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US-20260125706-A1 - ENGINEERED MUSCLE TARGETING COMPOSITIONS

US20260125706A1US 20260125706 A1US20260125706 A1US 20260125706A1US-20260125706-A1

Abstract

Described herein are targeting moieties that can be capable of specifically targeting muscle cells and can include an n-mer motif. In some embodiments, the n-mer motif contains an RGD motif. Also described herein are vector systems, particles, polypeptides that can encode and/or contain one or more targeting moieties. Also described herein are methods of delivering a cargo to a cell, such as a muscle cell, using one or more of the targeting moieties described herein.

Inventors

  • Pardis Sabeti
  • Mohammadsharif Tabebordbar
  • Simon Ye

Assignees

  • THE BROAD INSTITUTE, INC.
  • PRESIDENT AND FELLOWS OF HARVARD COLLEGE
  • MASSACHUSETTS INSTITUTE OF TECHNOLOGY

Dates

Publication Date
20260507
Application Date
20251114

Claims (20)

  1. 1 . A composition comprising an adeno-associated virus (AAV) which comprises a targeting moiety effective to target a muscle cell and a polynucleotide encoding a cargo polynucleotide wherein the targeting moiety comprises a viral capsid protein comprising an n-mer motif, which comprises XmRGDXn, wherein X is any amino acid, m is 3, and n is 4, and wherein the n-mer motif is (a) inserted between any two native amino acids of the viral capsid protein, (b) replaces one or more native viral capsid protein amino acids, or both (a) and (b).
  2. 2 . The composition of claim 1 , wherein the n-mer motif is located in variable region VIII (VR-VIII).
  3. 3 . The composition of claim 1 , wherein the n-mer motif is inserted in the N or C terminus of any amino acid in amino acids 581 to 593 corresponding to the wild type AAV9 capsid protein.
  4. 4 . The composition of claim 1 , wherein the n-mer motif is located in variable region IV (VR-IV).
  5. 5 . The composition of claim 1 , wherein the n-mer motif is located in the N or C terminus of any amino acid in amino acids 452 and 460 corresponding to the wild type AAV9 capsid protein.
  6. 6 . The composition of claim 1 , wherein the n-mer motif is 10 amino acids in length.
  7. 7 . The composition of claim 6 , wherein the viral capsid protein comprises an AAVRh74 capsid protein with one, two, or three additional amino acid substitution, insertion, or deletion.
  8. 8 . The composition of claim 6 , wherein the amino acid sequence of the viral capsid protein is different from the amino acid sequence of the wild type AAVRh74 capsid protein by 11 amino acids.
  9. 9 . The composition of claim 6 , wherein the amino acid sequence of the viral capsid protein is different from the amino acid sequence of the wild type AAVRh74 capsid protein by 12 amino acids.
  10. 10 . The composition of claim 3 , wherein the n-mer motif is 10 amino acids, and wherein the viral capsid protein comprises an AAVRh74 capsid protein with one, two, or three additional amino acid substitution, insertion, or deletion.
  11. 11 . The composition of claim 6 , wherein the viral capsid protein comprises an AAV9 capsid protein with one, two, or three additional amino acid substitution, insertion or deletion.
  12. 12 . The composition of claim 11 , wherein the amino acid sequence of the viral capsid protein is different from the amino acid sequence of the wild type AAV9 capsid protein by 11 amino acids.
  13. 13 . The composition of claim 11 , wherein the amino acid sequence of the viral capsid protein is different from the amino acid sequence of the wild type AAV9 capsid protein by 12 amino acids.
  14. 14 . The composition of claim 5 , wherein the n-mer motif is 10 amino acids in length, and wherein the viral capsid protein comprises an AAV9 capsid protein with one, two, or three additional amino acid substitution, insertion, or deletion.
  15. 15 . The composition of claim 1 , wherein the cargo comprises a micro-dystrophin protein.
  16. 16 . The composition of claim 13 , wherein the cargo polynucleotide is operably linked to a promoter.
  17. 17 . The composition of claim 16 , wherein the promoter comprises a muscle-specific promoter.
  18. 18 . The composition of claim 17 , wherein the muscle-specific promoter comprises a MHCK7 promoter.
  19. 19 . A method of targeting a muscle cell in a subject in need thereof having a muscular dystrophy comprising administering the composition of claim 1 to the subject, wherein the cargo is expressed in the muscle cell.
  20. 20 . A method of targeting a muscle cell in a subject in need thereof having a muscular dystrophy comprising administering the composition of claim 6 to the subject, wherein the cargo is expressed in the muscle cell.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 17/764,509, filed on Mar. 28, 2022, which is the U.S. National Stage Application under 35 U.S.C. § 371 of Patent Cooperation Treaty Application No.: PCT/US2020/056133, filed on Oct. 16, 2020. Patent Cooperation Treaty Application No.: PCT/US2020/056133 claims the benefit of and priority to U.S. Provisional Patent Application Nos. 62/916,207, filed on Oct. 16, 2019; entitled “Engineered Adeno-Associated Virus Capsids”; 62/916,221, filed on Oct. 16, 2019, entitled “Engineered Adeno-Associated Virus Capsids”; 63/018,454, filed on Apr. 30, 2020, entitled “Engineered Adeno-Associated Virus Capsids”; and 63/055,252, filed on Jul. 22, 2020, entitled “Engineered Muscle Targeting Compositions”. The contents of the above-identified applications are hereby fully incorporated herein by reference in their entireties. SEQUENCE LISTING The contents of the electronic sequence listing (“BROD-5005US-CON3_ST26.xml”; size is 11,165,381 bytes and it was created on Nov. 14, 2025) is herein incorporated by reference in its entirety. TECHNICAL FIELD The subject matter disclosed herein is generally directed to muscle targeting compositions including, but not limited to, recombinant adeno-associated virus (AAV) vectors and systems thereof, compositions, and uses thereof. BACKGROUND Recombinant AAVs (rAAVs) are the most commonly used delivery vehicles for gene therapy and gene editing. Nonetheless, rAAVs that contain natural capsid variants have limited cell tropism. Indeed, rAAVs used today mainly infect the liver after systemic delivery. Further, the transduction efficiency of conventional rAAVs in other cell-types, tissues, and organs by these conventional rAAVs with natural capsid variants is limited. Therefore, AAV-mediated polynucleotide delivery for diseased that affect cells, tissues, and organs other than the liver (e.g. nervous system, skeletal muscle, and cardiac muscle) typically requires an injection of a large dose of virus (typically about 1×1014 vg/kg), which often results in liver toxicity. Furthermore, because large doses are required when using conventional rAAVs, manufacturing sufficient amounts of a therapeutic rAAV needed to dose adult patients is extremely challenging. Additionally, due to differences in gene expression and physiology, mouse and primate models respond differently to viral capsids. Transduction efficiency of different virus particles varies between different species, and as a result, preclinical studies in mice often do not accurately reflect results in primates, including humans. As such, there exists a need for improved rAAVs for use in the treatment of various genetic diseases. SUMMARY Described in certain example embodiments herein are compositions comprising a targeting moiety effective to target a muscle cell, wherein the targeting moiety comprises an n-mer motif, and a cargo, wherein the cargo is coupled to or is otherwise associated with the targeting moiety. In certain example embodiments, the n-mer motif comprises an RGD motif or a non-RGD n-mer motif. In certain example embodiments, the RGD motif has a formula of XmRGDXn, wherein m is 0-4 amino acids, wherein n is 0-15 amino acids, wherein X is any amino acid, and wherein each X amino acid present is independently selected from the others from the group consisting of: any amino acid. In certain example embodiments, the RGD motif has the formula RGDXn, wherein n is 4 or 5, wherein X is any amino acid, and wherein each X amino acid present is independently selected from the others from the group consisting of: any amino acid. In certain example embodiments, the n-mer motif is any one of SEQ ID NO: 13-50, 1277-2493, 3737-4979, 6647-8313, 8314-8502, or 8692-8889. In certain example embodiments, the targeting moiety comprises a polypeptide, a polynucleotide, a lipid, a polymer, a sugar, or a combination thereof. In certain example embodiments, the targeting moiety comprises a viral protein. In certain example embodiments, the viral protein is a capsid protein. In certain example embodiments, the viral protein is an adeno associated virus (AAV) protein. In certain example embodiments, the n-mer motif is located between two amino acids of the viral protein such that the n-mer motif is external to a viral capsid of which the viral capsid protein is part. In certain example embodiments, the n-mer motif is inserted between any two contiguous amino acids between amino acids 262-269, 327-332, 382-386, 452-460, 488-505, 527-539, 545-558, 581-593, 704-714, or any combination thereof in an AAV9 capsid polypeptide or in an analogous position in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, or AAV rh.10 capsid polypeptide. In certain example embodiments, the n-mer motif is inserted between amino acids 588 and 589 in an AAV9 capsid polypeptide or in an analogous position in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV