCRASDGLYSNLAWYQQKPGKAPKL




LIYDAKNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFW




GTPLTFGQGTKVEIK









In some embodiments, the VL comprises a sequence of any one of the sequences listed in Table B. Each of the Fabs described in Table B may have a heavy chain constant region that comprises or consist of the sequence:









(SEQ ID NO: 4)



ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS



 



GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN



 



HKPSNTKVDKRVEPKSCDKTH.













TABLE B







VH chains with CDR mutation combinations in bold.









SEQ




ID




NO.
Fab
VH SEQUENCE





35
Fab0002
EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQ




RLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSE




DTAVYYCARGTRAYHYWGQGTMVTVSS


 


36
Fab0060
EVQLVESGGGLVQPGGSLRLSCATSGYTFTSYWVHWVRQAPGK




GLEWVAEINPTNGRTNYIEKFKSRITLSVDKSKSTVYLQMNSLRA




EDTAVYYCARGTRAYHYWGQGTLVTVSS


 


37
Fab0061
EVQLVESGGGLVQPGGSLRLSCATSGYTFTSYWMHWVRQAPGK




GLEWVAEINPTNGRVNYIEKFKSRITLSVDKSKSTVYLQMNSLRA




EDTAVYYCARGTRAYHYWGQGTLVTVSS


 


37
Fab0062
EVQLVESGGGLVQPGGSLRLSCATSGYTFTSYWMHWVRQAPGK




GLEWVAEINPTNGRVNYIEKFKSRITLSVDKSKSTVYLQMNSLRA




EDTAVYYCARGTRAYHYWGQGTLVTVSS


 


37
Fab0063
EVQLVESGGGLVQPGGSLRLSCATSGYTFTSYWMHWVRQAPGK




GLEWVAEINPTNGRVNYIEKFKSRITLSVDKSKSTVYLQMNSLRA




EDTAVYYCARGTRAYHYWGQGTLVTVSS


 


37
Fab0064
EVQLVESGGGLVQPGGSLRLSCATSGYTFTSYWMHWVRQAPGK




GLEWVAEINPTNGRVNYIEKFKSRITLSVDKSKSTVYLQMNSLRA




EDTAVYYCARGTRAYHYWGQGTLVTVSS


 


38
Fab0065
EVQLVESGGGLVQPGGSLRLSCATSGYTFTSYWVHWVRQAPGK




GLEWVAEINPTNGRSNYIEKFKSRITLSVDKSKSTVYLQMNSLRA




EDTAVYYCARGTRAYHYWGQGTLVTVSS


 


39
Fab0066
EVQLVESGGGLVQPGGSLRLSCATSGYTFTSYWVHWVRQAPGK




GLEWVAEINPTNGRVNYIEKFKSRITLSVDKSKSTVYLQMNSLRA




EDTAVYYCARGTRAYHYWGQGTLVTVSS


 


36
Fab0067
EVQLVESGGGLVQPGGSLRLSCATSGYTFTSYWVHWVRQAPGK




GLEWVAEINPTNGRTNYIEKFKSRITLSVDKSKSTVYLQMNSLRA




EDTAVYYCARGTRAYHYWGQGTLVTVSS


 


39
Fab0068
EVQLVESGGGLVQPGGSLRLSCATSGYTFTSYWVHWVRQAPGK




GLEWVAEINPTNGRVNYIEKFKSRITLSVDKSKSTVYLQMNSLRA




EDTAVYYCARGTRAYHYWGQGTLVTVSS


 


38
Fab0069
EVQLVESGGGLVQPGGSLRLSCATSGYTFTSYWVHWVRQAPGK




GLEWVAEINPTNGRSNYIEKFKSRITLSVDKSKSTVYLQMNSLRA




EDTAVYYCARGTRAYHYWGQGTLVTVSS


 


40
Fab0070
EVQLVESGGGLVQPGGSLRLSCATSGFTFTSYWMHWVRQAPGK




GLEWVAEINPTNGRTNYIEKFKSRITLSVDKSKSTVYLQMNSLRA




EDTAVYYCARGTRAYHYWGQGTLVTVSS


 


41
Fab0071
EVQLVESGGGLVQPGGSLRLSCATSGYTFNSYWMHWVRQAPGK




GLEWVAEINPTNGRTNYIEKFKSRITLSVDKSKSTVYLQMNSLRA




EDTAVYYCARGTRAYHYWGQGTLVTVSS


 


42
Fab0072
EVQLVESGGGLVQPGGSLRLSCATSGYTFNSYWMHWVRQAPGK




GLEWVAEINPTNGRFNYIEKFKSRITLSVDKSKSTVYLQMNSLRA




EDTAVYYCARGTRAYHYWGQGTLVTVSS


 


43
Fab0073
EVQLVESGGGLVQPGGSLRLSCATSGYTFKSYWMHWVRQAPGK




GLEWVAEINPTNGRTNYIEKFKSRITLSVDKSKSTVYLQMNSLRA




EDTAVYYCARGTRAYHYWGQGTLVTVSS


 


43
Fab0074
EVQLVESGGGLVQPGGSLRLSCATSGYTFKSYWMHWVRQAPGK




GLEWVAEINPTNGRTNYIEKFKSRITLSVDKSKSTVYLQMNSLRA




EDTAVYYCARGTRAYHYWGQGTLVTVSS









Tables C-H show the CDR1, CDR2, and CDR3 variants from VL and VH with the combined beneficial mutations.







TABLE C







VL CDR1 variant









SEQ ID NO:
Fab
CDR1





6
Fab0060
RASDGLYSNLA


 


6
Fab0061
RASDGLYSNLA


 


6
Fab0062
RASDGLYSNLA


 


6
Fab0063
RASDGLYSNLA


 


6
Fab0064
RASDGLYSNLA


 


7
Fab0065
RASDNLYRNLA


 


6
Fab0066
RASDGLYSNLA


 


7
Fab0067
RASDNLYRNLA


 


6
Fab0068
RASDGLYSNLA


 


6
Fab0069
RASDGLYSNLA


 


8
Fab0070
RASDKLYSNLA


 


6
Fab0071
RASDGLYSNLA


 


7
Fab0072
RASDNLYRNLA


 


7
Fab0073
RASDNLYRNLA


 


6
Fab0074
RASDGLYSNLA
















TABLE D







VL CDR2 variants









SEQ ID NO:
Fab
CDR2





13
Fab0060
DATKLAS


 


13
Fab0061
DATKLAS


 


11
Fab0062
DAFNLAS


 


12
Fab0063
DATRLAS


 


14
Fab0064
DAKNLAS


 


13
Fab0065
DATKLAS


 


11
Fab0066
DAFNLAS


 


13
Fab0067
DATKLAS


 


13
Fab0068
DATKLAS


 


13
Fab0069
DATKLAS


 


9
Fab0070
DATLLAS


 


10
Fab0071
DARNLAS


 


10
Fab0072
DARNLAS


 


13
Fab0073
DATKLAS


 


14
Fab0074
DAKNLAS
















TABLE E







VL CDR3 variant









SEQ ID NO:
Fab
CDR3





15
Fab0060
QHFWGTPLT


 


15
Fab0061
QHFWGTPLT


 


15
Fab0062
QHFWGTPLT


 


15
Fab0063
QHFWGTPLT


 


15
Fab0064
QHFWGTPLT


 


15
Fab0065
QHFWGTPLT


 


15
Fab0066
QHFWGTPLT


 


15
Fab0067
QHFWGTPLT


 


15
Fab0068
QHFWGTPLT


 


15
Fab0069
QHFWGTPLT


 


15
Fab0070
QHFWGTPLT


 


15
Fab0071
QHFWGTPLT


 


15
Fab0072
QHFWGTPLT


 


15
Fab0073
QHFWGTPLT


 


15
Fab0074
QHFWGTPLT
















TABLE F







VH CDR1 variants











SEQ ID NO:
Fab
CDR1







19
Fab0060
GYTFTSYWVH



 



20
Fab0061
GYTFTSYWMH



 



20
Fab0062
GYTFTSYWMH



 



20
Fab0063
GYTFTSYWMH



 



20
Fab0064
GYTFTSYWMH



 



19
Fab0065
GYTFTSYWVH



 



19
Fab0066
GYTFTSYWVH



 



19
Fab0067
GYTFTSYWVH



 



19
Fab0068
GYTFTSYWVH



 



19
Fab0069
GYTFTSYWVH



 



18
Fab0070
GFTFTSYWMH



 



16
Fab0071
GYTFNSYWMH



 



16
Fab0072
GYTFNSYWMH



 



17
Fab0073
GYTFKSYWMH



 



17
Fab0074
GYTFKSYWMH

















TABLE G







VH CDR2 variants









SEQ ID NO:
Fab
CDR2





23
Fab0060
EINPTNGRTNYIEKFKS


 


21
Fab0061
EINPTNGRVNYIEKFKS


 


21
Fab0062
EINPTNGRVNYIEKFKS


 


21
Fab0063
EINPTNGRVNYIEKFKS


 


21
Fab0064
EINPTNGRVNYIEKFKS


 


24
Fab0065
EINPTNGRSNYIEKFKS


 


21
Fab0066
EINPTNGRVNYIEKFKS


 


23
Fab0067
EINPTNGRTNYIEKFKS


 


21
Fab0068
EINPTNGRVNYIEKFKS


 


24
Fab0069
EINPTNGRSNYIEKFKS


 


23
Fab0070
EINPTNGRTNYIEKFKS


 


23
Fab0071
EINPTNGRTNYIEKFKS


 


22
Fab0072
EINPTNGRFNYIEKFKS


 


23
Fab0073
EINPTNGRTNYIEKFKS


 


23
Fab0074
EINPTNGRTNYIEKFKS
















TABLE H







VH CDR3 variants











SEQ ID NO:
Fab
CDR3







25
Fab0060
GTRAYHY



 



25
Fab0061
GTRAYHY



 



25
Fab0062
GTRAYHY



 



25
Fab0063
GTRAYHY



 



25
Fab0064
GTRAYHY



 



25
Fab0065
GTRAYHY



 



25
Fab0066
GTRAYHY



 



25
Fab0067
GTRAYHY



 



25
Fab0068
GTRAYHY



 



25
Fab0069
GTRAYHY



 



25
Fab0070
GTRAYHY



 



25
Fab0071
GTRAYHY



 



25
Fab0072
GTRAYHY



 



25
Fab0073
GTRAYHY



 



25
Fab0074
GTRAYHY










In some embodiments, the Fab binds TfR1. In some embodiments, the Fab binds TfR1 with an affinity of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nM. In some embodiments, the Fab binds TfR1 with an affinity of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nM. In some embodiments, the Fab binds TfR1 with an affinity of at least about 1 nM. In some embodiments, the Fab binds TfR1 with a KD value of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nM. In some embodiments, the Fab binds TfR1 with a KD value of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nM. In some embodiments, the Fab binds TfR1 with a KD value of at least about 1 nM.
In some embodiments, the Fab is conjugated to an RNAi agent disclosed herein. In some embodiments, the RNAi agent is conjugated to the Fab using a covalent or non-covalent bond, ionic bond, hydrogen bond, hydrophobic interaction, peptide, polymer, or a nucleic acid binding protein. In some embodiments, the RNAi agent is conjugated to the Fab using a covalent bond. In some embodiments, the RNAi agent is conjugated to the Fab via a lysine residue or a cysteine residue. In some embodiments, the RNAi agent is conjugated to the Fab via a lysine residue. In some embodiments, the RNAi agent is conjugated to the Fab via a cysteine residue. In some embodiments, the RNAi agent is conjugated to the Fab in a site-specific manner. In some embodiments, the RNAi agent is conjugated to the Fab in a non-site-specific manner.
In some embodiments, the RNAi agent is conjugated to the Fab at the 5′ terminus or the 3′ terminus of the RNAi agent. In some embodiments, the RNAi agent is conjugated to the Fab at the 5′ terminus of the RNAi agent. In some embodiments, the RNAi agent is conjugated to the Fab at the 3′ terminus of the RNAi agent. In some embodiments, the RNAi agent is conjugated to the Fab at the 5′ terminus or the 3′ terminus of the sense strand of the RNAi agent. In some embodiments, the RNAi agent is conjugated to the Fab at the 5′ terminus of the sense strand of the RNAi agent. In some embodiments, the RNAi agent is conjugated to the Fab at the 3′ terminus of the sense strand of the RNAi agent.
Pharmaceutical Compositions and Formulations
The MAPT RNAi agents disclosed herein can be prepared as pharmaceutical compositions or formulations (also referred to herein as “medicaments”). In some embodiments, pharmaceutical compositions include at least one MAPT RNAi agent. These pharmaceutical compositions are particularly useful in the inhibition of the expression of MAPT mRNA in a target cell, a group of cells, a tissue, or an organism. The pharmaceutical compositions can be used to treat a subject having a disease, disorder, or condition that would benefit from reduction in the level of the target mRNA, or inhibition in expression of the target gene. The pharmaceutical compositions can be used to treat a subject at risk of developing a disease or disorder that would benefit from reduction of the level of the target mRNA or an inhibition in expression the target gene. In one embodiment, the method includes administering a MAPT RNAi agent linked to an antigen binding protein as described herein, to a subject to be treated. In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and/or delivery polymers) are added to the pharmaceutical compositions that include a MAPT RNAi agent, thereby forming a pharmaceutical formulation or medicament suitable for in vivo delivery to a subject, including a human.
The MAPT RNAi agents disclosed herein can be prepared as pharmaceutical compositions or formulations (also referred to herein as “medicaments”). In some embodiments, pharmaceutical compositions include at least one MAPT RNAi agent. These pharmaceutical compositions are particularly useful in the inhibition of the expression of MAPT mRNA in a target cell, a group of cells, a tissue, or an organism. The pharmaceutical compositions can be used to treat a subject having a disease, disorder, or condition that would benefit from reduction in the level of the target mRNA, or inhibition in expression of the target gene. The pharmaceutical compositions can be used to treat a subject at risk of developing a disease or disorder that would benefit from reduction of the level of the target mRNA or an inhibition in expression the target gene. In one embodiment, the method includes administering a MAPT RNAi agent linked to a PK/PD modulator as described herein, to a subject to be treated. In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and/or delivery polymers) are added to the pharmaceutical compositions that include a MAPT RNAi agent, thereby forming a pharmaceutical formulation or medicament suitable for in vivo delivery to a subject, including a human.
The pharmaceutical compositions that include a MAPT RNAi agent and methods disclosed herein decrease the level of the target mRNA in a cell, group of cells, tissue, organ, or subject, including by administering to the subject a therapeutically effective amount of a herein described MAPT RNAi agent, thereby inhibiting the expression of MAPT mRNA in the subject. In some embodiments, the subject has been previously identified or diagnosed as having a disease or disorder that can be mediated at least in part by a reduction in MAPT gene expression. In some embodiments, the subject has been previously diagnosed with having one or more neurodegenerative diseases such as Alzheimer's disease, Frontotemporal lobar degeneration dementia (FTLD), Progressive supranuclear palsy, and other tauopathies. In some embodiments the neurodegenerative disease is Alzheimer's Disease.
In some embodiments the subject has been previously diagnosed with having neurodegenerative disease.
Embodiments of the present disclosure include pharmaceutical compositions for delivering a MAPT RNAi agent to a CNS cell in vivo. Such pharmaceutical compositions can include, for example, a MAPT RNAi agent conjugated to an antigen binding protein. In other embodiments, a pharmaceutical composition can include a MAPT RNAi agent conjugated to a lipid moiety.
In some embodiments, the described pharmaceutical compositions including a MAPT RNAi agent are used for treating or managing clinical presentations in a subject that would benefit from the inhibition of expression of MAPT. In some embodiments, a therapeutically or prophylactically effective amount of one or more of pharmaceutical compositions is administered to a subject in need of such treatment. In some embodiments, administration of any of the disclosed MAPT RNAi agents can be used to decrease the number, severity, and/or frequency of symptoms of a disease in a subject.
In some embodiments, the described MAPT RNAi agents are optionally combined with one or more additional (i.e., second, third, etc.) therapeutics. A second therapeutic can be another MAPT RNAi agent (e.g., a MAPT RNAi agent that targets a different sequence within a MAPT gene). In some embodiments, a second therapeutic can be an RNAi agent that targets the MAPT gene. An additional therapeutic can also be a small molecule drug, antibody, antibody fragment, and/or aptamer. The MAPT RNAi agents, with or without the one or more additional therapeutics, can be combined with one or more excipients to form pharmaceutical compositions.
The described pharmaceutical compositions that include a MAPT RNAi agent can be used to treat at least one symptom in a subject having a disease or disorder that would benefit from reduction or inhibition in expression of MAPT mRNA. In some embodiments, the subject is administered a therapeutically effective amount of one or more pharmaceutical compositions that include a MAPT RNAi agent thereby treating the symptom. In other embodiments, the subject is administered a prophylactically effective amount of one or more MAPT RNAi agents, thereby preventing or inhibiting the at least one symptom.
In some embodiments, one or more of the described MAPT RNAi agents are administered to a mammal in a pharmaceutically acceptable carrier or diluent. In some embodiments, the mammal is a human.
The route of administration is the path by which a MAPT RNAi agent is brought into contact with the body. In general, methods of administering drugs, oligonucleotides, and nucleic acids including the CNS, for treatment of a mammal are well known in the art and can be applied to administration of the compositions described herein. The MAPT RNAi agents disclosed herein can be administered via any suitable route in a preparation appropriately tailored to the particular route. Thus, in some embodiments, the herein described pharmaceutical compositions are administered via inhalation, intranasal administration, intratracheal administration, or oropharyngeal aspiration administration. In some embodiments, the pharmaceutical compositions can be administered by injection, for example, intravenously, intramuscularly, intracutaneously, subcutaneously, intracerebroventricularly, intraarticularly, intraocularly, or intraperitoneally, or topically.
The pharmaceutical compositions including a MAPT RNAi agent described herein can be delivered to a cell, group of cells, tissue, or subject using oligonucleotide delivery technologies known in the art. In general, any suitable method recognized in the art for delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with the compositions described herein. For example, delivery can be by local administration, (e.g., direct injection, implantation, or topical administering), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intracerebroventricular, intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration. In some embodiments, the compositions are administered via inhalation, intranasal administration, oropharyngeal aspiration administration, or intratracheal administration. For example, in some embodiments, it is desired that the MAPT RNAi agents described herein inhibit the expression of a MAPT gene in the CNS.
In some embodiments, the pharmaceutical compositions described herein comprise one or more pharmaceutically acceptable excipients. The pharmaceutical compositions described herein are formulated for administration to a subject.
As used herein, a pharmaceutical composition or medicament includes a pharmacologically effective amount of at least one of the described therapeutic compounds and one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than the Active Pharmaceutical Ingredient (API, therapeutic product, e.g., MAPT RNAi agent) that are intentionally included in the drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage. Excipients can act to a) aid in processing of the drug delivery system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in product identification, and/or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.
Excipients include, but are not limited to: absorption enhancers, anti-adherents, anti-foaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor® EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Formulations suitable for intra-articular administration can be in the form of a sterile aqueous preparation of the drug that can be in microcrystalline form, for example, in the form of an aqueous microcrystalline suspension. Liposomal formulations or biodegradable polymer systems can also be used to present the drug for both intra-articular and ophthalmic administration.
The active compounds can be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
The MAPT RNAi agents can be formulated in compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
A pharmaceutical composition can contain other additional components commonly found in pharmaceutical compositions. Such additional components include, but are not limited to: anti-pruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamine, diphenhydramine, etc.). It is also envisioned that cells, tissues, or isolated organs that express or comprise the herein defined RNAi agents may be used as “pharmaceutical compositions.” As used herein, “pharmacologically effective amount,” “therapeutically effective amount,” or simply “effective amount” refers to that amount of an RNAi agent to produce a pharmacological, therapeutic, or preventive result.
In some embodiments, MAPT RNAi agent pharmaceutical compositions may contain salts such as sodium chloride, calcium chloride, magnesium chloride, potassium chloride, sodium phosphate dibasic, sodium phosphate monobasic, or combinations thereof.
In some embodiments, the methods disclosed herein further comprise the step of administering a second therapeutic or treatment in addition to administering an RNAi agent disclosed herein. In some embodiments, the second therapeutic is another MAPT RNAi agent (e.g., a MAPT RNAi agent that targets a different sequence within the MAPT target). In other embodiments, the second therapeutic can be a small molecule drug, an antibody, an antibody fragment, and/or an aptamer.
In some embodiments, described herein are compositions that include a combination or cocktail of at least two MAPT RNAi agents having different sequences. In some embodiments, the two or more MAPT RNAi agents are each separately and independently linked to antigen binding proteins. In some embodiments, the two or more MAPT RNAi agents are each separately and independently linked to lipids.
Described herein are compositions for delivery of MAPT RNAi agents to central nervous system (CNS) cells. Furthermore, compositions for delivery of MAPT RNAi agents to cells, including neurons, astrocytes, microglia and endothelial cells, in vivo, are generally described herein.
Generally, an effective amount of a MAPT RNAi agent disclosed herein will be in the range of from about 0.0001 to about 20 mg/kg of body weight dose, e.g., from about 0.001 to about 5 mg/kg of body weight dose. In some embodiments, an effective amount of a MAPT RNAi agent will be in the range of from about 0.01 mg/kg to about 3.0 mg/kg of body weight per dose. In some embodiments, an effective amount of a MAPT RNAi agent will be in the range of from about 0.03 mg/kg to about 2.0 mg/kg of body weight per dose. In some embodiments, an effective amount of a MAPT RNAi agent will be in the range of from about 0.01 to about 1.0 mg/kg. In some embodiments, an effective amount of a MAPT RNAi agent will be in the range of from about 0.50 to about 1.0 mg/kg. In some embodiments, a fixed dose of MAPT RNAi agent is administered to the subject. In some embodiments the dose administered to the human subject is between about 1.0 mg and about 750 mg. In some embodiments, the dose of MAPT RNAi agent administered to the human subject is between about 10 mg and about 450 mg. In some embodiments, the dose of MAPT RNAi agent administered to the human subject is between about 25 mg and about 450 mg. In some embodiments, the dose of MAPT RNAi agent administered to the human subject is about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, or about 450 mg. The amount administered will also likely depend on such variables as the overall health status of the patient, the relative biological efficacy of the compound delivered, the formulation of the drug, the presence and types of excipients in the formulation, and the route of administration. Also, it is to be understood that the initial dosage administered can be increased beyond the above upper level to rapidly achieve the desired blood-level or tissue level, or the initial dosage can be smaller than the optimum. In some embodiments, a dose is administered daily. In some embodiments, a dose is administered weekly. In further embodiments, a dose is administered bi-weekly, tri-weekly, once monthly, or once quarterly (i.e., once every three months).
For treatment of disease or for formation of a medicament or composition for treatment of a disease, the pharmaceutical compositions described herein including a MAPT RNAi agent can be combined with an excipient or with a second therapeutic agent or treatment including, but not limited to: a second or other RNAi agent, a small molecule drug, an antibody, an antibody fragment, peptide, and/or an aptamer.
The described MAPT RNAi agents, when added to pharmaceutically acceptable excipients or adjuvants, can be packaged into kits, containers, packs, or dispensers.
Methods of Treatment and Inhibition of MAPT Gene Expression
The MAPT RNAi agents disclosed herein can be used to treat a subject (e.g., a human or other mammal) having a disease or disorder that would benefit from administration of the RNAi agent. In some embodiments, the RNAi agents disclosed herein can be used to treat a subject (e.g., a human) that would benefit from a reduction and/or inhibition in expression of MAPT mRNA and/or a reduction in MAPT protein.
In some embodiments, the RNAi agents disclosed herein can be used to treat a subject (e.g., a human) having a disease or disorder for which the subject would benefit from reduction in MAPT protein, including but not limited to, Alzheimer's disease, Frontotemporal lobar degeneration dementia (FTLD), Progressive supranuclear palsy, and other tauopathies. Treatment of a subject can include therapeutic and/or prophylactic treatment. The subject is administered a therapeutically effective amount of any one or more MAPT RNAi agents described herein. The subject can be a human, patient, or human patient. The subject may be an adult, adolescent, child, or infant. Administration of a pharmaceutical composition described herein can be to a human being or animal.
Mutant MAPT activity is known to promote neurodegenerative disorders. In some embodiments, the described MAPT RNAi agents are used to treat at least one symptom mediated at least in part by a reduction in mutant MAPT protein levels, in a subject. The subject is administered a therapeutically effective amount of any one or more of the described MAPT RNAi agents. In some embodiments, the subject is administered a prophylactically effective amount of any one or more of the described RNAi agents, thereby treating the subject by preventing or inhibiting the at least one symptom.
In certain embodiments, the present disclosure provides methods for treatment of diseases, disorders, conditions, or pathological states mediated at least in part by MAPT gene expression, in a patient in need thereof, wherein the methods include administering to the patient any of the MAPT RNAi agents described herein.
In some embodiments, the MAPT RNAi agents are used to treat or manage a clinical presentation or pathological state in a subject, wherein the clinical presentation or pathological state is mediated at least in part by a reduction in MAPT gene expression. The subject is administered a therapeutically effective amount of one or more of the MAPT RNAi agents or MAPT RNAi agent-containing compositions described herein. In some embodiments, the method comprises administering a composition comprising a MAPT RNAi agent described herein to a subject to be treated.
In a further aspect, the disclosure features methods of treatment (including prophylactic or preventative treatment) of diseases or symptoms that may be addressed by a reduction in MAPT protein levels, the methods comprising administering to a subject in need thereof a MAPT RNAi agent that includes an antisense strand comprising the sequence of any of the sequences in Table 2, Table 3, or Table 9. Also described herein are compositions for use in such methods.
The described MAPT RNAi agents and/or compositions that include MAPT RNAi agents can be used in methods for therapeutic treatment of disease or conditions caused by enhanced or elevated MAPT protein levels. Such methods include administration of a MAPT RNAi agent as described herein to a subject, e.g., a human or animal subject.
In another aspect, the disclosure provides methods for the treatment (including prophylactic treatment) of a pathological state (such as a condition or disease) mediated at least in part by MAPT gene expression, wherein the methods include administering to a subject a therapeutically effective amount of an RNAi agent that includes an antisense strand comprising the sequence of any of the sequences in Table 2, Table 3, or Table 9.
In some embodiments, methods for inhibiting expression of a MAPT gene are disclosed herein, wherein the methods include administering to a cell an RNAi agent that includes an antisense strand comprising the sequence of any of the sequences in Table 2, Table 3, or Table 9.
In some embodiments, methods for the treatment (including prophylactic treatment) of a pathological state mediated at least in part by MAPT gene expression are disclosed herein, wherein the methods include administering to a subject a therapeutically effective amount of an RNAi agent that includes a sense strand comprising the sequence of any of the sequences in Table 2, Table 4, Table 5, Table 6, or Table 9.
In some embodiments, methods for inhibiting expression of a MAPT gene are disclosed herein, wherein the methods comprise administering to a cell an RNAi agent that includes a sense strand comprising the sequence of any of the sequences in Table 2, Table 4, Table 5, Table 6, or Table 9.
In some embodiments, methods for the treatment (including prophylactic treatment) of a pathological state mediated at least in part by MAPT gene expression are disclosed herein, wherein the methods include administering to a subject a therapeutically effective amount of an RNAi agent that includes a sense strand comprising the sequence of any of the sequences in Table 4, Table 5, Table 6, or Table 9, and an antisense strand comprising the sequence of any of the sequences in Table 3 or Table 9.
In some embodiments, methods for inhibiting expression of a MAPT gene are disclosed herein, wherein the methods include administering to a cell an RNAi agent that includes a sense strand comprising the sequence of any of the sequences in Table 4, Table 5, Table 6, or Table 9, and an antisense strand comprising the sequence of any of the sequences in Table 3 or Table 9.
In some embodiments, methods of inhibiting expression of a MAPT gene are disclosed herein, wherein the methods include administering to a subject a MAPT RNAi agent that includes a sense strand consisting of the nucleobase sequence of any of the sequences in Table 4, Table 5, Table 6, or Table 9, and the antisense strand consisting of the nucleobase sequence of any of the sequences in Table 3 or Table 9. In other embodiments, disclosed herein are methods of inhibiting expression of a MAPT gene, wherein the methods include administering to a subject a MAPT RNAi agent that includes a sense strand consisting of the modified sequence of any of the modified sequences in Table 4, Table 5, Table 6, or Table 9, and the antisense strand consisting of the modified sequence of any of the modified sequences in Table 3 or Table 9.
In some embodiments, methods for inhibiting expression of a MAPT gene in a cell are disclosed herein, wherein the methods include administering one or more MAPT RNAi agents comprising a duplex structure of one of the duplexes set forth in Tables 7, 8, and 9.
In some embodiments, the gene expression level and/or mRNA level of a MAPT gene in certain CNS cells of subject to whom a described MAPT RNAi agent is administered is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99%, relative to the subject prior to being administered the MAPT RNAi agent or to a subject not receiving the MAPT RNAi agent. In some embodiments, the MAPT protein levels in certain CNS cells of a subject to whom a described MAPT RNAi agent is administered is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99%, relative to the subject prior to being administered the MAPT RNAi agent or to a subject not receiving the MAPT RNAi agent. The gene expression level, protein level, and/or mRNA level in the subject may be reduced in a cell, group of cells, and/or tissue of the subject. In some embodiments, the MAPT mRNA levels in certain CNS cells subject to whom a described MAPT RNAi agent has been administered is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% relative to the subject prior to being administered the MAPT RNAi agent or to a subject not receiving the MAPT RNAi agent.
A reduction in gene expression, mRNA, and protein levels can be assessed by any methods known in the art. Reduction or decrease in MAPT protein levels are collectively referred to herein as a decrease in, reduction of, or inhibition of MAPT expression. The Examples set forth herein illustrate known methods for assessing inhibition of MAPT gene expression, including but not limited to determining MAPT protein levels.
Cells, Tissues, Organs, and Non-Human Organisms
Cells, tissues, organs, and non-human organisms that include at least one of the MAPT RNAi agents described herein are contemplated. The cell, tissue, organ, or non-human organism is made by delivering the RNAi agent to the cell, tissue, organ, or non-human organism.
ADDITIONAL ILLUSTRATIVE EMBODIMENTS
Provided here are certain additional illustrative embodiments of the disclosed technology. These embodiments are illustrative only and do not limit the scope of the present disclosure or of the claims attached hereto.
Embodiment 1. An RNAi agent for inhibiting expression of a microtubule associated protein tau (MAPT) gene, comprising:


    
    
        an antisense strand comprising at least 17 contiguous nucleotides differing by 0 or 1 nucleotides from any one of the sequences provided in Table 2 or Table 3; and
        a sense strand comprising a nucleotide sequence that is at least partially complementary to the antisense strand.

Embodiment 2. The RNAi agent of embodiment 1, wherein the antisense strand comprises nucleotides 2-18 of any one of the sequences provided in Table 2 or Table 3.

Embodiment 3. The RNAi agent of embodiment 1 or embodiment 2, wherein the sense strand comprises a nucleotide sequence of at least 17 contiguous nucleotides differing by 0 or 1 nucleotides from any one of the sequences provided in Table 2 or Table 4, and wherein the sense strand has a region of at least 85% complementarity over the 17 contiguous nucleotides to the antisense strand.

Embodiment 4. The RNAi agent of any one of embodiments 1-3, wherein at least one nucleotide of the RNAi agent is a modified nucleotide or includes a modified internucleoside linkage.

Embodiment 5. The RNAi agent of any one of embodiments 1-4, wherein all or substantially all of the nucleotides are modified nucleotides.

Embodiment 6. The RNAi agent of any one of embodiments 4-5, wherein the modified nucleotide is selected from the group consisting of: 2′-O-methyl nucleotide, 2′-fluoro nucleotide, 2′-deoxy nucleotide, 2′,3′-seco nucleotide mimic, locked nucleotide, 2′-F-arabino nucleotide, 2′-methoxyethyl nucleotide, abasic nucleotide, ribitol, inverted nucleotide, inverted 2′-O-methyl nucleotide, inverted 2′-deoxy nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, vinyl phosphonate-containing nucleotide, cyclopropyl phosphonate-containing nucleotide, and 3′-O-methyl nucleotide.

Embodiment 7. The RNAi agent of embodiment 5, wherein all or substantially all of the nucleotides are modified with 2′-O-methyl nucleotides, 2′-fluoro nucleotides, or combinations thereof.

Embodiment 8. The RNAi agent of any one of embodiments 1-7, wherein the antisense strand comprises the nucleotide sequence of any one of the modified sequences provided in Table 3.

Embodiment 9. The RNAi agent of any one of embodiments 1-8, wherein the sense strand comprises the nucleotide sequence of any one of the modified sequences provided in Table 4.

Embodiment 10. The RNAi agent of embodiment 1, wherein the antisense strand comprises the nucleotide sequence of any one of the modified sequences provided in Table 3 and the sense strand comprises the nucleotide sequence of any one of the modified sequences provided in Table 4.

Embodiment 11. The RNAi agent of any one of embodiments 1-10, wherein the sense strand is between 18 and 30 nucleotides in length, and the antisense strand is between 18 and 30 nucleotides in length.

Embodiment 12. The RNAi agent of embodiment 11, wherein the sense strand and the antisense strand are each between 18 and 27 nucleotides in length.

Embodiment 13. The RNAi agent of embodiment 12, wherein the sense strand and the antisense strand are each between 18 and 24 nucleotides in length.

Embodiment 14. The RNAi agent of embodiment 13, wherein the sense strand and the antisense strand are each 21 nucleotides in length.

Embodiment 15. The RNAi agent of embodiment 14, wherein the RNAi agent has two blunt ends.

Embodiment 16. The RNAi agent of any one of embodiments 1-15, wherein the sense strand comprises one or two terminal caps.

Embodiment 17. The RNAi agent of any one of embodiments 1-16, wherein the sense strand comprises one or two inverted abasic residues.

Embodiment 18. The RNAi agent of embodiment 1, wherein the RNAi agent is comprised of a sense strand and an antisense strand that form a duplex having the structure of any one of the duplexes in Table 7A, Table 7B, Table 8, Table 9A, or Table 10.

Embodiment 19. The RNAi agent of embodiment 18, wherein all or substantially all of the nucleotides are modified nucleotides.

Embodiment 20. The RNAi agent of embodiment 1, comprising an antisense strand that consists of, consists essentially of, or comprises a nucleotide sequence that differs by 0 or 1 nucleotides from any one of the following sequences (5′→3′):

    
    











(SEQ ID NO: 314)



UAGUCUACCAUGUCGAUGC;



 



(SEQ ID NO: 132)



UUGCUUUUACUGACCAUGC;



 



(SEQ ID NO: 247)



UGCCUAAUGAGCCACACUU;



 



(SEQ ID NO: 743)



UAGUCUACCAUGUCGAUGCUG;



 



(SEQ ID NO: 746)



UUGCUUUUACUGACCAUGCGA;



or



 



(SEQ ID NO: 766)



UGCCUAAUGAGCCACACUUGG.






Embodiment 21. The RNAi agent of embodiment 20, wherein the sense strand consists of, consists essentially of, or comprises a nucleotide sequence that differs by 0 or 1 nucleotides from one of the following nucleotide sequences (5′→3′):










(SEQ ID NO: 540)



GCAUCGACAUGGUAGACUA;



 



(SEQ ID NO: 358)



GCAUGGUCAGUAAAAGCAA;



 



(SEQ ID NO: 777)



CAGCAUCGACAUGGUAGACUA;



 



(SEQ ID NO: 781)



UCGCAUGGUCAGUAAAAGCAA;



or



 



(SEQ ID NO: 779)



CCAAGUGUGGCUCAUUAGGUA.






Embodiment 22. The RNAi agent of embodiment 20 or 21, wherein all or substantially all of the nucleotides are modified nucleotides.

Embodiment 23. The RNAi agent of embodiment 1, comprising an antisense strand that comprises, consists of, or consists essentially of a modified nucleotide sequence that differs by 0 or 1 nucleotides from one of the following nucleotide sequences (5′→3′):










(SEQ ID NO: 592)



cPrpusAfsgucuAfccauGfuCfgAfugcussg;



 



(SEQ ID NO: 553)



cPrpusUfsgcuUUNAUfuacuGfaCfcAfugcgssa;



or



 



(SEQ ID NO: 584)



cPrpusGfsccuaAfugagCfcAfcAfcuugsg;






    
    
        wherein a represents 2′-O-methyl adenosine, c represents 2′-O-methyl cytidine, g represents 2′-O-methyl guanosine, u represents 2′-O-methyl uridine; Af represents 2′-fluoro adenosine, Cf represents 2′-fluoro cytidine, Gf represents 2′-fluoro guanosine, Uf represents 2′-fluoro uridine; cPrpu represents 5′-cyclopropyl phosphonate-2′-O-methyl uridine; UUNA represents 2′,3′-seco-uridine-3′-phosphate; s represents a phosphorothioate linkage; and
        wherein all or substantially all of the nucleotides on the sense strand are modified nucleotides.

Embodiment 24. The RNAi agent of embodiment 1, wherein the sense strand comprises, consists of, or consists essentially of a modified nucleotide sequence that differs by 0 or 1 nucleotides from one of the following nucleotide sequences (5′→3′):

    
    











(SEQ ID NO: 598)



cagcaucgAfcAfUfgguagacua;



 



(SEQ ID NO: 605)



ucgcauggUfcAfguaaaagcaa;



or



 



(SEQ ID NO: 603)



ccaaguGfuGfgCfucauuaggua;






wherein a represents 2′-O-methyl adenosine, c represents 2′-O-methyl cytidine, g represents 2′-O-methyl guanosine, u represents 2′-O-methyl uridine; Af represents 2′-fluoro adenosine, Cf represents 2′-fluoro cytidine, Gf represents 2′-fluoro guanosine, and Uf represents 2′-fluoro uridine.

Embodiment 25. The RNAi agent of any one of embodiments 20-24, wherein the sense strand further includes inverted abasic residues at the 3′ terminal end of the nucleotide sequence, at the 5′ end of the nucleotide sequence, or at both.

Embodiment 26. The RNAi agent of any one of the preceding embodiments, wherein the RNAi agent is conjugated to a targeting ligand and has the duplex structure of AC005033, AC004265, AC912671, AC004123, AC004125, AC007414, or AC009806.

Embodiment 27. The RNAi agent of any one of embodiments 1-25, wherein the RNAi agent is linked to an antigen binding protein.

Embodiment 28. The RNAi agent of embodiment 27, wherein the antigen binding protein is an antibody fragment (Fab), wherein the Fab specifically binds to one or more epitopes on a transferrin receptor (TfR1).

Embodiment 29. The RNAi agent of embodiment 28, wherein the Fab comprises (i) 6 complementary determining regions (CDRs), (ii) 3 CDRs on the variable light chain (VL), and/or (iii) 3 CDRs on the variable heavy chain (VH).

Embodiment 30. The RNAi agent of embodiment 29, wherein the variable light chain has a VL CDR1 sequence selected from the group consisting of: RASDGLYSNLA (SEQ ID NO: 6), RASDNLYRNLA (SEQ ID NO: 7), and RASDKLYSNLA (SEQ ID NO: 8); a VL CDR2 sequence selected from the group consisting of: DATLLAS (SEQ ID NO: 9), DARNLAS (SEQ ID NO: 10), DAFNLAS (SEQ ID NO: 11), DATRLAS (SEQ ID NO: 12), DATKLAS (SEQ ID NO: 13), and DAKNLAS (SEQ ID NO: 14); and/or a VL CDR 3 sequence of QHFWGTPLT (SEQ ID NO: 15).

Embodiment 31. The RNAi agent of embodiment 29 or 30, wherein the variable light chain is selected from any one of the VL chains shown in Table A.

Embodiment 32. The RNAi agent of any one of embodiments 29-31, wherein the variable heavy chain has a VH CDR1 sequence selected from the group consisting of: GYTFNSYWMH (SEQ ID NO: 16), GYTFKSYWMH (SEQ ID NO: 17), GFTFTSYWMH (SEQ ID NO: 18), GYTFTSYWVH (SEQ ID NO: 19), and GYTFTSYWMH (SEQ ID NO: 20), a VH CDR2 sequence selected from the group consisting of: EINPTNGRVNYIEKFKS (SEQ ID NO: 21), EINPTNGRFNYIEKFKS (SEQ ID NO: 22), EINPTNGRTNYIEKFKS (SEQ ID NO: 23), and EINPTNGRSNYIEKFKS (SEQ ID NO: 24); and/or a VH CDR3 sequence of: GTRAYHY (SEQ ID NO: 25).

Embodiment 33. The RNAi agent of any one of embodiments 29-32, wherein the variable heavy chain is selected from any one of the VH chains shown in Table B.

Embodiment 34. The RNAi agent of any one of embodiments 28-33, wherein the Fab further comprises a light constant chain 1 (CL).

Embodiment 35. The RNAi agent of embodiment 34, wherein the light constant chain 1 (CL) sequence is:








(SEQ ID NO: 2)


RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS


 


GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV


 


TKSFNRGEC.






Embodiment 36. The RNAi agent of any one of embodiments 28-35, wherein the Fab further comprises a heavy constant chain 1 (CH).

Embodiment 37. The RNAi agent of embodiment 36, wherein the heavy constant chain 1 (CH) sequence is:








(SEQ ID NO: 4)


ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG


 


VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV


 


EPKSCDKTH.






Embodiment 38. The RNAi agent of any one of embodiments 28-37, wherein the antibody fragment (Fab) binds TfR1 with an affinity of at least 1 nM KD.

Embodiment 39. The RNAi agent of any one of embodiments 27-38, wherein the antigen binding protein is linked to the sense strand of the RNAi agent.

Embodiment 40. The RNAi agent of embodiment 39, wherein the antigen binding protein is linked to the 5′ end of the sense strand.

Embodiment 41. The RNAi agent of embodiment 39, wherein the antigen binding protein is linked to the 3′ end of the sense strand.

Embodiment 42. The RNAi agent of any one of embodiments 1-26, wherein the RNAi agent is linked to a lipid moiety.

Embodiment 43. The RNAi agent of embodiment 42, wherein the lipid moiety is conjugated to the sense strand.

Embodiment 44. The RNAi agent of embodiment 43, wherein the lipid moiety is conjugated to the 5′ terminal end of the sense strand.

Embodiment 45. A conjugate comprising the RNAi agent of any one of embodiments 1-26 conjugated to an antibody fragment (Fab) that specifically binds to one or more epitopes on a transferrin receptor (TfR1).

Embodiment 46. The conjugate of embodiment 45, wherein the Fab comprises (i) 6 complementary determining regions (CDRs), (ii) 3 CDRs on the variable light chain (VL), or (iii) 3 CDRs on the variable heavy chain (VH).

Embodiment 47. The conjugate of embodiment 46, wherein the variable light chain has a VL CDR1 sequence selected from the group consisting of: RASDGLYSNLA (SEQ ID NO: 6), RASDNLYRNLA (SEQ ID NO: 7), and RASDKLYSNLA (SEQ ID NO: 8); a VL CDR2 sequence selected from the group consisting of: DATLLAS (SEQ ID NO: 9), DARNLAS (SEQ ID NO: 10), DAFNLAS (SEQ ID NO: 11), DATRLAS (SEQ ID NO: 12), DATKLAS (SEQ ID NO: 13), and DAKNLAS (SEQ ID NO: 14); and/or a VL CDR 3 sequence of QHFWGTPLT (SEQ ID NO: 15).

Embodiment 48. The conjugate of embodiment 46 or 47, wherein the variable light chain is selected from any one of the VL chains shown in Table A.

Embodiment 49. The conjugate of any one of embodiments 46-48, wherein the variable heavy chain has a VH CDR1 sequence selected from the group consisting of: GYTFNSYWMH (SEQ ID NO: 16), GYTFKSYWMH (SEQ ID NO: 17), GFTFTSYWMH (SEQ ID NO: 18), GYTFTSYWVH (SEQ ID NO: 19), and GYTFTSYWMH (SEQ ID NO: 20), a VH CDR2 sequence selected from the group consisting of: EINPTNGRVNYIEKFKS (SEQ ID NO: 21), EINPTNGRFNYIEKFKS (SEQ ID NO: 22), EINPTNGRTNYIEKFKS (SEQ ID NO: 23), and EINPTNGRSNYIEKFKS (SEQ ID NO: 24); and/or a VH CDR3 sequence of: GTRAYHY (SEQ ID NO: 25).

Embodiment 50. The conjugate of any one of embodiments 46-49, wherein the variable heavy chain is selected from any one of the VH chains shown in Table B.

Embodiment 51. The conjugate of any one of embodiments 45-50, wherein the Fab further comprises a light constant chain 1 (CL).

Embodiment 52. The conjugate of embodiment 51, wherein the light constant chain 1 (CL) sequence is:








(SEQ ID NO: 2)


RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS


 


GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV


 


TKSFNRGEC.






Embodiment 53. The conjugate of any one of embodiments 45-52, wherein the Fab further comprises a heavy constant chain 1 (CH).

Embodiment 54. The conjugate of embodiment 53, wherein the heavy constant chain 1 (CH) sequence is:








(SEQ ID NO: 4)


ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG


 


VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV


 


EPKSCDKTH.






Embodiment 55. The conjugate of any one of embodiments 45-54, wherein the antibody fragment (Fab) binds TfR1 with an affinity of at least 1 nM KD.

Embodiment 56. The conjugate of embodiment any one of embodiments 45-55, wherein the RNAi agent is conjugated to the Fab using a covalent or non-covalent bond, ionic bond, hydrogen bond, hydrophobic interaction, peptide, polymer, or a nucleic acid binding protein.

Embodiment 57. The conjugate of any one of embodiments 45-56, wherein the RNAi agent is conjugated to the Fab through a linker comprising a structure selected from the group consisting of:





    
    
        wherein A represents a point of attachment to the Fab, and R represents a point of attachment to the RNAi agent portion of the conjugate.

Embodiment 58. A composition comprising the RNAi agent of any one of embodiments 1-44, or the conjugate of any one of embodiments 45-57, wherein the composition further comprises a pharmaceutically acceptable excipient.

Embodiment 59. The composition of embodiment 58, further comprising a second RNAi agent capable of inhibiting the expression of MAPT gene expression.

Embodiment 60. The composition of any one of embodiments 58-59, further comprising one or more additional therapeutics.

Embodiment 61. The composition of any one of embodiments 58-60, wherein the RNAi agent is a sodium salt.

Embodiment 62. The composition of any one of embodiments 58-61, wherein the pharmaceutically acceptable excipient is water for injection.

Embodiment 63. The composition of any one of embodiments 58-62, wherein the pharmaceutically acceptable excipient is a buffered saline solution.

Embodiment 64. A method for inhibiting expression of a MAPT gene in a cell, the method comprising introducing into a cell an effective amount of an RNAi agent of any one of embodiments 1-44, the conjugate of any one of embodiments 45-57, or the composition of any one of embodiments 58-63.

Embodiment 65. The method of embodiment 64, wherein the cell is within a subject.

Embodiment 66. The method of embodiment 65, wherein the subject is a human subject.

Embodiment 67. The method of any one of embodiments 64-66, wherein following the administration of the RNAi agent the MAPT gene expression is inhibited by at least about 30%.

Embodiment 68. A method of treating one or more symptoms or diseases that are mediated at least in part by MAPT activity and/or MAPT gene expression, the method comprising administering to a human subject in need thereof a therapeutically effective amount of the RNAi agent of any one of embodiments 1-44, the conjugate of any one of embodiments 45-57, or the composition of any one of embodiments 58-63.

Embodiment 69. The method of embodiment 68, wherein the disease is a neurodegenerative disease.

Embodiment 70. The method of embodiment 69, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Frontotemporal lobar degeneration dementia (FTLD), Progressive supranuclear palsy, and other tauopathies.

Embodiment 71. The method of embodiment 70, wherein the disease is Alzheimer's disease.

Embodiment 72. The method of any one of embodiments 64-71, wherein the RNAi agent is administered at a dose of about 0.01 mg/kg to about 5.0 mg/kg of body weight of the subject.

Embodiment 73. The method of any one of embodiments 64-71, wherein the RNAi agent is administered at a dose of about 0.03 mg/kg to about 2.0 mg/kg of body weight of the subject.

Embodiment 74. The method of any one of embodiments 64-73, wherein the RNAi agent is administered in two or more doses.

Embodiment 75. The RNAi agent of any one of embodiments 1-44, or the conjugate of any one of embodiments 45-57, for use in the treatment of a disease, disorder, or symptom that is mediated at least in part by MAPT activity and/or MAPT gene expression.

Embodiment 76. The composition according to any one of embodiments 58-63, for use in the treatment of a disease, disorder, or symptom that is mediated at least in part by MAPT activity and/or MAPT gene expression.

Embodiment 77. The composition according to any one of embodiments 58-63, for use in the manufacture of a medicament for the treatment of a disease, disorder, or symptom that is mediated at least in part by MAPT activity and/or MAPT gene expression.

Embodiment 78. The composition of any one of embodiments 76-77, wherein the disease is a neurodegenerative disease.

Embodiment 79. A method of making an RNAi agent of any one of embodiments 1-44, comprising annealing a sense strand and an antisense strand to form a double-stranded ribonucleic acid molecule.

Embodiment 80. The RNAi agent of any one of embodiments 1-44, the conjugate of any one of embodiments 45-57, or the composition of any one of embodiments 58-63 for use in inhibiting expression of a MAPT gene in a cell.

Embodiment 81. The RNAi agent, conjugate, or composition of embodiment 80, wherein the cell is within a subject.

Embodiment 82. The RNAi agent, conjugate, or composition of embodiment 81, wherein the subject is a human subject.

Embodiment 83. The RNAi agent, conjugate, or composition of any one of embodiments 80-82, wherein following the administration of the RNAi agent the MAPT gene expression is inhibited by at least about 30%.

Embodiment 84. The RNAi agent of any one of embodiments 1-44, the conjugate of any one of embodiments 45-57, or the composition of any one of embodiments 58-63 for use in treating one or more symptoms or diseases that are mediated at least in part by MAPT activity and/or MAPT gene expression.

Embodiment 85. The RNAi agent, conjugate or composition of embodiment 84, wherein the disease is a neurodegenerative disease.

Embodiment 86. The RNAi agent, conjugate or composition of embodiment 85, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Frontotemporal lobar degeneration dementia (FTLD), Progressive supranuclear palsy, and other tauopathies.

Embodiment 87. The RNAi agent, conjugate or composition of embodiment 86, wherein the disease is Alzheimer's disease.

Embodiment 88. The RNAi agent, conjugate or composition of any one of embodiments 80-87, wherein the RNAi agent is administered at a dose of about 0.01 mg/kg to about 5.0 mg/kg of body weight of the subject.

Embodiment 89. The RNAi agent, conjugate or composition of any one of embodiments 80-87, wherein the RNAi agent is administered at a dose of about 0.03 mg/kg to about 2.0 mg/kg of body weight of the subject.

Embodiment 90. The RNAi agent, conjugate or composition of any one of embodiments 80-89, wherein the RNAi agent is administered in two or more doses.

Embodiment 91. The RNAi agent of any one of embodiments 42-44, wherein the lipid is selected from the group consisting of:

    
    




















 


LP128


 








 


LP132


 








 


LP183


 








 


LP183-(NH-C6)s


 








 


LP183r


 








 


LP183r-(C5)s


 








 


LP200


 








 


LP232


 








 


LP-233


 








 


LP242


 








 


LP243


 








 


LP245


 








 


LP249


 








 


LP257


 








 


LP259


 








 


LP260


 








 


LP262


 








 


LP269


 








 


LP273


 








 


LP274


 








 


LP276


 








 


LP283


 








 


LP286


 








 


LP287


 








 


LP289


 








 


LP290


 








 


LP293


 








 


LP296


 








 


LP300


 








 


LP303


 








 


LP304


 








 


LP310


 








 


LP383


 








 


LP395


 








 


LP396


 








 


LP409


 








 


LP429


 








 


LP430


 








 


LP431


 








 


LP435


 








 


LP439


 








 


LP440


 








 


LP441


 








 


LP456


 








 


LP462


 








 


LP463


 








 


LP464


 








 


LP465


 








 


LP466


 








 


(2C8C12)s


 








 


(2C6C10)s


 








 


HO-C16s


 








 


C16, and


 








 


C22s









Embodiment 92. The RNAi agent of any one of embodiments 42-43, wherein the lipid moiety is present on the 2′ position of a sense strand nucleotide.

Embodiment 93. The RNAi agent of embodiment 92, wherein the sense strand comprises a nucleotide selected from the group consisting of:


















aC16


 






aC16s


 






uC16


 






uC16s


 






cC16


 






cC16s


 






gC16


 


and



 






gC16s









EXAMPLES
Example 1. Synthesis of MAPT RNAi Agents
MAPT RNAi agent duplexes disclosed herein were synthesized in accordance with the following:
A. Synthesis.
The sense and antisense strands of the MAPT RNAi agents were synthesized according to phosphoramidite technology on solid phase used in oligonucleotide synthesis. Depending on the scale, a MerMade96E® (Bioautomation), a MerMade12® (Bioautomation), or an OP Pilot 100 (GE Healthcare) was used. Syntheses were performed on a solid support made of controlled pore glass (CPG, 500 Å or 600 Å, obtained from Prime Synthesis, Aston, PA, USA). All RNA and 2′-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA). Specifically, the 2′-O-methyl phosphoramidites that were used included the following: (5′-O-dimethoxytrityl-N6-(benzoyl)-2′-O-methyl-adenosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, 5′-O-dimethoxy-trityl-N4-(acetyl)-2′-O-methyl-cytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, (5′-O-dimethoxytrityl-N2-(isobutyryl)-2′-O-methyl-guanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, and 5′-O-dimethoxytrityl-2′-O-methyl-uridine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite. The 2′-deoxy-2′-fluoro-phosphoramidites carried the same protecting groups as the 2′-O-methyl RNA amidites. 5′-dimethoxytrityl-2′-O-methyl-inosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidites were purchased from Glen Research (Virginia). The inverted abasic (3′-O-dimethoxytrityl-2′-deoxyribose-5′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidites were purchased from ChemGenes (Wilmington, MA, USA). The following UNA phosphoramidites were used: 5′-(4,4′-Dimethoxytrityl)-N6-(benzoyl)-2′,3′-seco-adenosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5′-(4,4′-Dimethoxytrityl)-N-acetyl-2′,3′-seco-cytosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diiso-propyl)]-phosphoramidite, 5′-(4,4′-Dimethoxytrityl)-N-isobutyryl-2′,3′-seco-guanosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5′-(4,4′-Dimethoxy-trityl)-2′,3′-seco-uridine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diiso-propyl)]-phosphoramidite. TFA aminolink phosphoramidites were also commercially purchased (ThermoFisher). Linker L6 was purchased as propargyl-PEG5-NHS from BroadPharm (catalog #BP-20907) and coupled to the NH2-C6 group from an aminolink phosphoramidite to form -L6-C6-, using standard coupling conditions. The linker Alk-cyHex was similarly commercially purchased from Lumiprobe (alkyne phosphoramidite, 5′-terminal) as a propargyl-containing compound phosphoramidite compound to form the linker -Alk-cyHex-. In each case, phosphorothioate linkages were introduced as specified using the conditions set forth herein. The cyclopropyl phosphonate phosphoramidites were synthesized in accordance with International Patent Application Publication No. WO 2017/214112 (see also Altenhofer et. al., Chem. Communications (Royal Soc. Chem.), 57(55):6808-6811 (July 2021)).
B. Cleavage and Deprotection of Support Bound Oligomer.
After finalization of the solid phase synthesis, the dried solid support was treated with a 1:1 volume solution of 40 wt. % methylamine in water and 28% to 31% ammonium hydroxide solution (Aldrich) for 1.5 hours at 30° C. The solution was evaporated and the solid residue was reconstituted in water (see below).
C. Purification.
Crude oligomers were purified by anionic exchange HPLC using a TSKgel SuperQ-5PW 13 μm column and Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0 and contained 20% Acetonitrile and buffer B was the same as buffer A with the addition of 1.5 M sodium chloride. UV traces at 260 nm were recorded. Appropriate fractions were pooled then run on size exclusion HPLC using a GE Healthcare XK 16/40 column packed with Sephadex G-25 fine with a running buffer of 100 mM ammonium bicarbonate, pH 6.7 and 20% Acetonitrile or filtered water. Alternatively, pooled fractions were desalted and exchanged into an appropriate buffer or solvent system via tangential flow filtration.
D. Annealing.
Complementary strands were mixed by combining equimolar RNA solutions (sense and antisense) in 1×PBS (Phosphate-Buffered Saline, 1×, Corning, Cellgro) to form the RNAi agents. Some RNAi agents were lyophilized and stored at −15 to −25° C. Duplex concentration was determined by measuring the solution absorbance on a UV-Vis spectrometer in 1×PBS. The solution absorbance at 260 nm was then multiplied by a conversion factor (0.050 mg/(mL-cm)) and the dilution factor to determine the duplex concentration.
E. Conjugation of RNAi Agents to Fabs.
RNAi agents described herein comprising a free amine were conjugated to L20-p




using standard amide reaction chemistry following cleavage from the solid phase. To a solution of Fab in PBS (0.2 umol, 1.0-10.0 mg/mL in PBS) was added a freshly prepared solution of (tris(2-carboxyethyl)phosphine) hydrochloride (TCEP-HCl) in PBS (5-20 eq, 70 mM). The reaction was held overnight at room temperature and covered from light. The next day, TCEP was removed by loading the reaction mixture on a PD-10 desalting column equilibrated with PBS and eluted with PBS. The concentration of Fab in the eluate was determined using the theoretical absorptivity factor at 280 nm. A solution of L20-modified sense strand in sodium phosphate buffer was prepared, and the concentration was determined using the theoretical absorptivity factor at 260 nm. To the desalted Fab solution was added L20-modified sense strand (1-1.3 eq, 0.5-2.5 mM), and the reaction was mixed end-over-end. Analysis by SEC Method 1 and AIEX Method 1 show a mixture of starting Fab, DAR1, and DAR2. After 1 hour, a solution of CP-1113-p (see Table 10 for structure) in DMSO and added to the reaction mixture (3 eq, 36 mM). After 1 hour, a solution of L-cysteine in PBS was added to the reaction mixture (6-10 eq, 165 mM). Finally, the conjugate was annealed by addition of antisense strand (1.2-1.5 eq, 0.5-2.5 mM). The conjugate was purified by an AKTA Pure FPLC system equipped with 20 mM tris pH 8 (Buffer A), 20 mM tris 1500 mM NaCl (Buffer B), and a 5×200 mm column packed with Tosoh SuperQ 5PW (20 micron). The crude reaction mixture was pump loaded onto the column and eluted with a gradient of 10-40% Buffer B. DART and DAR2 fractions were differentiated by SEC Method 1, AEX Method 1, and Nanodrop 260/280 readings. DART fractions were pooled and buffer exchanged to PBS using a PD-10 desalting column. The purified conjugate was analyzed by SEC Method 1 and eluted as a monomeric peak with a retention time of 13.2 minutes.

SEC Method 1














Mobile phases
Phosphate Buffered Saline pH 7.4


Column
Superdex 200 Increase 10/300 GL



Cytiva PN 29219757


Column
25° C.


temperature


Autosampler
ambient


Injection volume
40 uL of 1 mg/mL protein (variable)


Flow rate
1.0 mL/min isocratic


Wavelength
PDA 190-450 nm; monitor 230 nm, 260 nm, 280 nm


Run time
30 minutes









AIEX Method 1














Mobile phases
C: 20 mM tris pH 8.0,



D: 20 mM tris 1500 mM NaCl pH 8.0


Column
ProPac SAX-10 4 mm × 250 mm, 10 um



Thermo Fisher Scientific PN 054997


Column
30° C.


temperature


Autosampler
5° C.


Injection volume
20 μl of 0.2 mg/mL oligo (variable)


Flow rate
1.0 mL/min (variable)


Wavelength
PDA 190-450 nm; monitor



230 nm, 260 nm, 280 nm


Run time
12.5 minutes














Time(min)
Event
Value





Gradient
0
D. Conc
0



0.10
D. Conc
0



0.11
D. Conc
25



10.11
D. Conc
75



10.11
T. Flow
1



10.12
D. Conc
0



10.12
T. Flow
1.5



12.50
Controller
Stop









RNAi agents described herein comprising a free amine were conjugated to L1026-p:




following cleavage from the solid phase according to the following procedure:

To a solution of Fab0070 (28 mg, 0.59 umol, 5.55 mg/mL in PBS) was added a freshly prepared solution of TCEP-HCl in PBS (5 eq, 70 mM, 42 uL). The reduction was mixed end-over-end at ambient temperature for 15 minutes then held at 5° C. overnight without agitation. The next day, TCEP was removed by loading the reaction mixture on two PD-10 desalting columns (Cytiva) equilibrated with 20 mM tris 50 mM NaCl pH 7.6 (alternatively, 20 mM tris pH 8 or PBS buffer can be used) and eluted with the same buffer. The concentration of the Fab in the eluate was determined using the theoretical absorptivity factor at 280 nm. A solution of L-1026-modified sense strand in 10 mM sodium phosphate buffer pH 6.0-6.5 was prepared, and the concentration was determined using the theoretical absorptivity factor at 260 nm. To the desalted Fab solution was added L-1026-modified CS915332 (1.15 eq, 2.75 mM, 240 uL), and the reaction was mixed end-over-end at ambient temperature. Analysis by SEC Method 1 and AIEX Method 1 show a mixture of starting Fab0070, DART product, and DAR2 product. After 30 m, a solution of L-cysteine in 20 mM tris 50 mM NaCl pH 7.6 (alternatively, some L-1026 conjugates have been prepared in 20 mM tris pH 8 or PBS buffer solutions) was added to the reaction mixture (10 eq, 165 mM, 36 uL). After 30 m, the conjugate was annealed by addition of antisense strand (1.3 eq, 1.45 mM in water, 529 uL). The conjugate was purified by an AKTA Pure FPLC system equipped with 20 mM tris pH 8 (Buffer A), 20 mM tris 1500 mM NaCl (Buffer B), and a 5×200 mm column packed with Tosoh SuperQ 5PW (20 micron). The crude reaction mixture was loaded onto the column and eluted with a gradient of 10-40% Buffer B. DART and DAR2 fractions were differentiated by SEC Method 1, AIEX Method 1, and UV-Vis 260/280 measurements. DART fractions were pooled and buffer exchanged to PBS using two PD-10 columns. The purified conjugate was analyzed by SEC Method 1 and eluted as a monomeric peak with a retention time of 7.2 minutes.
SEC Method 1
















Mobile phases
2x Phosphate Buffered Saline pH 7.4



Column
ACQUITY UPLC Protein BEH SEC




Column, 200 Å, 1.7 μm, 4.6




mm × 300 mm




Waters PN 186005226



Column
30° C.



temperature



Autosampler
ambient



Injection volume
2-5 uL



Flow rate
0.3 mL/min



Wavelength
PDA 190-450 nm



Run time
20 minutes










AIEX Method 1














Mobile phases
C: 20 mM tris pH 8.0,



D: 20 mM tris 1500 mM NaCl pH 8.0


Column
ProPac SAX-10 4 mm × 250 mm, 10 um



Thermo Fisher Scientific PN 054997


Column
30° C.


temperature


Autosampler
5° C.


Injection volume
5-20 μl


Flow rate
1.0 mL/min (variable)


Wavelength
PDA 190-450 nm


Run time
12.5 minutes














Time(min)
Event
Value





Gradient
0
D. Conc
10



0.10
D. Conc
10



0.11
D. Conc
25



10.11
D. Conc
75



10.11
T. Flow
1



10.12
D. Conc
10



10.12
T. Flow
1.5



12.50
Controller
Stop









F. Synthesis of Lipids

Synthesis of LP293-p





To a solution of compound 1 (73 mg), NEt3 (0.112 mL), and COMU (126 mg) in DMF was added compound 2 (48.9 mg) under ambient conditions. The reaction was stirred until full conversion was observed by LC-MS. Conversion was not able to be clearly observed by LC-MS, and instead, reaction was allowed to stir for 30 min. until bright yellow color (before the addition of compound 2) transitioned to a honey orange color and all material was observed to be mainly dissolved. The reaction mixture was then washed with water, extracted with DCM, dried over Na2SO4, filtered, and concentrated under vacuum. The residue was purified by CombiFlash® via DCM liquid-load onto a 12-g column with a gradient hexanes to 100% EtOAc in which product eluted at 30% B. The product was concentrated under vacuum to provide a white solid residue and confirmed by 1H NMR in CDCl3.
Synthesis of LP310-p




To the solution of 1 in DCM was added DIPEA (0.057 mL), COMU (0.077 g) and 2 (0.0300 g) at room temperature. After stirring at room temperature for 2 h, the reaction was quenched with 0.1N HCl. The organic layer was washed with brine. After removing the solvent, the residue was loaded on a 4 g column. Hexanes to 50% Hexanes in EtOAc as gradient was used to purify. Product was a white solid, 46 mg, 44%. LC-MS: calculated [M+H] 422.36, found 422.61.




The solution of 1(0.046 g) in 4N HCl/Dioxane (2 mL) was stirred at room temperature overnight. After removing the solvent in vacuo, the residue was placed under high vacuum for 3 h. Then the residue was dissolved in DCM at room temperature, then COMU (0.0700 g), DIPEA (0.038 mL) and 2 (0.036 g) were added at room temperature. After stirring at room temperature for 2 h, the solvent was removed in vacuo. The residue was loaded on a 4 g column. Hexanes to 50% Hexanes in EtOAc as gradient was used to purify. Product was a white solid, 21 mg, 38%. LC-MS: calculated [M+H] 514.29, found 514.61.
Synthesis of LP429-p




17-hydroxyhexadecanoic acid (6) (3.53 g, 12.3 mmol) was added to a 500 mL RBF. The flask was purged with nitrogen, then DCM (150 mL) was added followed by acetic anhydride (18.6 mL, 197 mmol) and pyridine (30.8 mL, 382 mmol). The reaction was stirred overnight. The reaction mixture was concentrated and azeotroped 3 times with toluene to remove residual pyridine, acetic acid, acetic anhydride. The residue was then stirred in 100 mL of a 1:1 THF/aq. NaHCO3 mixture for 24 hours. About half of the THF was removed via rotary evaporator and the mixture was diluted with water and acidified with 3 M HCl until a pH of 1. The mixture became very foamy during the acidification. The product was collected by filtration and dried in vacuo to yield 3.22 g (80% yield) of compound 5 as a white solid. The product was not purified further.




Compound 5 (3.47 g, 10.6 mmol) was dissolved in THF (55 mL) and cooled to −15 to −20° C. in a methanol/ice bath. Once cooled, N-methyl morpholine (1.4 mL, 12.7 mmol) and ethyl chloroformate (1.2 mL, 12.7 mmol) were added. The reaction was stirred at −15 for 30 minutes. After 30 minutes a solution of sodium azide (1.72 grams, 26.4 mmol) in water (6.6 mL) was added and the reaction was stirred for 30 minutes at −5°-0° C. in a water/salt/ice bath. The reaction mixture was diluted with EtOAc (20 mL) and water (20 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2×50 mL), the combined organic layers were washed with water (50 mL), brine (50 mL), dried over sodium sulfate and concentrated to a white solid. Proton NMR showed no remaining starting material based on protons alpha to the carbonyl. The solid was dissolved in toluene (55 mL) and heated to 65° C. until gas evolution stopped (about 30 minutes). The reaction was cooled to room temperature and N-hydroxy succinimide (1.22 g, 10.5 mmol) was added followed by pyridine (0.85 mL, 10.5 mmol). Proton NMR indicated not all the isocyanate was consumed after 2 hours, additional 2 eq of N-hydroxy succinimide (2.43 g, 21.1 mmol) was added. The reaction was stirred overnight. No isocyanate remained by proton NMR after stirring overnight. The reaction mixture was concentrated, the resulting white powder was dissolved in EtOAc (100 mL) and poured into 300 mL hexanes. The precipitate was collected by filtration. Proton NMR of the product showed residual N-hydroxy succinimide. The product was dissolved in DCM and purified by silica gel chromatography 65:35 Hexanes:EtOAc to 0:100 Hexanes:EtOAc. Product began eluting at 50% EtOAc and dragged on the column. Fractions containing product were combined to yield 2.25 g (48% yield) of compound 7 as a white solid.




Compound 7 (1.00 g, 2.27 mmol) was added to a solution of 6-amino-1-hexanol (0.266 g, 2.27 mmol) and NEt3 (0.95 mL, 6.81 mmol) in DCM (50 mL). A white ppt formed. No SM remained by LC-MS after 18 hours. The reaction was concentrated by rotary evaporator, the residue was dissolved in about 8 mL of ethyl acetate and was cool to −20° C. in a freezer. A precipitate formed and settled at the bottom of the flask. The EtOAc was decanted off twice and the precipitate was collected and dried under vacuum to yield 0.95 grams (94% yield) of compound 8 as a white powder.




In a 100 mL RBF compound 8 (0.95 g, 2.14 mmol) was dried by 3 successive evaporations of toluene. Diisopropylammonium tetrazolide (0.146 g, 0.86 mmol) and 4 angstrom molecular sieves were added to the flask. The flask was purged and backfilled with nitrogen 3 times, and the solids were dissolved in DCM (50 mL). The mixture was stirred for 30 minutes. After 30 minutes 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (0.98 g, 3.25 mmol) was added and the reaction was stirred for 18 hours. After 18 hours, LC-MS indicated no starting alcohol remained. The reaction was transferred to a separatory funnel, washed with sat. aq. NaHCO3 (2×40 mL), water (40 mL), brine (40 mL), dried over magnesium sulfate and concentrated to dryness. Hexanes was added to the flask and the residue was stirred in hexanes for 2 hours to yield a white precipitate. The white solid was collected by filtration, washed with hexanes (2×20 mL), and dried under vacuum to yield 1.2 grams (87% yield) of compound 9 as a white solid.
Synthesis of LP462-p




To a round bottom flask containing 2099-117 (1 eq) was added anhydrous THF (30 mL) and the solution was cooled to −20° C. Ethyl chloroformate (1.2) and N-methylmorpholine (1.2 eq) were added to the solution and the solution was stirred at −20° C. to −10° C. for 30 minutes. A solution of sodium azide (2.5 eq) in 1.5 mL of water was added to the reaction and the reaction was stirred at −7° C. for 90 minutes. The reaction was diluted with EtOAc. The aq. layer was separated and extracted 2 additional times with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated to a clear liquid. The liquid was dissolved in toluene (30 mL) and heated to 65° C. for 1 hour, when no additional nitrogen gas formation was observed. Next, the solution was concentrated under reduced pressure and then dissolved in 30 mL of anhydrous DCM. 6-amino-1-hexanol (3 eq) and pyridine (1 eq) were added to the reaction mixture and stirring was continued for 12 hours. The mixture was concentrated under reduced pressure onto celite and purified via CombiFlash chromatography using 5% methanol in 95% DCM to give compound 1 as an oil in 51% yield. LC-MS [M+H2O]+ 717.4538 m/z, observed 717.4530.
Compound 1 (1 eq) was rotovaped twice with toluene before charging anhydrous DCM (10 mL) to the reaction flask. The suspension was stirred 900 RPM under N2 at ambient temperature with molecular sieves. 2-Cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite (1.5 eq) was added to the suspension, followed by diisopropylammonium tetrazolide (0.4 eq). After 12 hours, TEA (300 uL) was added, and the reaction mixture was dry loaded onto celite. The product was purified using hexanes: ethyl acetate+1% TEA (60:40) to give LP462-p as an oil in 64% yield. LC-MS [M+H]+ 916.5538 m/z, observed 916.5543.
Conjugation of Lipid PK/PD Modulator Precursors
Either prior to or after annealing, one or more lipid PK/PD modulator precursors can be linked to the RNAi agents disclosed herein. The following describes the general conjugation process used to link lipid PK/PD modulator precursors to the constructs set forth in the Examples depicted herein.
A. Conjugation of Activated Ester PK/PD Modulators
The following procedure was used to conjugate PK/PD modulators having an activated ester moiety such as TFP (tetrafluorophenoxy) or PNP (para-nitrophenol) to an RNAi agent with an amine-functionalized sense strand, such as C6-NH2, NH2-C6, or (NH2-C6). An annealed RNAi Agent dried by lyophilization was dissolved in DMSO and 10% water (v/v %) at 25 mg/mL. Then 50-100 equivalents of TEA and 3 equivalents of activated ester PK/PD modulator were added to the solution. The solution was allowed to react for 1-2 hours, while monitored by RP-HPLC-MS (mobile phase A 100 mM HFIP, 14 mM TEA; mobile phase B: acetonitrile on an Waters™ XBridge C18 column, Waters Corp.)
The product was then precipitated by adding 12 mL acetonitrile and 0.4 mL PBS and centrifuging the solid to a pellet. The pellet was then re-dissolved in 0.4 mL of 1×PBS and 12 mL of acetonitrile. The resulting pellet was dried on high vacuum for one hour.
B. Conjugation of Phosphoramidite PK/PD Modulators
PK/PD modulators having a phosphoramidite moiety may be attached on resin using typical oligonucleotide manufacturing conditions.
C. Hydrolysis of PK/PD Modulators
Certain PK/PD modulators are hydrolyzed in the cleavage and deprotection conditions described in Example 1, above. For example LP-429p and LP-462p include moieties that are hydrolyzed under the cleavage and deprotection conditions.
Example 2. In Vivo Knockdown of MAPT in in Cynomolgus Monkeys
On Study day 0, cynomolgus monkeys were injected with either artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or a compound formulation containing either 3 mg/kg at a concentration of 1.5 mg/mL and a volume of 2 mL/kg, 15 mg/kg at a concentration of 7.5 mg/mL and a volume of 2 mL/kg, or 30 mg/kg at a concentration of 7.5 mg/mL and a volume of 4 mL/kg of AC007414 in aCSF according to Table 11 below:







TABLE 11







Dosing groups for the non-human primates of Example 2.










Group ID
Animals dosed







Group 1 (aCSF)
n = 4



Group 2 (3 × 3 mg/kg AC007414)
n = 4



Group 3 (3 × 15 mg/kg AC007414)
n = 4



Group 4 (3 × 30 mg/kg AC007414)
n = 4










Four (n=4) monkeys were dosed in each group. Monkeys were injected subcutaneously on days 0, 7, and 14. On study day 29, animals from each group were euthanized and brain and spinal cord tissue was collected from each animal. Samples were analyzed by qPCR for MAPT mRNA knockdown. Samples were analyzed by JESS for protein knockdown. Average mRNA knockdown for frontal cortex, hippocampus and thoracic spinal cord for each group, relative to Group 1, are shown in Table 12 below:







TABLE 12





Relative expression of MAPT mRNA in various tissues analyzed


by qPCR for each of the dosing groups of Example 2.




















Hippocampus
Frontal Cortex




Group Average (n = 4)
Group Average (n = 4)
















Rel.
Error
Error
Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)
Exp.
(Low)
(High)





1
aCSF
1.000
0.144
0.168
1.000
0.287
0.402


2
AC007414
0.265
0.058
0.073
0.191
0.054
0.075



(3 mg/kg)


3
AC007414
0.226
0.041
0.049
0.209
0.098
0.183



(15 mg/kg)


4
AC007414
0.200
0.054
0.075
0.164
0.055
0.082



(30 mg/kg)














Thoracic Spinal Cord




Group Average (n = 4)













Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)





1
aCSF
1.000
0.192
0.238


2
AC007414
0.372
0.068
0.083



(3 mg/kg)


3
AC007414
0.252
0.023
0.025



(15 mg/kg)


4
AC007414
0.257
0.012
0.012



(30 mg/kg)









Average protein knockdown for frontal cortex, hippocampus and thoracic spinal cord for each group, relative to Group 1, are shown in FIG. 1.
As can be seen in Table 12 and FIG. 1, MAPT RNAi agent linked to an anti-Transferrin Fab achieved dose dependent and deep knockdown in CNS tissues when injected subcutaneously. The results demonstrate that treatment of MAPT-related diseases and disorder may be mitigated by RNAi agents administered subcutaneously.
Example 3. In Vivo Knockdown of MAPT in in Cynomolgus Monkeys
Cynomolgus monkeys (Macaca fascicularis) were injected with either phosphate buffered saline (PBS) or a compound formulation containing 3.0 mg/kg at a concentration of 1.5 mg/mL of AC007414 (formulated in PBS, dose volume 2.0 mL/kg) according to Table 13 below.







TABLE 13







Dosing groups for the non-human primates of Example 3.











Animals




Group ID
Dosed
Dosing Days
Sacrifice Day





Group 1 PBS
n = 4
SC injection:
Day 43




Day 1, 8, 15


Group 2 3.0 mg/kg
n = 4
SC injection:
Day 43


AC007414

Day 1, 8, 15


Group 3 3.0 mg/kg
n = 4
SC injection:
Day 99


AC007414

Day 1, 8, 15


Group 4 3.0 mg/kg
n = 4
SC injection: Day 1
Day 29


AC007414









Four (n=4) male monkeys were dosed in each group. Animals were dosed, via subcutaneous (SC) injection, on Day 1, Day 8, and Day 15 (Group 4 animals were dosed only on Day 1). In accordance with Table 13 above, on study Day 29, 43, or 99, animals in each test Group were sacrificed. The animals were randomized and assigned to groups using a computer-based procedure.
Prior to dosing, the dose formulations were removed from refrigerated storage and allowed to equilibrate at room temperature for at least 10 minutes. Dose sites were shaved prior to dosing and remarked as necessary throughout the study. Dose one (Day 1) was delivered to the animals' left scapular region, dose two (Day 8) was delivered to the right scapular region and dose three (Day 15) was delivered to the animals' left scapular region. The SC injection site on Day 1 did not overlap with the injection sites of Day 8 or Day 15. Each dose was given using a syringe with 23-25 gauge needle.
Upon sacrifice, animals from each group were euthanized and brain and spinal cord tissue was collected from each animal. Samples were analyzed by qPCR for MAPT mRNA knockdown. Samples were analyzed by JESS for protein knockdown. Average mRNA knockdown for frontal cortex, hippocampus, thoracic spinal cord, temporal cortex, caudate, and putamen for each group, with cPPIB as endogenous gene, normalized to Group 1 animals dosed with PBS, are shown in Tables 14a and 14b below. Groups 2 and 4 were normalized to a first assay of Group 1, shown in Table 14a, and Group 3 was normalized to a separate assay of Group 1, shown in Table 14b.







TABLE 14a





Relative expression of MAPT mRNA in various tissues analyzed


by qPCR for dosing groups 1, 2 and 4 of Example 3.




















Hippocampus
Frontal Cortex




Group Average (n = 4)
Group Average (n = 4)
















Rel.
Error
Error
Rel.
Error
Error


Group #
Sac Day
Exp.
Low
High
Exp.
Low
High





1. PBS
Day 43
1.000
0.235
0.308
1.000
0.249
0.332


2. 3x 3.0 mg/kg AC007414
Day 43
0.339
0.110
0.163
0.245
0.048
0.060


4. 1x 3.0 mg/kg AC007414
Day 29
0.413
0.121
0.171
0.383
0.104
0.143















Thoracic Spinal Cord
Temporal Cortex




Group Average (n = 4)
Group Average (n = 4)
















Rel.
Error
Error
Rel.
Error
Error


Group #
Sac Day
Exp.
Low
High
Exp.
Low
High





1. PBS
Day 43
1.000
0.346
0.528
1.000
0.090
0.099


2. 3x 3.0 mg/kg AC007414
Day 43
0.336
0.036
0.041
0.283
0.057
0.072


4. 1x 3.0 mg/kg AC007414
Day 29
0.466
0.110
0.144
0.375
0.070
0.085















Caudate
Putamen




Group Average (n = 4)
Group Average (n = 4)
















Rel.
Error
Error
Rel.
Error
Error


Group #
Sac Day
Exp.
Low
High
Exp.
Low
High





1. PBS
Day 43
1.000
0.193
0.239
1.000
0.076
0.082


2. 3x 3.0 mg/kg AC007414
Day 43
0.278
0.026
0.029
0.297
0.042
0.049


4. 1x 3.0 mg/kg AC007414
Day 29
0.434
0.060
0.070
0.413
0.063
0.074
















TABLE 14b





Relative expression of MAPT mRNA in various tissues analyzed


by qPCR for dosing groups 1 and 3 of Example 3.




















Hippocampus
Frontal Cortex




Group Average (n = 4)
Group Average (n = 4)
















Rel.
Error
Error
Rel.
Error
Error


Group #
Sac Day
Exp.
Low
High
Exp.
Low
High





1. PBS
Day 43
1.000
0.237
0.311
1.000
0.279
0.387


3. 3x 3.0 mg/kg AC007414
Day 99
0.419
0.124
0.177
0.474
0.155
0.230















Thoracic Spinal Cord
Temporal Cortex




Group Average (n = 4)
Group Average (n = 4)
















Rel.
Error
Error
Rel.
Error
Error


Group #
Sac Day
Exp.
Low
High
Exp.
Low
High





1. PBS
Day 43
1.000
0.335
0.503
1.000
0.106
0.119


3. 3x 3.0 mg/kg AC007414
Day 99
0.485
0.060
0.069
0.359
0.107
0.153















Caudate
Putamen




Group Average (n = 4)
Group Average (n = 4)
















Rel.
Error
Error
Rel.
Error
Error


Group #
Sac Day
Exp.
Low
High
Exp.
Low
High





1. PBS
Day 43
1.000
0.188
0.232
1.000
0.128
0.147


3. 3x 3.0 mg/kg AC007414
Day 99
0.437
0.091
0.115
0.430
0.039
0.043









As can be seen in Tables 14a and 14b, MAPT RNAi agent AC007414 linked to an anti-Transferrin Fab achieved MAPT knockdown in CNS tissues when injected subcutaneously. MAPT mRNA inhibition was observed out to at least Day 99. Most notably, at Day 43, animals dosed with 3× subcutaneous injections of AC007414 at 3.0 mg/kg achieved inhibition of MAPT mRNA in the hippocampus (˜66% inhibition, 0.339 relative to control), frontal cortex (˜75% inhibition, 0.245 relative to control), thoracic spinal cord (˜66% inhibition, 0.336 relative to control), temporal cortex (˜72% inhibition, 0.283 relative to control), caudate (˜72% inhibition, 0.278 relative to control), and putamen (˜70% inhibition, 0.297 relative to control). Average protein knockdown for frontal cortex, hippocampus and thoracic spinal cord for each group, relative to Group 1, are shown in FIG. 2. Similarly, MAPT RNAi agent AC007414 achieved MAPT protein knockdown in CNS tissues out to at least Day 99. Most notably, at Day 43, animals dosed with 3× subcutaneous injections of AC007414 at 3.0 mg/kg achieved inhibition of MAPT protein in the hippocampus (˜77%), frontal cortex (˜66%), and thoracic spinal cord (˜39%).
Example 4. In Vivo Knockdown of MAPT in Transgenic PS19 Mice
On Study day 1, PS19 mice were injected with either 10 μL artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or 10 μL of compound formulation at a concentration of 10 mg/mL in aCSF for groups 2-17, according to Table 15 below:







TABLE 15







Dosing groups for the mice of Example 4.









Group ID
Animals dosed
AC Duplex Number





Group 1 (aCSF)
n = 4
N/A


Group 2 100 μg AD13081
n = 4
AC911178


Group 3 100 μg AD13082
n = 4
AC911179


Group 4 100 μg AD13083
n = 4
AC911180


Group 5 100 μg AD13084
n = 4
AC911181


Group 6 100 μg AD13085
n = 4
AC911182


Group 7 100 μg AD13086
n = 4
AC911183


Group 8 100 μg AD13087
n = 4
AC911184


Group 9 100 μg AD13088
n = 4
AC911185


Group 10 100 μg AD13089
n = 4
AC911186


Group 11 100 μg AD13090
n = 4
AC911187


Group 12 100 μg AD13091
n = 4
AC911188


Group 13 100 μg AD13092
n = 4
AC911189


Group 14 100 μg AD13093
n = 4
AC911190


Group 15 100 μg AD13094
n = 4
AC911191


Group 16 100 μg AD13095
n = 4
AC911192


Group 17 100 μg AD13096
n = 4
AC911193









Four (n=4) mice were dosed in each group. Mice were injected intracerebroventricularly on day 1. On day 8, mice were euthanized and the left half of the brain and thoracic spinal cord were collected and stored in 1000 NBF. Tissue samples were taken from the thoracic spinal cord, cortex, and cerebellum. Samples were analyzed by qPCR for MAPT mRNA knockdown. Average results for each group are shown in Table 16 below:







TABLE 16





Relative expression of MAPT mRNA in various tissues analyzed


by qPCR for each of the dosing groups of Example 4.




















Cortex
Hippocampus




Group Average (n = 4)
Group Average (n = 4)
















Rel.
Error
Error
Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)
Exp.
(Low)
(High)





1
aCSF
1.000
0.122
0.139
1.000
0.228
0.296


2
100 μg AC911178
0.877
0.180
0.226
0.912
0.254
0.352


3
100 μg AC911179
0.830
0.128
0.152
0.901
0.137
0.162


4
100 μg AC911180
0.998
0.240
0.315
0.667
0.045
0.048


5
100 μg AC911181
0.704
0.256
0.402
0.803
0.145
0.177


6
100 μg AC911182
0.826
0.104
0.119
0.924
0.118
0.135


7
100 μg AC911183
0.698
0.156
0.202
0.781
0.139
0.169


8
100 μg AC911184
0.787
0.250
0.366
0.943
0.208
0.268


9
100 μg AC911185
0.810
0.148
0.181
0.870
0.095
0.107


10
100 μg AC911186
0.967
0.112
0.126
0.994
0.044
0.046


11
100 μg AC911187
0.397
0.107
0.147
0.626
0.083
0.095


12
100 μg AC911188
0.862
0.142
0.169
1.066
0.161
0.190


13
100 μg AC911189
0.913
0.151
0.181
0.942
0.148
0.175


14
100 μg AC911190
0.695
0.256
0.405
1.110
0.137
0.157


15
100 μg AC911191
0.675
0.232
0.354
1.170
0.191
0.229


16
100 μg AC911192
0.854
0.227
0.310
1.058
0.160
0.189


17
100 μg AC911193
0.834
0.127
0.150
0.963
0.146
0.172














Thoracic Spinal Cord




Group Average (n = 4)













Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)





1
aCSF
1.000
0.201
0.252


2
100 μg AC911178
0.742
0.145
0.180


3
100 μg AC911179
0.726
0.169
0.220


4
100 μg AC911180
0.920
0.093
0.104


5
100 μg AC911181
0.555
0.074
0.086


6
100 μg AC911182
0.833
0.122
0.143


7
100 μg AC911183
0.632
0.164
0.221


8
100 μg AC911184
0.875
0.204
0.266


9
100 μg AC911185
0.665
0.188
0.263


10
100 μg AC911186
0.894
0.094
0.106


11
100 μg AC911187
0.829
0.210
0.281


12
100 μg AC911188
0.401
0.057
0.067


13
100 μg AC911189
0.762
0.213
0.296


14
100 μg AC911190
0.884
0.068
0.074


15
100 μg AC911191
0.909
0.165
0.201


16
100 μg AC911192
0.748
0.208
0.289


17
100 μg AC911193
0.604
0.063
0.071









As shown in Table 16, AC911187 demonstrated the greatest knockdown of hMAPT in the cortex and cerebellum, and AC911188 demonstrated the greatest knockdown of hMAPT in the thoracic spinal cord.
Example 5. In Vivo Knockdown of MAPT in Transgenic PS19 Mice
On Study day 1, PS19 mice were injected with either 10 μL artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or 10 μL of compound formulation at a concentration of 10 mg/mL in aCSF for groups 2-17, according to Table 17 below:







TABLE 17







Dosing groups for the mice of Example 5.









Group ID
Animals dosed
AC Duplex Number





Group 1 (aCSF)
n = 4
N/A


Group 2 100 μg AD13097
n = 4
AC911194


Group 3 100 μg AD13098
n = 4
AC911195


Group 4 100 μg AD13099
n = 4
AC911196


Group 5 100 μg AD13100
n = 4
AC911197


Group 6 100 μg AD13101
n = 4
AC911198


Group 7 100 μg AD13102
n = 4
AC911199


Group 8 100 μg AD13103
n = 4
AC911200


Group 9 100 μg AD13104
n = 4
AC911201


Group 10 100 μg AD13105
n = 4
AC911202


Group 11 100 μg AD13106
n = 4
AC911203


Group 12 100 μg AD13107
n = 4
AC911204


Group 13 100 μg AD13108
n = 4
AC911205


Group 14 100 μg AD13109
n = 4
AC911206


Group 15 100 μg AD13110
n = 4
AC911207


Group 16 100 μg AD13111
n = 4
AC911208


Group 17 100 μg AD13112
n = 4
AC911209









Four (n=4) mice were dosed in each group. Mice were injected intracerebroventricularly on day 1. On day 8, mice were euthanized and the left half of the brain and thoracic spinal cord were collected and stored in 1000 NBF. Tissue samples were taken from the thoracic spinal cord, cortex, and cerebellum. Samples were analyzed by qPCR for MAPT mRNA knockdown. Average results for each group are shown in Table 18 below:







TABLE 18





Relative expression of MAPT mRNA in various tissues analyzed


by qPCR for each of the dosing groups of Example 5.




















Cortex
Cerebellum




Group Average (n = 4)
Group Average (n = 4)
















Rel.
Error
Error
Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)
Exp.
(Low)
(High)





1
aCSF
1.000
0.184
0.226
1.000
0.088
0.096


2
100 μg AC911194
1.045
0.164
0.195
1.212
0.261
0.332


3
100 μg AC911195
0.831
0.200
0.264
0.822
0.087
0.098


4
100 μg AC911196
1.059
0.204
0.253
1.052
0.122
0.138


5
100 μg AC911197
1.293
0.227
0.275
0.857
0.174
0.219


6
100 μg AC911198
1.074
0.104
0.115
0.699
0.065
0.072


7
100 μg AC911199
0.695
0.139
0.174
0.412
0.135
0.202


8
100 μg AC911200
1.075
0.139
0.160
0.675
0.171
0.229


9
100 μg AC911201
0.688
0.093
0.107
0.491
0.041
0.045


10
100 μg AC911202
1.027
0.176
0.213
0.774
0.081
0.091


11
100 μg AC911203
0.870
0.087
0.096
0.913
0.206
0.266


12
100 μg AC911204
0.766
0.187
0.247
0.837
0.108
0.124


13
100 μg AC911205
1.025
0.121
0.138
0.837
0.069
0.075


14
100 μg AC911206
0.936
0.149
0.178
0.732
0.192
0.260


15
100 μg AC911207
0.945
0.118
0.134
1.007
0.239
0.313


16
100 μg AC911208
0.961
0.172
0.209
0.963
0.194
0.242


17
100 μg AC911209
0.477
0.070
0.083
0.640
0.211
0.315














Thoracic Spinal Cord




Group Average (n = 4)













Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)





1
aCSF
1.000
0.094
0.103


2
100 μg AC911194
1.196
0.265
0.341


3
100 μg AC911195
0.615
0.074
0.084


4
100 μg AC911196
1.025
0.230
0.297


5
100 μg AC911197
1.152
0.187
0.223


6
100 μg AC911198
0.799
0.178
0.229


7
100 μg AC911199
0.455
0.150
0.223


8
100 μg AC911200
0.878
0.236
0.323


9
100 μg AC911201
0.553
0.101
0.124


10
100 μg AC911202
1.113
0.250
0.322


11
100 μg AC911203
0.802
0.135
0.162


12
100 μg AC911204
0.633
0.109
0.132


13
100 μg AC911205
0.943
0.145
0.172


14
100 μg AC911206
0.815
0.178
0.227


15
100 μg AC911207
0.969
0.155
0.184


16
100 μg AC911208
0.963
0.303
0.443


17
100 μg AC911209
0.384
0.070
0.086









As shown in Table 18, AC911209 demonstrated the greatest knockdown of hMAPT in the cortex and thoracic spinal cord, and AC911199 demonstrated the greatest knockdown of hMAPT in the cerebellum.
Example 6. In Vivo Knockdown of MAPT in Transgenic PS19 Mice
On Study day 1, PS19 mice were injected with either 10 μL artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or 10 μL of compound formulation at a concentration of 7 mg/mL in aCSF for groups 2-13, according to Table 19 below:







TABLE 19







Dosing groups for the mice of Example 6.









Group ID
Animals dosed
AC Duplex Number





Group 1 (aCSF)
n = 4
N/A


Group 2 70 μg AD13112
n = 4
AC911209


Group 3 70 μg AD13904
n = 4
AC912001


Group 4 70 μg AD13905
n = 4
AC912002


Group 5 70 μg AD13906
n = 4
AC912003


Group 6 70 μg AD13907
n = 4
AC912004


Group 7 70 μg AD13908
n = 4
AC912005


Group 8 70 μg AD13909
n = 4
AC912006


Group 9 70 μg AD13910
n = 4
AC912007


Group 10 70 μg AD13911
n = 4
AC912008


Group 11 70 μg AD13912
n = 4
AC912009


Group 12 70 μg AD13913
n = 4
AC912010


Group 13 70 μg AD13914
n = 4
AC912011









Four (n=4) mice were dosed in each group. Mice were injected intracerebroventricularly on day 1. On day 8, mice were euthanized and the left half of the brain and thoracic spinal cord were collected and stored in 1000 NBF. Tissue samples were taken from the thoracic spinal cord, cortex, and hippocampus. Samples were analyzed by qPCR for MAPT mRNA knockdown. Average results for each group are shown in Table 20 below:







TABLE 20





Relative expression of MAPT mRNA in various tissues analyzed


by qPCR for each of the dosing groups of Example 6.




















Cortex
Hippocampus




Group Average (n = 4)
Group Average (n = 4)
















Rel.
Error
Error
Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)
Exp.
(Low)
(High)





1
aCSF
1.000
0.308
0.446
1.000
0.229
0.296


2
70 μg AC911209
0.836
0.152
0.186
0.686
0.212
0.307


3
70 μg AC912001
0.748
0.105
0.122
0.687
0.077
0.087


4
70 μg AC912002
0.839
0.212
0.283
0.640
0.152
0.200


5
70 μg AC912003
0.669
0.152
0.196
0.400
0.099
0.132


6
70 μg AC912004
0.811
0.221
0.303
0.554
0.158
0.221


7
70 μg AC912005
0.554
0.161
0.228
0.420
0.152
0.239


8
70 μg AC912006
0.503
0.228
0.419
0.450
0.250
0.561


9
70 μg AC912007
0.505
0.201
0.335
0.400
0.112
0.156


10
70 μg AC912008
0.617
0.164
0.223
0.401
0.160
0.267


11
70 μg AC912009
0.731
0.140
0.173
0.580
0.106
0.130


12
70 μg AC912010
0.785
0.209
0.285
0.723
0.200
0.276


13
70 μg AC912011
0.640
0.183
0.256
0.460
0.129
0.180














Thoracic Spinal Cord




Group Average (n = 4)













Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)





1
aCSF
1.000
0.393
0.647


2
70 μg AC911209
0.961
0.146
0.172


3
70 μg AC912001
0.851
0.096
0.108


4
70 μg AC912002
0.754
0.145
0.179


5
70 μg AC912003
0.911
0.214
0.279


6
70 μg AC912004
0.934
0.105
0.118


7
70 μg AC912005
0.557
0.112
0.141


8
70 μg AC912006
0.823
0.362
0.646


9
70 μg AC912007
0.580
0.215
0.342


10
70 μg AC912008
0.650
0.133
0.167


11
70 μg AC912009
0.576
0.069
0.078


12
70 μg AC912010
1.016
0.236
0.307


13
70 μg AC912011
0.742
0.065
0.071









As shown in Table 20, AC912006 demonstrated the greatest knockdown of hMAPT in the cortex, with AC912007 showing similar knockdown, AC912007 and AC912003 demonstrated the greatest knockdown in the hippocampus, and AC912005 demonstrated the greatest knockdown of hMAPT in the thoracic spinal cord.
Example 7. In Vivo Knockdown of MAPT in Transgenic PS19 Mice
On Study day 1, PS19 mice were injected with either 10 μL artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or 10 μL of compound formulation at a concentration of either 3 mg/mL for groups, 2, 4, 6, 8, 10 and 12 or 7 mg/mL for groups 3, 5, 7, 9, 11, and 13 in aCSF, according to Table 21 below:







TABLE 21







Dosing groups for the mice of Example 7.









Group ID
Animals dosed
AC Duplex Number





Group 1 (aCSF)
n = 4
N/A


Group 2 30 μg AD13908
n = 4
AC912005


Group 3 70 μg AD13908
n = 4
AC912005


Group 4 30 μg AD13909
n = 4
AC912006


Group 5 70 μg AD13909
n = 4
AC912006


Group 6 30 μg AD13910
n = 4
AC912007


Group 7 70 μg AD13910
n = 4
AC912007


Group 8 30 μg AD13911
n = 4
AC912008


Group 9 70 μg AD13911
n = 4
AC912008


Group 10 30 μg AD13912
n = 4
AC912009


Group 11 70 μg AD13912
n = 4
AC912009


Group 12 30 μg AD13914
n = 4
AC912011


Group 13 70 μg AD13914
n = 4
AC912011









Four (n=4) mice were dosed in each group. Mice were injected intracerebroventricularly on day 1. On day 8, mice were euthanized and the left half of the brain and thoracic spinal cord were collected and stored in 1000 NBF. Tissue samples were taken from the thoracic spinal cord, cortex, and hippocampus. Samples were analyzed by qPCR for MAPT mRNA knockdown. Average results for each group are shown in Table 22 below:







TABLE 22





Relative expression of MAPT mRNA in various tissues analyzed


by qPCR for each of the dosing groups of Example 7.




















Cortex
Hippocampus




Group Average (n = 4)
Group Average (n = 4)
















Rel.
Error
Error
Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)
Exp.
(Low)
(High)





1
aCSF
1.000
0.103
0.115
1.000
0.214
0.272


2
30 μg AC912005
0.549
0.279
0.567
0.544
0.264
0.511


3
70 μg AC912005
0.397
0.073
0.090
0.369
0.060
0.071


4
30 μg AC912006
0.763
0.100
0.114
0.577
0.194
0.293


5
70 μg AC912006
0.771
0.194
0.259
0.435
0.055
0.063


6
30 μg AC912007
0.414
0.274
0.811
0.308
0.201
0.574


7
70 μg AC912007
0.469
0.160
0.243
0.329
0.176
0.377


8
30 μg AC912008
0.514
0.083
0.100
0.439
0.075
0.090


9
70 μg AC912008
0.409
0.152
0.242
0.250
0.105
0.181


10
30 μg AC912009
0.800
0.088
0.098
0.611
0.120
0.150


11
70 μg AC912009
0.374
0.150
0.249
0.281
0.062
0.080


12
30 μg AC912011
0.690
0.135
0.167
0.500
0.164
0.245


13
70 μg AC912011
0.574
0.099
0.120
0.503
0.143
0.200














Thoracic Spinal Cord




Group Average (n = 4)













Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)





1
aCSF
1.000
0.181
0.220


2
30 μg AC912005
0.804
0.286
0.443


3
70 μg AC912005
0.427
0.061
0.072


4
30 μg AC912006
0.703
0.043
0.046


5
70 μg AC912006
0.429
0.137
0.200


6
30 μg AC912007
0.475
0.172
0.270


7
70 μg AC912007
0.468
0.058
0.066


8
30 μg AC912008
0.657
0.110
0.132


9
70 μg AC912008
0.260
0.103
0.170


10
30 μg AC912009
0.698
0.073
0.082


11
70 μg AC912009
0.390
0.128
0.190


12
30 μg AC912011
0.725
0.063
0.069


13
70 μg AC912011
0.533
0.096
0.117









As shown in Table 22, AC912008 demonstrated dose-dependent knockdown in all tissues while achieving the greatest knockdown in both the hippocampus and thoracic spinal cord at the 70 μg dose level.
Example 8. In Vivo Knockdown of MAPT in Transgenic PS19 Mice
On Study day 1, PS19 mice were injected with either 10 μL artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or 10 μL of compound formulation at a concentration of 5 mg/mL for groups 2-9 in aCSF, according to Table 23 below:







TABLE 23







Dosing groups for the mice of Example 8.










Group ID
Animals dosed







Group 1 (aCSF)
n = 4



Group 2 50 μg AC912008
n = 4



Group 3 50 μg AC912669
n = 4



Group 4 50 μg AC912670
n = 4



Group 5 50 μg AC912671
n = 4



Group 6 50 μg AC912672
n = 4



Group 7 50 μg AC912673
n = 4



Group 8 50 μg AC912674
n = 4



Group 9 50 μg AC912675
n = 4










Four (n=4) mice were dosed in each group. Mice were injected intracerebroventricularly on day 1. On day 8, mice were euthanized and the left half of the brain and thoracic spinal cord were collected and stored in 1000 NBF. Tissue samples were taken from the thoracic spinal cord, cortex, and hippocampus. Samples were analyzed by qPCR for MAPT mRNA knockdown. Average results for each group are shown in Table 24 below:







TABLE 24





Relative expression of MAPT mRNA in various tissues analyzed


by qPCR for each of the dosing groups of Example 8.




















Cortex
Hippocampus




Group Average (n = 4)
Group Average (n = 4)
















Rel.
Error
Error
Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)
Exp.
(Low)
(High)





1
aCSF
1.000
0.110
0.123
1.000
0.167
0.200


2
50 μg AC912008
0.536
0.093
0.113
0.722
0.164
0.212


3
50 μg AC912669
0.423
0.060
0.069
0.505
0.183
0.287


4
50 μg AC912670
0.406
0.047
0.053
0.512
0.116
0.150


5
50 μg AC912671
0.311
0.097
0.141
0.418
0.202
0.393


6
50 μg AC912672
0.499
0.085
0.102
0.536
0.115
0.147


7
50 μg AC912673
0.481
0.150
0.218
0.554
0.221
0.367


8
50 μg AC912674
0.548
0.096
0.117
0.859
0.244
0.340


9
50 μg AC912675
0.535
0.177
0.264
0.641
0.200
0.291














Thoracic Spinal Cord




Group Average (n = 4)













Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)





1
aCSF
1.000
0.082
0.090


2
50 μg AC912008
0.497
0.081
0.097


3
50 μg AC912669
0.375
0.048
0.056


4
50 μg AC912670
0.379
0.057
0.067


5
50 μg AC912671
0.374
0.061
0.073


6
50 μg AC912672
0.502
0.089
0.108


7
50 μg AC912673
0.461
0.047
0.052


8
50 μg AC912674
0.443
0.052
0.059


9
50 μg AC912675
0.463
0.149
0.219









As shown in Table 24, AC912671 demonstrated the greatest knockdown in all tissues.
Example 9. In Vivo Knockdown of MAPT in Transgenic PS19 Mice
On Study day 1, PS19 mice were injected with either 10 μL artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or 10 μL of compound formulation at a concentration of 5 mg/mL for groups 2-8 in aCSF, according to Table 25 below:







TABLE 25







Dosing groups for the mice of Example 9.










Group ID
Animals dosed







Group 1 (aCSF)
n = 4



Group 2 50 μg AC912671
n = 4



Group 3 50 μg AC003990
n = 4



Group 4 50 μg AC003991
n = 4



Group 5 50 μg AC003992
n = 4



Group 6 50 μg AC003993
n = 4



Group 7 50 μg AC003994
n = 4



Group 8 50 μg AC003995
n = 4










Four (n=4) mice were dosed in each group. Mice were injected intracerebroventricularly on day 1. On day 8, mice were euthanized and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the thoracic spinal cord, cortex, and hippocampus. Samples were analyzed by qPCR for MAPT mRNA knockdown. Average results for each group are shown in Table 26 below:







TABLE 26





Relative expression of MAPT mRNA in various tissues analyzed by qPCR.




















Cortex
Hippocampus




Group Average (n = 4)
Group Average (n = 4)
















Rel.
Error
Error
Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)
Exp.
(Low)
(High)





1
aCSF
1.000
0.180
0.220
1.000
0.134
0.155


2
50 μg AC912671
0.453
0.129
0.180
0.319
0.025
0.027


3
50 μg AC003990
0.278
0.075
0.104
0.147
0.042
0.059


4
50 μg AC003991
0.745
0.186
0.248
0.538
0.142
0.193


5
50 μg AC003992
0.409
0.038
0.042
0.290
0.082
0.115


6
50 μg AC003993
1.046
0.250
0.329
0.887
0.251
0.349


7
50 μg AC003994
0.455
0.089
0.111
0.356
0.061
0.074


8
50 μg AC003995
0.393
0.096
0.127
0.308
0.118
0.192














Thoracic Spinal Cord




Group Average (n = 4)













Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)





1
aCSF
1.000
0.111
0.125


2
50 μg AC912671
0.387
0.130
0.196


3
50 μg AC003990
0.336
0.083
0.111


4
50 μg AC003991
0.800
0.103
0.118


5
50 μg AC003992
0.422
0.054
0.063


6
50 μg AC003993
0.984
0.190
0.235


7
50 μg AC003994
0.358
0.111
0.160


8
50 μg AC003995
0.402
0.100
0.132









As shown in Table 26, AC003990 demonstrated the greatest knockdown in all tissues.
Example 10. In Vivo Knockdown of MAPT in Transgenic PS19 Mice
On Study day 1, PS19 mice were injected with either 10 μL artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or 10 μL of compound formulation at a concentration of 30 mg/mL for groups 2-7 in aCSF, according to Table 27 below:







TABLE 27







Dosing groups for the mice of Example 10.










Group ID
Animals dosed







Group 1 (aCSF)
n = 4



Group 2 300 μg AC912671
n = 4



Group 3 300 μg AC004123
n = 4



Group 4 300 μg AC004124
n = 4



Group 5 300 μg AC004125
n = 4



Group 6 300 μg AC004126
n = 4



Group 7 300 μg AC004130
n = 4










Four (n=4) mice were dosed in each group. Mice were injected intracerebroventricularly on day 1. On day 15, mice were euthanized and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the thoracic spinal cord, temporal cortex, and hippocampus. Samples were analyzed by qPCR for MAPT mRNA knockdown. Average results for each group are shown in Table 28 below:







TABLE 28





Relative expression of MAPT mRNA in various tissues analyzed by qPCR.




















Temporal Cortex
Hippocampus




Group Average (n = 4)
Group Average (n = 4)
















Rel.
Error
Error
Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)
Exp.
(Low)
(High)





1
aCSF
1.000
0.175
0.212
1.000
0.181
0.222


2
300 μg AC912671
0.178
0.055
0.080
0.161
0.040
0.054


3
300 μg AC004123
0.121
0.030
0.040
0.099
0.011
0.013


4
300 μg AC004124
0.207
0.029
0.034
0.210
0.042
0.053


5
300 μg AC004125
0.173
0.048
0.067
0.169
0.042
0.055


6
300 μg AC004126
0.160
0.028
0.034
0.167
0.037
0.047


7
300 μg AC004130
0.229
0.052
0.067
0.240
0.074
0.106














Thoracic Spinal Cord




Group Average (n = 4)













Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)





1
aCSF
1.000
0.170
0.206


2
300 μg AC912671
0.345
0.181
0.380


3
300 μg AC004123
0.229
0.042
0.051


4
300 μg AC004124
0.228
0.027
0.031


5
300 μg AC004125
0.242
0.023
0.026


6
300 μg AC004126
0.238
0.028
0.031


7
300 μg AC004130
0.427
0.031
0.033









As shown in Table 28, AC004123 demonstrated the greatest knockdown in the temporal cortex and hippocampus, and AC004124 demonstrated the greatest knockdown in the thoracic spinal cord on day 15.
Example 11. In Vivo Knockdown of MAPT in Transgenic PS19 Mice
On Study day 1, PS19 mice were injected with either 10 μL artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or 10 μL of compound formulation at a concentration of 30 mg/mL for groups 2-7 in aCSF, according to Table 29 below:







TABLE 29







Dosing groups for the mice of Example 11.










Group ID
Animals dosed







Group 1 (aCSF)
n = 4



Group 2 300 μg AC912671
n = 4



Group 3 300 μg AC004123
n = 4



Group 4 300 μg AC004124
n = 4



Group 5 300 μg AC004125
n = 4



Group 6 300 μg AC004126
n = 4



Group 7 300 μg AC004130
n = 4










Four (n=4) mice were dosed in each group. Mice were injected intracerebroventricularly on day 1. On day 29, mice were euthanized and the left half of the brain and thoracic spinal cord were collected and stored in 1000 NBF. Tissue samples were taken from the thoracic spinal cord, temporal cortex, and hippocampus. Samples were analyzed by qPCR for MAPT mRNA knockdown. Average results for each group are shown in Table 30 below:







TABLE 30





Relative expression of MAPT mRNA in various tissues analyzed


by qPCR for each of the dosing groups of Example 11.




















Temporal Cortex
Hippocampus




Group Average (n = 4)
Group Average (n = 4)
















Rel.
Error
Error
Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)
Exp.
(Low)
(High)





1
aCSF
1.000
0.421
0.726
1.000
0.405
0.679


2
300 μg AC912671
0.235
0.078
0.117
0.284
0.143
0.290


3
300 μg AC004123
0.190
0.096
0.195
0.134
0.064
0.122


4
300 μg AC004124
0.139
0.034
0.045
0.131
0.060
0.112


5
300 μg AC004125
0.304
0.158
0.329
0.356
0.197
0.444


6
300 μg AC004126
0.154
0.056
0.087
0.269
0.100
0.158


7
300 μg AC004130
0.113
0.064
0.149
0.310
0.115
0.182














Thoracic Spinal Cord




Group Average (n = 4)













Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)





1
aCSF
1.000
0.357
0.555


2
300 μg AC912671
0.256
0.163
0.447


3
300 μg AC004123
0.367
0.234
0.646


4
300 μg AC004124
0.367
0.137
0.220


5
300 μg AC004125
0.235
0.124
0.261


6
300 μg AC004126
0.389
0.157
0.264


7
300 μg AC004130
0.318
0.119
0.191









As shown in Table 30, AC004130 demonstrated the greatest knockdown in the temporal cortex, AC004124 demonstrated the greatest knockdown in the hippocampus, and AC004125 demonstrated the greatest knockdown in the thoracic spinal cord.
Example 12. In Vivo Knockdown of MAPT in Transgenic PS19 Mice
On Study day 1, PS19 mice were injected with either 10 μL artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or 10 μL of compound formulation at a concentration of 30 mg/mL for groups 2-7 in aCSF, according to Table 31 below:







TABLE 31







Dosing groups for the mice of Example 12.










Group ID
Animals dosed







Group 1 (aCSF)
n = 4



Group 2 300 μg AC912671
n = 4



Group 3 300 μg AC004123
n = 4



Group 4 300 μg AC004124
n = 4



Group 5 300 μg AC004125
n = 4



Group 6 300 μg AC004126
n = 4



Group 7 300 μg AC004130
n = 4










Four (n=4) mice were dosed in each group. Mice were injected intracerebroventricularly on day 1. On day 57, mice were euthanized and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the frontal cortex, temporal cortex, thoracic spinal cord, and hippocampus. Samples were analyzed by qPCR for MAPT mRNA knockdown. Average results for each group are shown in Table 32 below:







TABLE 32





Relative expression of MAPT mRNA in various tissues analyzed by qPCR.




















Frontal Cortex
Temporal Cortex




Group Average (n = 4)
Group Average (n = 4)
















Rel.
Error
Error
Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)
Exp.
(Low)
(High)





1
aCSF
1.000
0.212
0.268
1.000
0.181
0.220


2
300 ug AC912671
0.336
0.070
0.088
0.267
0.057
0.073


3
300 ug AC004123
0.169
0.082
0.157
0.135
0.068
0.136


4
300 ug AC004124
0.228
0.089
0.145
0.221
0.086
0.140


5
300 ug AC004125
0.133
0.077
0.184
0.129
0.081
0.220


6
300 ug AC004126
0.287
0.101
0.157
0.324
0.154
0.294


7
300 ug AC004130
0.215
0.038
0.046
0.252
0.084
0.127















Thoracic Spinal Cord
Hippocampus




Group Average (n = 4)
Group Average (n = 4)
















Rel.
Error
Error
Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)
Exp.
(Low)
(High)





1
aCSF
1.000
0.042
0.044
1.000
0.156
0.184


2
300 ug AC912671
0.231
0.028
0.032
0.198
0.053
0.073


3
300 ug AC004123
0.300
0.088
0.124
0.077
0.038
0.075


4
300 ug AC004124
0.286
0.144
0.291
0.095
0.020
0.026


5
300 ug AC004125
0.145
0.080
0.178
0.101
0.053
0.113


6
300 ug AC004126
0.319
0.097
0.139
0.260
0.100
0.163


7
300 ug AC004130
0.281
0.032
0.036
0.157
0.044
0.061









As shown in Table 32, AC004125 demonstrated the greatest knockdown in the frontal cortex, temporal cortex, and thoracic spinal cord. AC004123 demonstrated the greatest knockdown in the hippocampus at day 57.
Example 13. In Vivo Knockdown of MAPT in Transgenic PS19 Mice
On Study day 1, PS19 mice were injected with either 10 μL artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or 10 μL of compound formulation at a concentration of 30 mg/mL for groups 2-7 in aCSF, according to Table 33 below:







TABLE 33







Dosing groups for the mice of Example 13.










Group ID
Animals dosed







Group 1 (aCSF)
n = 4



Group 2 300 μg AC912671
n = 4



Group 3 300 μg AC004123
n = 4



Group 4 300 μg AC004124
n = 4



Group 5 300 μg AC004125
n = 4



Group 6 300 μg AC004126
n = 4



Group 7 300 μg AC004130
n = 4










Four (n=4) mice were dosed in each group. Mice were injected intracerebroventricularly on day 1. On day 85, mice were euthanized and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the frontal cortex, temporal cortex, thoracic spinal cord, and hippocampus. Samples were analyzed by qPCR for MAPT mRNA knockdown. Average results for each group are shown in Table 34 below:







TABLE 34





Relative expression of MAPT mRNA in various tissues analyzed by qPCR.




















Frontal Cortex
Temporal Cortex




Group Average (n = 4)
Group Average (n = 4)
















Rel.
Error
Error
Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)
Exp.
(Low)
(High)





1
aCSF
1.000
0.173
0.209
1.000
0.201
0.252


2
300 ug AC912671
0.640
0.190
0.271
0.523
0.118
0.152


3
300 ug AC004123
0.258
0.079
0.113
0.333
0.046
0.053


4
300 ug AC004124
0.221
0.092
0.158
0.218
0.120
0.269


5
300 ug AC004125
0.290
0.096
0.144
0.231
0.135
0.324


6
300 ug AC004126
0.358
0.175
0.340
0.619
0.183
0.261


7
300 ug AC004130
0.281
0.113
0.190
0.286
0.142
0.282















Thoracic Spinal Cord
Hippocampus




Group Average (n = 4)
Group Average (n = 4)
















Rel.
Error
Error
Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)
Exp.
(Low)
(High)





1
aCSF
1.000
0.069
0.074
1.000
0.170
0.205


2
300 ug AC912671
0.579
0.160
0.221
0.366
0.128
0.196


3
300 ug AC004123
0.423
0.057
0.066
0.351
0.087
0.116


4
300 ug AC004124
0.365
0.079
0.101
0.203
0.090
0.163


5
300 ug AC004125
0.526
0.060
0.068
0.188
0.084
0.153


6
300 ug AC004126
0.463
0.101
0.130
0.510
0.195
0.317


7
300 ug AC004130
0.353
0.041
0.047
0.218
0.110
0.222









As shown in Table 34, AC004124 demonstrated the greatest knockdown in the frontal cortex, temporal cortex, AC004130 demonstrated the greatest knockdown in the thoracic spinal cord, and AC004125 demonstrated the greatest knockdown in the hippocampus at day 85.
Example 14. In Vivo Knockdown of MAPT in Transgenic PS19 Mice
On Study day 1, PS19 mice were injected with either 10 μL artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or 10 μL of compound formulation at a concentration of 0.3 mg/mL for groups 2, 7, 12 and 17, 1 mg/mL for groups 3, 8, 13 and 18, 3 mg/mL for groups 4, 9, 14 and 19, 10 mg/mL for groups 5, 10, 15 and 20, and 30 mg/mL for groups 6, 11, 16, and 21 in aCSF, according to Table 35 below:







TABLE 35







Dosing groups for the mice of Example 14.










Group ID
Animals dosed







Group 1 (aCSF)
n = 4



Group 2 3 μg AC912671
n = 4



Group 3 10 μg AC912671
n = 4



Group 4 30 μg AC912671
n = 4



Group 5 100 μg AC912671
n = 4



Group 6 300 μg AC912671
n = 4



Group 7 3 μg AC004123
n = 4



Group 8 10 μg AC004123
n = 4



Group 9 30 μg AC004123
n = 4



Group 10 100 μg AC004123
n = 4



Group 11 300 μg AC004123
n = 4



Group 12 3 μg AC004125
n = 4



Group 13 10 μg AC004125
n = 4



Group 14 30 μg AC004125
n = 4



Group 15 100 μg AC004125
n = 3



Group 16 300 μg AC004125
n = 4



Group 17 3 μg AC004130
n = 4



Group 18 10 μg AC004130
n = 4



Group 19 30 μg AC004130
n = 4



Group 20 100 μg AC004130
n = 4



Group 21 300 μg AC004130
n = 4










Four (n=4) mice were dosed in each group except for group 15, in which three (n=3) mice were dosed. Mice were injected intracerebroventricularly on day 1. On day 15, mice were euthanized and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the thoracic spinal cord, cortex, and hippocampus. Samples were analyzed by qPCR for MAPT mRNA knockdown. Average results for each group are shown in Table 36 below:







TABLE 36





Relative expression of MAPT mRNA in various tissues analyzed by qPCR.




















Cortex
Hippocampus




Group Average (n = 4)
Group Average (n = 4)
















Rel.
Error
Error
Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)
Exp.
(Low)
(High)





1
aCSF
1.000
0.148
0.174
1.000
0.202
0.254


2
3 μg AC912671
0.897
0.330
0.523
0.756
0.257
0.390


3
10 μg AC912671
0.908
0.142
0.168
0.770
0.161
0.204


4
30 μg AC912671
0.502
0.215
0.376
0.343
0.143
0.244


5
100 μg AC912671
0.232
0.083
0.130
0.182
0.080
0.144


6
300 μg AC912671
0.219
0.100
0.185
0.152
0.058
0.093


7
3 μg AC004123
0.771
0.250
0.370
0.762
0.341
0.618


8
10 μg AC004123
0.662
0.211
0.311
0.530
0.142
0.195


9
30 μg AC004123
0.438
0.141
0.208
0.359
0.140
0.230


10
100 μg AC004123
0.373
0.157
0.270
0.261
0.102
0.166


11
300 μg AC004123
0.137
0.065
0.124
0.114
0.069
0.176


12
3 μg AC004125
0.565
0.127
0.163
0.636
0.148
0.193


13
10 μg AC004125
0.596
0.155
0.209
0.465
0.175
0.281


14
30 μg AC004125
0.421
0.154
0.244
0.311
0.072
0.093


15
100 μg AC004125
0.182
0.045
0.060
0.126
0.024
0.030


16
300 μg AC004125
0.177
0.051
0.072
0.144
0.041
0.057


17
3 μg AC004130
0.658
0.291
0.523
0.698
0.294
0.508


18
10 μg AC004130
0.816
0.104
0.119
0.684
0.075
0.084


19
30 μg AC004130
0.428
0.093
0.118
0.435
0.151
0.232


20
100 μg AC004130
0.181
0.021
0.024
0.257
0.050
0.062


21
300 μg AC004130
0.200
0.113
0.258
0.186
0.103
0.230














Thoracic Spinal Cord




Group Average (n = 4)













Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)





1
aCSF
1.000
0.117
0.132


2
3 μg AC912671
0.982
0.172
0.208


3
10 μg AC912671
0.880
0.114
0.131


4
30 μg AC912671
0.504
0.154
0.221


5
100 μg AC912671
0.212
0.068
0.100


6
300 μg AC912671
0.251
0.103
0.175


7
3 μg AC004123
0.862
0.141
0.168


8
10 μg AC004123
0.784
0.050
0.053


9
30 μg AC004123
0.550
0.063
0.071


10
100 μg AC004123
0.276
0.034
0.039


11
300 μg AC004123
0.204
0.095
0.176


12
3 μg AC004125
0.709
0.156
0.199


13
10 μg AC004125
0.868
0.196
0.252


14
30 μg AC004125
0.511
0.111
0.143


15
100 μg AC004125
0.285
0.018
0.019


16
300 μg AC004125
0.234
0.084
0.131


17
3 μg AC004130
0.901
0.136
0.161


18
10 μg AC004130
0.925
0.148
0.176


19
30 μg AC004130
0.653
0.142
0.181


20
100 μg AC004130
0.390
0.199
0.406


21
300 μg AC004130
0.421
0.088
0.112









As shown in Table 36, each group showed a general dose-dependent response to the administered RNAi agent. AC004125 demonstrated deep knockdown in the cortex and hippocampus, especially at the 100 μg dose level.
Example 15. In Vivo Knockdown of MAPT in in Cynomolgus Monkeys
On Study day 1, cynomolgus monkeys were injected with either artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or a compound formulation containing 15 mg of a MAPT RNAi agent formulated in aCSF according to Table 37 below:







TABLE 37







Dosing groups for the non-human primates of Example 15.










Group ID
Animals dosed







Group 1 (aCSF)
n = 4



Group 2 15 mg AC005033
n = 6



Group 3 15 mg AC004265
n = 6



Group 4 15 mg AC912671
n = 7



Group 5 15 mg AC004123
n = 4



Group 6 15 mg AC004125
n = 7



Group 7 15 mg AC004130
n = 6










Four (n=4) monkeys were dosed in group 1, six (n=6) monkeys were dosed in groups 2, 3, and 7, and seven (n=7) monkeys were dosed in groups 3 and 6. Monkeys were injected intrathecally on day 1. On study day 85, animals from each group were euthanized and brain and spinal cord tissue was collected from each animal.
Intrathecal injection in NHPs is a challenging procedure and mis-dosing is commonly observed due to the limited space and accessibility leading to improper placement of the injection needle and leakage of the test article. To adjust for mis-dosing in analysis of protein and expression levels, mis-dosing criteria was defined such that improperly dosed animals were excluded.
The mis-dosing criteria was solely based on tissue distribution of siRNA compound. Cynomolgus monkeys were determined mis-dosed and excluded from the analysis, if approximately 5000 or more of the brain tissue regions analyzed have compound concentrations lower than 25% of group mean. Out of the overall thirty-six (36) NHP which received test article, ten (10) animals were identified as mis-dosed and the protein expression level data were excluded from the analysis. Two animals were excluded from Group 2, five animals were excluded from group 3, four animals were excluded from group 4, two animals were excluded from group 5, three animals were excluded from group 6, and two animals were excluded from group 7 for misdosing.
Samples were analyzed by qPCR for MAPT mRNA knockdown. Samples were analyzed by JESS for protein knockdown. Average mRNA knockdown for frontal cortex, temporal cortex, hippocampus and thoracic spinal cord for each group, relative to Group 1, are shown in Table 42 below:







TABLE 38





Relative expression of MAPT mRNA in various tissues analyzed by qPCR.




















Frontal Cortex
Temporal Cortex




Group Average
Group Average
















Rel.
Error
Error
Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)
Exp.
(Low)
(High)





1
aCSF
1.000
0.559
1.269
1.000
0.240
0.317


2
15 mg AC005033
1.365
0.625
1.153
0.803
0.325
0.546


3
15 mg AC004265
2.079
0.523
0.700
1.207
0.630
1.318


4
15 mg AC912671
1.201
0.541
0.985
0.822
0.475
1.128


5
15 mg AC004123
0.502
0.241
0.463
0.468
0.205
0.366


6
15 mg AC004125
0.936
0.403
0.709
0.481
0.215
0.387


7
15 mg AC004130
0.976
0.300
0.434
0.641
0.252
0.415















Thoracic Spinal Cord
Hippocampus




Group Average
Group Average
















Rel.
Error
Error
Rel.
Error
Error


Group #
Description
Exp.
(Low)
(High)
Exp.
(Low)
(High)





1
aCSF
1.000
0.190
0.234
1.000
0.147
0.173


2
15 mg AC005033
0.335
0.136
0.229
1.465
0.451
0.652


3
15 mg AC004265
0.685
0.227
0.340
1.821
0.502
0.694


4
15 mg AC912671
0.415
0.219
0.461
0.980
0.421
0.739


5
15 mg AC004123
0.165
0.110
0.333
0.801
0.341
0.593


6
15 mg AC004125
0.203
0.110
0.242
0.861
0.249
0.350


7
15 mg AC004130
0.447
0.191
0.333
1.029
0.314
0.452









Average protein knockdown for frontal cortex, temporal cortex, hippocampus and thoracic spinal cord for each group, relative to Group 1, are shown in FIG. 3.
Protein analysis by JESS shows that unexpectedly high knockdown was observed in the tissues of animals dosed in group 6 (AC004125).
Example 16. In Vivo Administration of MAPT RNAi Agents in Cynomolgus Monkeys
MAPT RNAi agents were evaluated in vivo in Cynomolgus monkeys. On Days 1, 8, and 15, four (n=4) male Cynomolgus monkeys for each test group were dosed with MAPT RNAi agents formulated in PBS at 3.0 mg/kg (adjusted for individual animal body weight), 2.0 ml/kg dose volume, at 1.5 mg/ml dose concentration, or dosed with PBS. Each dose was administered via subcutaneous (SC) injection, and each dose was administered based on each respective animal's most recent body weight. The dosing was in accordance with the following Table 39.







TABLE 39







Dosing for Cynomolgus monkeys of Example 16.










Group ID, Dose

Sacrifice
# of Animals


(RNAi Agent)
Dosing Route
Day
(n=)





1. PBS
SC Injection on
Day 43
n = 4



Day 1, 8, 15


2. 3.0 mg/kg AC009806
SC Injection on
Day 43
n = 4



Day 1, 8, 15


3. 3.0 mg/kg AC007414
SC Injection on
Day 43
n = 4



Day 1, 8, 15









The test animals were male, naïve, Cynomolgus monkeys (Macaca fascicularis). The test animals were acclimated to laboratory housing, per facility and acclimation standard operating procedures, for at least 3 days prior to the initiation of dosing. The test animals were randomized and assigned to groups using a computer-based procedure prior to transfer into the study.
Each animal from Groups 1-3 was dosed on Day 1, 8, and 15. The RNAi agent test articles were administered via subcutaneous (SC) administration with a syringe and needle in the mid-scapular region. Dose sites were shaved before dosing and remarked as necessary throughout the study. Dose one (on Day 1) was delivered to the left scapular region, dose two (on Day 8) was delivered to the right scapular region, and dose three (on Day 15) was delivered to the left scapular region. Each subcutaneous dose was delivered using a syringe with 23-25-gauge needle.
The test animals' individual body weights were recorded once pre-treatment Day −7, and then weekly through the duration of the study, and once prior to necropsy.
Cerebrospinal fluid (CSF), ˜1.0 mL, was collected on Day −7 and on day of necropsy for all Groups. At Day 43, the animals were euthanized. From the test animals, the following tissues were collected: left and right brain hemisphere, spinal cord, dorsal root ganglion (DRG).
MAPT mRNA transcript expression was analyzed via qPCR in the tissues, with cPPIB as endogenous control gene, normalized to Group 1 cynos dosed with PBS. The MAPT mRNA transcript expression data is shown in the following Table 40.







TABLE 40





MAPT mRNA expression in cyno tissues.

















Day 43










Frontal Cortex
Temporal Cortex














Rel. Exp.
Std. Dev
Rel. Exp.
Std. Dev


Group ID
Sac Day
MAPT
+/−
MAPT
+/−





1. PBS
Day 43
1.039
0.301
1.012
0.173


2. 3.0 mg/kg AC009806
Day 43
0.324
0.082
0.200
0.040


3. 3.0 mg/kg AC007414
Day 43
0.378
0.050
0.215
0.026













Caudate
Putamen














Rel. Exp.
Std. Dev
Rel. Exp.
Std. Dev


Group ID
Sac Day
MAPT
+/−
MAPT
+/−





1. PBS
Day 43
1.018
0.219
1.006
0.122


2. 3.0 mg/kg AC009806
Day 43
0.305
0.066
0.291
0.053


3. 3.0 mg/kg AC007414
Day 43
0.377
0.023
0.261
0.0544













Hippocampus
Cerebellum














Rel. Exp.
Std. Dev
Rel. Exp.
Std. Dev


Group ID
Sac Day
MAPT
+/−
MAPT
+/−





1. PBS
Day 43
1.002
0.078
1.006
0.123


2. 3.0 mg/kg AC009806
Day 43
0.266
0.003
0.715
0.102


3. 3.0 mg/kg AC007414
Day 43
0.307
0.036
0.766
0.079













Thoracic Spinal Cord
Motor Cortex














Rel. Exp.
Std. Dev
Rel. Exp.
Std. Dev


Group ID
Sac Day
MAPT
+/−
MAPT
+/−





1. PBS
Day 43
1.007
0.144
1.012
0.183


2. 3.0 mg/kg AC009806
Day 43
0.291
0.050
0.213
0.028


3. 3.0 mg/kg AC007414
Day 43
0.282
0.039
0.222
0.023













Medulla
Pons














Rel. Exp.
Std. Dev
Rel. Exp.
Std. Dev


Group ID
Sac Day
MAPT
+/−
MAPT
+/−





1. PBS
Day 43
1.017
0.215
1.022
0.240


2. 3.0 mg/kg AC009806
Day 43
0.243
0.029
0.289
0.066


3. 3.0 mg/kg AC007414
Day 43
0.287
0.049
0.261
0.018













Thalamus
Midbrain














Rel. Exp.
Std. Dev
Rel. Exp.
Std. Dev


Group ID
Sac Day
MAPT
+/−
MAPT
+/−





1. PBS
Day 43
1.002
0.077
1.006
0.122


2. 3.0 mg/kg AC009806
Day 43
0.321
0.085
0.269
0.036


3. 3.0 mg/kg AC007414
Day 43
0.352
0.035
0.335
0.097









MAPT RNAi agents achieved MAPT mRNA transcript knockdown out to at least Day 43 in the brain. The most notable and significant knockdown was observed in the motor cortex, with 3×3.0 mg/kg AC009806 subcutaneous (SC) dose achieving ˜˜79% MAPT transcript inhibition (0.213) on Day 43.
MAPT protein expression was analyzed via Jess protein assay in the cyno tissues, normalized to Group 1 cynos dosed with PBS. The MAPT protein expression data is shown in the following Table 41.







TABLE 41





MAPT protein expression in cyno tissues.

















Day 43










Frontal Cortex
Temporal Cortex














Rel. Exp.
Std. Dev
Rel. Exp.
Std. Dev


Group ID
Sac Day
MAPT
+/−
MAPT
+/−





1. PBS
Day 43
102.062
23.674
102.260
25.583


2. 3.0 mg/kg AC009806
Day 43
33.905
12.750
26.527
8.780


3. 3.0 mg/kg AC007414
Day 43
33.647
15.521
31.366
4.909













Caudate
Putamen














Rel. Exp.
Std. Dev
Rel. Exp.
Std. Dev


Group ID
Sac Day
MAPT
+/−
MAPT
+/−





1. PBS
Day 43
100.521
12.221
102.840
25.380


2. 3.0 mg/kg AC009806
Day 43
37.884
7.318
45.583
9.483


3. 3.0 mg/kg AC007414
Day 43
45.084
2.200
52.174
5.785












Hippocampus














Rel. Exp.
Std. Dev



Group ID
Sac Day
MAPT
+/−







1. PBS
Day 43
104.294
34.024



2. 3.0 mg/kg AC009806
Day 43
42.938
14.751



3. 3.0 mg/kg AC007414
Day 43
59.876
20.621










MAPT RNAi agents achieved MAPT protein knockdown out to at least Day 43 in CNS tissues. The most notable and significant knockdown was observed in the temporal cortex, with 3×3.0 mg/kg AC009806 subcutaneous (SC) dose achieving ˜73% MAPT protein inhibition (26.527 relative to control) on Day 43.
Example 17. In Vivo Knockdown of MAPT in in Cynomolgus Monkeys
Cynomolgus monkeys were injected with either artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or a compound formulation containing 1.7 mg, 5 mg, or 15 mg of a MAPT RNAi agent formulated in aCSF according to Tables 42 and 43 below:







TABLE 42







Dosing groups for the non-human primates of Example 17.











# Animals
Dose
Dose


Group ID
Dosed (n=)
Concentration
Volume





Group 1 aCSF
n = 4
N/A
2 mL











Group 2 1.7 mg AC004125
n = 6
0.85
mg/mL
2 mL


Group 3 5 mg AC004125
n = 6
2.5
mg/mL
2 mL


Group 4 15 mg AC004125
n = 6
7.5
mg/mL
2 mL









Four (n=4) monkeys were dosed in group 1, six (n=6) monkeys were dosed in groups 2, 3, and 4. The test animals were cynomolgus monkeys (Macaca fascicularis), non-naïve, male, 3-5 years of age, and 3-5 kg in weight.







TABLE 43







Dosing groups for the non-human primates of Example 19.











Distribution of animals per surgical



Animals
procedure (n=)














Group ID
Dosed
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6

















Group 1 aCSF
n = 4
4







Group 2 1.7 mg AC004125
n = 6

4
2


Group 3 5 mg AC004125
n = 6


2
4


Group 4 15 mg AC004125
n = 6




4
2









Monkeys were dosed on the days listed in Table 43. Table 43 details the number of animals dosed on the indicated study days for each respective test group. For example, for Group 2, four (4) cynos were dosed on Day 2, and two (2) cynos were dosed on Day 3.
Cynos were dosed via intrathecal injection. On study Day 85, animals from each group were euthanized and brain and spinal cord tissue was collected from each animal.
Intrathecal injection in NHPs is a challenging procedure and mis-dosing is commonly observed due to the limited space and accessibility leading to improper placement of the injection needle and leakage of the test article. To adjust for mis-dosing in analysis of protein and expression levels, mis-dosing criteria was defined such that improperly dosed animals were excluded.
The mis-dosing criteria were solely based on tissue distribution of RNAi agent compound. Cynomolgus monkeys were determined mis-dosed and excluded from the analysis, if approximately 50% or more of the brain tissue regions analyzed have compound concentrations lower than 25% of group mean. Out of the overall twenty-two (22) cynos which received test article, nine (9) cynos were identified as mis-dosed and both the mRNA transcript and protein expression level data were excluded from the analysis. Three (3) animals were excluded from Group 2, three (3) animals were excluded from Group 3, and three (3) animals were excluded from Group 4, for misdosing.
Average mRNA knockdown for frontal cortex, temporal cortex, hippocampus, thoracic spinal cord, cerebellum, and caudate for each group, with PPIB as endogenous control gene, relative to Group 1, are shown in Table 44 below:







TABLE 44





Relative expression of MAPT mRNA in


various tissues analyzed by qPCR.

















Day 85










Frontal Cortex
Temporal Cortex












Rel. Exp.

Rel. Exp.



Group ID
MAPT
Std Dev.
MAPT
Std Dev.





1. aCSF
100.394
10.392
100.501
11.137


2. 1.7 mg AC004125
87.126
10.091
104.035
13.215


3. 5 mg AC004125
88.154
45.335
78.768
40.360


4. 15 mg AC004125
18.690
5.700
22.751
7.191













Hippocampus
Thoracis Spinal Cord












Rel. Exp.

Rel. Exp.



Group ID
MAPT
Std Dev.
MAPT
Std Dev.





1. aCSF
101.010
16.577
140.820
83.359


2. 1.7 mg AC004125
114.587
57.413
93.333
24.856


3. 5 mg AC004125
86.453
15.315
45.933
9.172


4. 15 mg AC004125
49.400
38.336
65.097
61.625













Cerebellum
Caudate












Rel. Exp.

Rel. Exp.



Group ID
MAPT
Std Dev.
MAPT
Std Dev.





1. aCSF
100.138
6.050
100.257
8.137


2. 1.7 mg AC004125
113.600
3.361
104.035
10.476


3. 5 mg AC004125
89.493
20.427
63.832
11.115


4. 15 mg AC004125
60.195
20.751
42.787
20.338









AC004125 achieved MAPT mRNA transcript inhibition in CNS tissues out to at least Day 85. The most notable and significant knockdown was observed in the frontal cortex, with a single 15 mg intrathecal dose achieving ˜81% MAPT inhibition (18.690 relative to control) on Day 85. A dose response was observed in the frontal cortex, temporal cortex, hippocampus, cerebellum, and caudate.
Average protein knockdown for frontal cortex, temporal cortex, hippocampus, thoracic spinal cord, cerebellum, and caudate for each group, relative to Group 1, are shown in Table 45 below:







TABLE 45





Relative expression of MAPT protein in various tissues analyzed


by JESS assay for each of the dosing groups of Example 17.

















Day 85










Frontal Cortex
Temporal Cortex












Rel. Exp.

Rel. Exp.



Group ID
MAPT
Std Dev.
MAPT
Std Dev.





1. aCSF
100.000
40.695
100.000
38.276


2. 1.7 mg AC004125
125.785
37.246
165.049
17.229


3. 5 mg AC004125
68.694
52.460
110.918
117.959


4. 15 mg AC004125
15.506
9.665
15.363
8.603













Hippocampus
Thoracis Spinal Cord












Rel. Exp.

Rel. Exp.



Group ID
MAPT
Std Dev.
MAPT
Std Dev.





1. aCSF
100.000
50.591
100.000
61.513


2. 1.7 mg AC004125
88.525
29.149
84.556
25.898


3. 5 mg AC004125
41.187
36.452
42.783
7.833


4. 15 mg AC004125
12.109
5.609
54.347
32.419













Cerebellum
Caudate












Rel. Exp.

Rel. Exp.



Group ID
MAPT
Std Dev.
MAPT
Std Dev.





1. aCSF
100.000
54.123
100.000
44.130


2. 1.7 mg AC004125
202.453
79.899
110.831
9.517


3. 5 mg AC004125
101.226
67.705
72.152
40.819


4. 15 mg AC004125
28.979
8.425
25.297
10.444









MAPT RNAi agent AC004125 achieved significant MAPT protein inhibition in CNS tissues out to at least Day 85. The most notable and significant knockdown was observed in the hippocampus, with a single 15 mg intrathecal dose achieving ˜88% MAPT inhibition (12.109 relative to control) on Day 85. A dose response was observed in the frontal cortex, temporal cortex, hippocampus, cerebellum, and caudate.
Example 18. In Vivo Knockdown of MAPT in Transgenic PS19 Mice
MAPT RNAi agents were evaluated in vivo in PS19 transgenic mice. On Day 1, four (n=4) male PS19 mice were dosed, via intracerebroventricular (ICV) injection, with either 300 ug MAPT RNAi agent at 10 μl dose volume (at 30 mg/ml concentration) or with 10 μl artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier). Dosing was in accordance with Table 46 below.







TABLE 46







Dosing groups mice of Example 18.













# Animals
Dose
Dose


Group ID
RNAi Agent
Dosed (n=)
Concentration
Volume





1
aCSF
n = 4
N/A
10 μL


2
300 ug AC005033
n = 4
30 mg/mL
10 μL


3
300 ug AC004265
n = 4
30 mg/mL
10 μL


4
300 ug AC912671
n = 4
30 mg/mL
10 μL









The transgenic PS19 mice (P301S Tg mice; also known as B6; C3-Tg(Pmp-MAPT*P301S)PS19Vle/J) express the P301S mutant form of human microtubule-associated protein tau (MAPT), driven by the mouse prion protein promoter (Prnp). The expression of the mutant human MAPT hMAPT is five-fold higher than expression of the endogenous mouse MAPT mMAPT protein.
On Day 15, mice were euthanized and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the thoracic spinal cord, cortex, and cerebellum. Samples were analyzed by qPCR for MAPT mRNA knockdown, with mPPIA as endogenous control gene, normalized to Group 1 mice dosed with aCSF. Average results for each group are shown in Table 47 below.







TABLE 47





Relative expression of MAPT mRNA in various tissues analyzed by qPCR.

















Day 15










Cortex
Thoracic Spinal Cord














Rel. Exp.
Error
Error
Rel. Exp.
Error
Error


Group ID
MAPT
Low
High
MAPT
Low
High





1. aCSF
1.000
0.290
0.408
1.000
0.251
0.336


2. 300 ug AC005033
0.134
0.043
0.062
0.290
0.033
0.038


3. 300 ug AC004265
0.802
0.522
1.498
0.082
0.026
0.038


4. 300 ug AC912671
0.140
0.044
0.064
0.141
0.036
0.048












Hippocampus













Rel. Exp.
Error
Error



Group ID
MAPT
Low
High







1. aCSF
1.000
0.223
0.286



2. 300 ug AC005033
0.110
0.020
0.024



3. 300 ug AC004265
0.046
0.020
0.035



4. 300 ug AC912671
0.052
0.018
0.029










AC005033 achieved the greatest knockdown in the cortex, while AC004265 achieved the greatest knockdown in the thoracic spinal cord and the hippocampus.
Example 19. In Vivo Knockdown of MAPT in Transgenic PS19 Mice
MAPT RNAi agents were evaluated in vivo in PS9 transgenic mice. On Day 1, four (n=4) male and female PS19 mice were dosed, via intracerebroventricular (ICV) injection, with either 300 ug MAPT RNAi agent at 10 μl dose volume (at 30 mg/ml concentration) or with 10 μl artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier). Dosing was in accordance with Table 48 below.







TABLE 48







Dosing groups mice of Example 19.













# Animals
Dose
Dose


Group ID
RNAi Agent
Dosed (n=)
Concentration
Volume





1
aCSF
n = 4
N/A
10 μL


2
300 ug AC005033
n = 4
30 mg/mL
10 μL


3
300 ug AC004265
n = 4
30 mg/mL
10 μL


4
300 ug AC912671
n = 4
30 mg/mL
10 μL









The transgenic PS19 mice (P301S Tg mice; also known as B6; C3-Tg(Pmp-MAPT*P301S)PS19Vle/J) express the P301S mutant form of human microtubule-associated protein tau (MAPT), driven by the mouse prion protein promoter (Prnp). The expression of the mutant human MAPT hMAPT is five-fold higher than expression of the endogenous mouse MAPT mMAPT protein.
On Day 29, mice were euthanized and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the thoracic spinal cord, cortex, and cerebellum. Samples were analyzed by qPCR for MAPT mRNA knockdown, with mPPIA as endogenous control gene, normalized to Group 1 mice dosed with aCSF. Average results for each group are shown in Table 49 below.







TABLE 49





Relative expression of MAPT mRNA in various tissues analyzed by qPCR.

















Day 29










Cortex
Thoracic Spinal Cord














Rel. Exp.
Error
Error
Rel. Exp.
Error
Error


Group ID
MAPT
Low
High
MAPT
Low
High





1. aCSF
1.000
0.243
0.320
1.000
0.159
0.189


2. 300 ug AC005033
0.252
0.082
0.122
0.327
0.087
0.119


3. 300 ug AC004265
0.253
0.095
0.152
0.154
0.077
0.155


4. 300 ug AC912671
0.147
0.052
0.081
0.219
0.063
0.089












Hippocampus













Rel. Exp.
Error
Error



Group ID
MAPT
Low
High







1. aCSF
1.000
0.638
1.761



2. 300 ug AC005033
0.284
0.063
0.081



3. 300 ug AC004265
0.211
0.047
0.060



4. 300 ug AC912671
0.219
0.043
0.054










AC912671 achieved the greatest knockdown in the cortex, while AC004265 achieved the greatest knockdown in the thoracic spinal cord and hippocampus.
Example 20. In Vivo Knockdown of MAPT in Transgenic PS19 Mice
MAPT RNAi agents were evaluated in vivo in PS19 transgenic mice. On Day 1, four (n=4) male and female PS19 mice were dosed, via intracerebroventricular (ICV) injection, with either 300 ug MAPT RNAi agent at 10 μl dose volume (at 30 mg/ml concentration) or with 10 μl artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier). Dosing was in accordance with Table 50 below.







TABLE 50







Dosing groups mice of Example 20.













# Animals
Dose
Dose


Group ID
RNAi Agent
Dosed (n=)
Concentration
Volume





1
aCSF
n = 4
N/A
10 μL


2
300 ug AC005033
n = 4
30 mg/mL
10 μL


3
300 ug AC004265
n = 4
30 mg/mL
10 μL


4
300 ug AC912671
n = 4
30 mg/mL
10 μL









The transgenic PS19 mice (P301S Tg mice; also known as B6; C3-Tg(Pmp-MAPT*P301S)PS19Vle/J) express the P301S mutant form of human microtubule-associated protein tau (MAPT), driven by the mouse prion protein promoter (Prnp). The expression of the mutant human MAPT hMAPT is five-fold higher than expression of the endogenous mouse MAPT mMAPT protein.
On Day 85, mice were euthanized and the left half of the brain and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the thoracic spinal cord, cortex, and cerebellum. Samples were analyzed by qPCR for MAPT mRNA knockdown, with mPPIA as endogenous control gene, normalized to Group 1 mice dosed with aCSF. Average results for each group are shown in Table 51 below.







TABLE 51





Relative expression of MAPT mRNA in various tissues analyzed by qPCR.

















Day 85










Temporal Cortex
Thoracic Spinal Cord














Rel. Exp.
Error
Error
Rel. Exp.
Error
Error


Group ID
MAPT
Low
High
MAPT
Low
High





1. aCSF
1.000
0.287
0.402
1.000
0.128
0.146


2. 300 ug AC005033
0.863
0.226
0.306
0.762
0.211
0.292


3. 300 ug AC004265
0.618
0.249
0.417
0.680
0.201
0.284


4. 300 ug AC912671
0.499
0.274
0.606
0.421
0.190
0.347












Hippocampus













Rel. Exp.
Error
Error



Group ID
MAPT
Low
High







1. aCSF
1.000
0.237
0.310



2. 300 ug AC005033
0.671
0.223
0.334



3. 300 ug AC004265
0.557
0.225
0.377



4. 300 ug AC912671
0.525
0.251
0.481










AC912671 achieved the greatest knockdown in all tissues analyzed.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.