Engineered muscle targeting compositions
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.
1. A composition comprising:
a targeting moiety effective to target a muscle cell, wherein the targeting moiety comprises an n-mer motif, wherein the n-mer motif is an RGD motif, wherein the RGD motif has a formula of X m RGDX n , wherein each instance of X is independently selected from any amino acid, m is 0-4 amino acids and n is 1-15 amino acids or m is 1-4 amino acids and n is 0-15 amino acids, wherein the targeting moiety comprises a viral capsid protein, wherein the n-mer motif is (a) inserted between any two contiguous amino acids of the viral capsid protein, (b) replaces one or more native viral capsid protein amino acids, or both (a) and (b); and
a cargo, wherein the cargo is coupled to or is otherwise associated with the targeting moiety.
2. The composition of claim 1 , wherein n is 4 or 5 amino acids.
3. The composition of claim 1 , wherein the n-mer motif is any one of SEQ ID NO: 13-50, 1277-2493, 3737-4979, 6647-8313, 8314-8502, or 8692-8889.
4. The composition of claim 1 , wherein the viral capsid protein is an adeno associated virus (AAV) capsid protein.
5. The composition of claim 4 , wherein the n-mer motif is located between two amino acids of the viral capsid protein such that the n-mer motif is external to a viral capsid of which the viral capsid protein is part, wherein 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, or 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.
6. The composition of claim 1 , wherein the composition is an engineered viral particle, or an engineered viral capsid, optionally an engineered AAV capsid and/or engineered AAV particle, wherein the optionally engineered AAV capsid and/or engineered AAV particle is optionally an engineered AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh.74, or AAV rh.10 viral particle or capsid.
7. The composition of claim 1 , wherein the cargo is capable of treating or preventing a muscle disease or disorder, optionally wherein the muscle disease or disorder is an auto immune disease; a cancer; a muscular dystrophy; a neuro-muscular disease; a sugar or glycogen storage disease; an expanded repeat disease; a dominant negative disease; a cardiomyopathy; a viral disease; a progeroid disease; or any combination thereof, and wherein the cargo is optionally 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 or polypeptides; 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.
8. The composition of claim 1 , wherein the cargo is capable of inducing exon skipping in a gene, optionally a dystrophin gene, or a mini- or micro-dystrophin gene, wherein the mini- or micro-dystrophin gene optionally comprises spectrin-like repeats 1, 1′, 2, 3, 16, 17, 20, 21, 22, 23, 24, or any combination thereof, and optionally an nNOS domain, an actin binding domain, one or more hinge regions, a dystroglycan binding domain, or any combination thereof.
9. The composition of claim 1 , wherein the cargo is operably coupled to a muscle specific promoter.
10. The composition of claim 7 , 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 FSHD, optionally wherein the myotonic dystrophy is a Type 1 or a Type 2 myotonic dystrophy, wherein the cardiomyopathy is dilated cardiomyopathy, hypertrophic cardiomyopathy, Duchene muscular dystrophy-associated cardiomyopathy, or Dannon disease, wherein the sugar or glycogen storage disease is a MPS type III disease or Pompe disease, optionally wherein the MPS type III disease, is MPS Type IIIA, IIIB, IIIC, or IIID, wherein the neuro-muscular disease is Charcot-Marie-Tooth disease or Friedreich's Ataxia, or any combination thereof.
11. The composition of claim 1 , wherein the composition has increased muscle cell potency, muscle cell specificity, reduced immunogenicity, or any combination thereof.
12. A vector system comprising:
a polynucleotide encoding the composition of claim 1 ;
optionally a cargo; and
optionally one or more regulatory elements operatively coupled to the polynucleotide encoding a targeting moiety, the cargo, or both.
13. The vector system of claim 12 , wherein n is 4 or 5, the n-mer motif is any one of SEQ ID NO: 13-50, 1277-2493, 3737-4979, 6647-8313, 8314-8502, or 8692-8889, or both.
14. The vector system of claim 12 , wherein the cargo is a cargo polynucleotide, is coupled to one or more of the one or more polynucleotides encoding the targeting moiety, or both.
15. The vector system of claim 12 , wherein the vector system is a viral vector system, optionally an AAV vector system, and is capable of producing virus particles, optionally AAV particles, comprising a viral capsid, optionally an AAV capsid, comprising the targeting moiety and that contain the optional cargo when present.
16. The vector system of any of claim 15 , wherein the AAV particles and/or AAV capsid are engineered AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh.74, or AAV rh.10 viral particle.
17. The vector system of claim 12 , wherein at least one of the one or more polynucleotides encoding the n-mer motif(s) is inserted between two codons corresponding to two amino acids of the viral protein such that at least one of the n-mer motifs is external to the viral capsid, optionally wherein the two codons correspond to 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, optionally 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.
18. The vector system of claim 12 , wherein the cargo is capable of treating or preventing a muscle disease or disorder, optionally wherein the muscle disease or disorder is an auto immune disease; a cancer; a muscular dystrophy; a neuro-muscular disease; a sugar or glycogen storage disease; an expanded repeat disease; a dominant negative disease; a cardiomyopathy; a viral disease; a progeroid disease; or any combination thereof, and wherein the cargo is optionally 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 or polypeptides; 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.
19. The vector system of claim 12 , wherein the cargo is capable of inducing exon skipping in a gene, optionally a dystrophin gene, or a mini- or micro-dystrophin gene, wherein the mini- or micro-dystrophin gene optionally comprises spectrin-like repeats 1, 1′, 2, 3, 16, 17, 20, 21, 22, 23, 24, or any combination thereof, and optionally an nNOS domain, an actin binding domain, one or more hinge regions, a dystroglycan binding domain, or any combination thereof.
20. The vector system of claim 18 , 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 FSHD, optionally wherein the myotonic dystrophy is a Type 1 or a Type 2 myotonic dystrophy, wherein the cardiomyopathy is dilated cardiomyopathy, hypertrophic cardiomyopathy, Duchene muscular dystrophy-associated cardiomyopathy, or Dannon disease, wherein the sugar or glycogen storage disease is a MPS type III disease or Pompe disease, optionally wherein the MPS type III disease, is MPS Type IIIA, IIIB, IIIC, or IIID, wherein the neuro-muscular disease is Charcot-Marie-Tooth disease or Friedreich's Ataxia, or any combination thereof.
21. A cell or pharmaceutical formulation comprising: the vector system of claim 12 .
22. A cell, pharmaceutical formulation, or viral particle, optionally an adeno associated virus (AAV) particle, comprising: the composition of claim 1 .
23. The cell, pharmaceutical formulation, or viral particle of claim 22 , wherein the viral particle has a muscle-specific tropism.
24. The cell, pharmaceutical formulation, or viral particle of claim 22 , wherein the cargo is capable of treating or preventing a muscle disease or disorder, optionally wherein the muscle disease or disorder is an auto immune disease; a cancer; a muscular dystrophy; a neuro-muscular disease; a sugar or glycogen storage disease; an expanded repeat disease; a dominant negative disease; a cardiomyopathy; a viral disease; a progeroid disease; or any combination thereof, and wherein the cargo is optionally 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 or polypeptides; 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.
25. The cell, pharmaceutical formulation, or viral particle of claim 22 , wherein the cargo is capable of inducing exon skipping in a gene, optionally a dystrophin gene, or a mini- or micro-dystrophin gene, wherein the mini- or micro-dystrophin gene optionally comprises spectrin-like repeats 1, 1′, 2, 3, 16, 17, 20, 21, 22, 23, 24, or any combination thereof, and optionally an nNOS domain, an actin binding domain, one or more hinge regions, a dystroglycan binding domain, or any combination thereof.
26. The cell, pharmaceutical formulation, or viral particle of claim 24 , 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 FSHD, optionally wherein the myotonic dystrophy is a Type 1 or a Type 2 myotonic dystrophy, wherein the cardiomyopathy is dilated cardiomyopathy, hypertrophic cardiomyopathy, Duchene muscular dystrophy-associated cardiomyopathy, or Dannon disease, wherein the sugar or glycogen storage disease is a MPS type III disease or Pompe disease, optionally wherein the MPS type III disease, is MPS Type IIIA, IIIB, IIIC, or IIID, wherein the neuro-muscular disease is Charcot-Marie-Tooth disease or Friedreich's Ataxia, or any combination thereof.
27. A method comprising:
administering, to a subject in need thereof, the composition of claim 1 or a pharmaceutical formulation thereof, wherein the wherein the cargo is capable of treating or preventing a muscle disease or disorder, optionally wherein the muscle disease or disorder is an auto immune disease; a cancer; a muscular dystrophy; a neuro-muscular disease; a sugar or glycogen storage disease; an expanded repeat disease; a dominant negative disease; a cardiomyopathy; a viral disease; a progeroid disease; or any combination thereof, and wherein the cargo is optionally 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 or polypeptides; 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.
28. The method of claim 27 , wherein the cargo is capable of inducing exon skipping in a gene, optionally a dystrophin gene, or a mini- or micro-dystrophin gene, wherein the mini- or micro-dystrophin gene optionally comprises spectrin-like repeats 1, 1′, 2, 3, 16, 17, 20, 21, 22, 23, 24, or any combination thereof, and optionally an nNOS domain, an actin binding domain, one or more hinge regions, a dystroglycan binding domain, or any combination thereof.
29. The method of claim 27 , 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 FSHD, optionally wherein the myotonic dystrophy is a Type 1 or a Type 2 myotonic dystrophy, wherein the cardiomyopathy is dilated cardiomyopathy, hypertrophic cardiomyopathy, Duchene muscular dystrophy-associated cardiomyopathy, or Dannon disease, wherein the sugar or glycogen storage disease is a MPS type III disease or Pompe disease, optionally wherein the MPS type III disease, is MPS Type IIIA, IIIB, IIIC, or IIID, wherein the neuro-muscular disease is Charcot-Marie-Tooth disease or Friedreich's Ataxia, or any combination thereof.
30. A method comprising:
administering, to a subject in need thereof, a cell, pharmaceutical formulation, or viral particle of claim 22 .