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US-12618082-B2 - Engineered muscle targeting compositions

US12618082B2US 12618082 B2US12618082 B2US 12618082B2US-12618082-B2

Abstract

Described herein are muscle-specific targeting moieties and compositions including the muscle specific targeting motifs. Also described herein are uses of the muscle-specific targeting motifs and compositions including the muscle specific targeting moieties. In some embodiments, the muscle-specific targeting moieties and compositions including the muscle specific targeting moieties can be used to direct delivery of a cargo to a muscle cell.

Inventors

  • Pardis Sabeti
  • Mohammadsharif Tabebordbar
  • Simon Ye
  • Kim Lagerborg
  • Alexandra Stanton
  • Amy Wagers

Assignees

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

Dates

Publication Date
20260505
Application Date
20220329

Claims (20)

  1. 1 . An engineered adeno-associated virus (AAV) capsid polypeptide comprising: an n-mer motif inserted in a surface variable region VR-VIII of the engineered AAV capsid polypeptide, wherein the n-mer motif comprises the amino acid sequence of any one of SEQ ID NOs: 2-7, 20-21, 28-33, 49, 51, 53, 55, 57, 59, 61, 64, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 91, 94, 96, 100, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 158, 160, 163, 166, 168, 170, 172, 174, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 199, 201, 203, 205, 207, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 282, 284, 286, 288, 290, 293, 295, 297, 299, 301, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 327, 329, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 401, 404, 406, 408, 411, 413, 418, 420, 423, 426, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 476, 478, 480, 482, 484, 487, 489, 492, 494, 497, 499, 501, 504, 506, 508, 510, 512, 514, 516, 519, 521, 523, 526, 529, 531, 533, 535, 538, 542, 544, 546, 548, 551, 553, 555, 558, 561, 563, 565, 567, 570, 572, 574, 576, 579, 582, 584, 586, 588, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 703, 707, 709, 711, 713, 715, 717, 720, 723, 726, 728, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 754, 756, 758, 760, 763, and 767.
  2. 2 . The engineered AAV capsid polypeptide of claim 1 , wherein the n-mer motif is inserted between any two amino acids in the surface variable region VR-VIII corresponding to amino acids 581-593 of an AAV9 capsid polypeptide of SEQ ID NO. 1 , or an analogous position in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74 or an AAV rh.10 capsid polypeptide.
  3. 3 . The engineered AAV capsid polypeptide of claim 2 , wherein the first three amino acids of the n-mer motif replace 0, 1, 2, or 3 amino acids of the variable region VIII of the wild-type AAV9 VP1 capsid polypeptide or a wild-type AAV VP1 capsid polypeptide of a different serotype.
  4. 4 . The engineered AAV capsid polypeptide of claim 1 , wherein the engineered AAV capsid polypeptide is an engineered AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh.74, or AAV rh.10 capsid polypeptide.
  5. 5 . The engineered AAV capsid polypeptide of claim 1 , wherein (a) the amino acid at position 588 in an AAV9 capsid polypeptide of SEQ ID NO. 1 or in an analogous position in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, or an AAV rh.10 capsid polypeptide is glycine or alanine, (b) the amino acid at position 592 in an AAV9 capsid polypeptide of SEQ ID NO. 1 or in an analogous position in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, or an AAV rh.10 capsid polypeptide is glutamine, or both (a) and (b).
  6. 6 . The engineered AAV capsid polypeptide of claim 1 , wherein the engineered AAV capsid polypeptide further comprises one or more mutations that result in a reduced or an eliminated uptake in a non-muscle cell as compared to an AAV capsid comprising an engineered AAV capsid polypeptide or a wild-type AAV capsid polypeptide that lack the one or more mutations, wherein the one or more mutations are in position 267, in position 269, in position 504, in position 505, in position 590, or any combination thereof in an AAV9 capsid polypeptide (SEQ ID NO: 1) or in one or more positions corresponding thereto in a non-AAV9 capsid polypeptide, wherein the non-AAV9 capsid polypeptide is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, or AAV rh.10 capsid polypeptide.
  7. 7 . The engineered AAV capsid polypeptide of claim 6 , wherein the engineered AAV capsid polypeptide is an engineered AAV9 capsid polypeptide comprising a mutation at position 267, position 269 or both of a wild-type AAV9 capsid polypeptide (SEQ ID NO: 1), wherein the mutation at position 267 is a G to A mutation and wherein the mutation at position 269 is an S to T mutation, wherein the engineered AAV capsid polypeptide is an engineered AAV9 capsid polypeptide comprising a mutation at position 590 of a wild-type AAV9 capsid polypeptide (SEQ ID NO: 1), wherein the mutation at position 509 is a Q to A mutation, or wherein the engineered AAV capsid polypeptide is an engineered AAV9 capsid polypeptide comprising a mutation at position 504, position 505, or both of a wild-type AAV9 capsid polypeptide (SEQ ID NO: 1), wherein the mutation at position 504 is a G to A mutation and wherein the mutation at position 505 is a P to A mutation.
  8. 8 . The engineered AAV capsid polypeptide of claim 6 , wherein the non-muscle cell is a liver cell.
  9. 9 . A cell, a pharmaceutical formulation, an engineered viral capsid, or an engineered viral particle comprising the engineered AAV capsid polypeptide of claim 1 .
  10. 10 . The cell, the pharmaceutical formulation, the engineered viral capsid, or the engineered viral particle of claim 9 , wherein the engineered viral capsid, the engineered viral particle, or both comprises modulated transduction via integrin heterodimer αVβ6, AAV receptor (AAVR), or both.
  11. 11 . The engineered AAV capsid polypeptide of claim 1 , wherein the n-mer motif is inserted between any two amino acids between amino acids 585 and 589 in an AAV9 capsid polypeptide or in an analogous position in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, AAV rh.10 capsid polypeptide.
  12. 12 . The engineered AAV capsid polypeptide of claim 1 , further comprising a cargo, wherein the cargo is coupled to or is otherwise associated with the engineered AAV capsid polypeptide.
  13. 13 . The engineered AAV capsid polypeptide of claim 12 , wherein the cargo is a morpholino, a peptide-linked morpholino, an antisense oligonucleotide, a PMO, a therapeutic transgene, a polynucleotide encoding a therapeutic polypeptide or peptide, a PPMO, one or more peptides, one or more polynucleotides encoding a CRISPR-Cas protein, a guide RNA, or both, a ribonucleoprotein, wherein the ribonucleoprotein comprises a CRISPR-Cas system molecule, a therapeutic transgene RNA, or other gene modifying or therapeutic RNA and/or protein, or any combination thereof.
  14. 14 . The engineered AAV capsid polypeptide of claim 12 , wherein the cargo treats or prevents a muscle disease or disorder selected from the group consisting of; an autoimmune disease, a cancer, a muscular dystrophy, a neuromuscular disease, a sugar or glycogen storage disease, an expanded repeat disease, a dominant negative disease, a cardiomyopathy, a viral disease, a progeroid disease, and a disease or disorder associated with a cell expressing both αVβ6 and AAVR; or any combination thereof.
  15. 15 . The engineered AAV capsid polypeptide of claim 14 , wherein the expanded repeat disease is Huntington's disease, a Myotonic Dystrophy, or Facioscapulohumeral muscular dystrophy (FSHD), wherein the muscular dystrophy is Duchene muscular dystrophy, Becker Muscular dystrophy, a Limb-Girdle muscular dystrophy, an Emery Dreifuss muscular dystrophy, a myotonic dystrophy, or FSH, wherein the myotonic dystrophy is Type 1 or Type 2, wherein the cardiomyopathy is dilated cardiomyopathy, hypertrophic cardiomyopathy, DMD-associated cardiomyopathy, or Dannon disease, wherein the sugar or glycogen storage disease is a MPS type III disease or Pompe disease, wherein the MPS type III disease, is MPS Type IIIA, IIIB, IIIC, or IIID, wherein the neuromuscular disease is Charcot-Marie-Tooth disease or Friedreich's Ataxia, or any combination thereof.
  16. 16 . The engineered AAV capsid polypeptide of claim 12 , wherein the cargo is capable of inducing exon skipping in a gene, optionally a dystrophin gene, or wherein the cargo is a mini- or micro-dystrophin gene and comprises spectrin-like repeats 1, 2, 3, and 24.
  17. 17 . A vector system comprising: a vector comprising: (a) one or more polynucleotides each encoding one or more engineered adeno-associated virus (AAV) capsid polypeptides, or (b) one or more polynucleotides encoding one or more engineered AAV capsid polypeptides and a cargo, wherein each of the one or more polypeptides of (a) and (b) comprises an n-mer motif inserted in a surface variable region VR-VIII of the one or more engineered AAV capsid polypeptides, wherein the n-mer motif comprises the amino acid sequence of any one of SEQ ID NO: 2-7, 20-21, 28-33, 49, 51, 53, 55, 57, 59, 61, 64, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 91, 94, 96, 100, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 158, 160, 163, 166, 168, 170, 172, 174, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 199, 201, 203, 205, 207, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 282, 284, 286, 288, 290, 293, 295, 297, 299, 301, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 327, 329, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 401, 404, 406, 408, 411, 413, 418, 420, 423, 426, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 476, 478, 480, 482, 484, 487, 489, 492, 494, 497, 499, 501, 504, 506, 508, 510, 512, 514, 516, 519, 521, 523, 526, 529, 531, 533, 535, 538, 542, 544, 546, 548, 551, 553, 555, 558, 561, 563, 565, 567, 570, 572, 574, 576, 579, 582, 584, 586, 588, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 703, 707, 709, 711, 713, 715, 717, 720, 723, 726, 728, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 754, 756, 758, 760, 763, and 767; and a regulatory element is operatively coupled to one or more polynucleotide(s), the cargo, or both.
  18. 18 . The vector system of claim 17 , wherein the cargo is a cargo polynucleotide and is operatively coupled to one or more of the one or more polynucleotides each encoding one or more engineered AAV capsid polypeptides.
  19. 19 . The vector system of claim 17 , wherein each of the one or more polypeptides of (a) and (b) comprise an engineered AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh.74, or AAV rh.10 capsid polypeptides.
  20. 20 . The vector system of claim 17 , wherein a polynucleotide encoding the n-mer motif is inserted between two codons corresponding to any two amino acids between amino acids 581-593 in an AAV9 capsid polypeptide or in an analogous position in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, AAV rh.10 capsid polypeptide; or is inserted between two codons corresponding to amino acids 588 and 589, in an AAV9 capsid polypeptide or in an analogous position thereto in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, AAV rh.10 capsid polypeptide, or is inserted between two codons corresponding to any two amino acids between amino acids 585 and 589 in an AAV9 capsid polypeptide or in an analogous position in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, AAV rh.10 capsid polypeptide.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 17/614,327, which is the U.S. National Phase Application under 35 U.S.C. § 371 of Patent Cooperation Treaty Application No.: PCT/US2021/042812, filed Jul. 22, 2021. Patent Cooperation Treaty Application No.: PCT/US2021/042812 claims the benefit of and priority to U.S. Provisional Patent Application No. 63/055,265, filed on Jul. 22, 2020, U.S. Provisional Patent Application No. 63/107,394, filed on Oct. 29, 2020, and U.S. Provisional Patent Application No. 63/183,038, filed on May 2, 2021. The entire contents of the above-identified applications are hereby fully incorporated herein by reference. SEQUENCE LISTING This application contains a sequence listing filed in electronic form as an ASCII.txt file entitled BROD-5215WP_ST25.txt, created on Jul. 13, 2021 and having a size of 186,097 bytes (188 KB on disk). The content of the sequence listing is incorporated herein in its entirety. TECHNICAL FIELD The subject matter disclosed herein is generally directed to muscle targeting compositions, including, 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 In certain example embodiments, described herein are compositions comprising: a targeting moiety effective to target a muscle cell, wherein the targeting moiety comprises one or more n-mer motif, wherein at least one n-mer motif of the one or more n-mer motifs comprises or consists of XmRGDXn, wherein Xm and Xn are each independently selected from any amino acid, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, or 9, and wherein m is 1-4; and optionally a cargo, wherein the cargo is coupled to or is otherwise associated with the targeting moiety. In certain example embodiments, the at least one n-mer motif is as in any one of Table 2, Table 3, FIG. 14, or any combination thereof. 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. 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 rh.74, or AAV rh.10 capsid polypeptide. In certain example embodiments, the composition is an engineered viral particle. In certain example embodiments, the engineered viral particle is an engineered AAV viral particle. In certain example embodiments, the AAV viral particle is an engineered AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh.74, or AAV rh.10 viral particle. In certain example embodiments, the n-mer motif is 3-15 amino acids. In certain example embodiments, the optional cargo is capable of treating or preventing a muscle disease or dis