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CN-121203037-B - AAV9 dominant mutant specifically targeting heart and muscle based on integrin engineering

CN121203037BCN 121203037 BCN121203037 BCN 121203037BCN-121203037-B

Abstract

The invention discloses an integrin-modified AAV9 dominant mutant specifically targeting heart and muscle. The invention provides an adeno-associated virus capsid protein comprising a targeting peptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs 1-28. According to the invention, according to the combination of RGD sequence and integrin, a diversified AAV capsid library is constructed by carrying random amino acid according to a polypeptide specific combination principle, and AAV with an adaptive capsid structure is screened by combining high flux, so that a novel adeno-associated virus vector with high heart and/or muscle transduction efficiency is successfully obtained, better targeting and transduction efficiency in heart and/or skeletal muscle are shown in an animal model, and off-target expression in non-target tissues is obviously reduced, thus having important scientific value and commercial prospect.

Inventors

  • WANG CHENG
  • FU YINGYING

Assignees

  • 北京因诺惟康医药科技有限公司

Dates

Publication Date
20260512
Application Date
20250926

Claims (13)

  1. 1. An adeno-associated viral capsid protein comprising a targeting peptide, wherein said adeno-associated viral capsid protein consists of the amino acid sequence shown in SEQ ID No. 39 or 40.
  2. 2. The adeno-associated virus capsid protein according to claim 1, wherein adeno-associated virus having capsid protein comprising said targeting peptide has a higher transduction efficiency for muscle and/or heart than adeno-associated virus employing capsid protein not comprising said targeting peptide.
  3. 3. A polynucleotide encoding the adeno-associated viral capsid protein of claim 1 or 2.
  4. 4. An adeno-associated virus comprising the adeno-associated virus capsid protein of claim 1 or 2 or the polynucleotide of claim 3.
  5. 5. The adeno-associated virus of claim 4, further comprising a transgene.
  6. 6. The adeno-associated virus of claim 5, wherein the transgene is a therapeutic transgene, a prophylactic transgene, or a diagnostic transgene.
  7. 7. A transgenic delivery vector comprising the adeno-associated virus of any one of claims 4-6.
  8. 8. A pharmaceutical composition comprising the adeno-associated virus according to any one of claims 4 to 6, or the transgenic delivery vector according to claim 7.
  9. 9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  10. 10. Use of an adeno-associated virus according to any one of claims 4-6, a transgene delivery vector according to claim 7, or a pharmaceutical composition according to claim 8 or 9 in the preparation of an agent for delivering a transgene to a cell, wherein the cell is from muscle and/or heart.
  11. 11. The use of claim 10, wherein the cell is from a subject.
  12. 12. The use of claim 11, wherein the subject is a mammalian subject.
  13. 13. The use of claim 11 or 12, wherein the subject has a muscle and/or heart disease.

Description

AAV9 dominant mutant specifically targeting heart and muscle based on integrin engineering Technical Field The invention relates to an integrin-modified AAV9 dominant mutant specifically targeting heart and muscle, belonging to the technical fields of biotechnology and genetic engineering. Background Recombinant adeno-associated virus (rAAV) vectors are one of the most potential in vivo gene therapy delivery tools at present, and are widely used in the treatment of various genetic and acquired diseases. AAV-mediated systemic muscle tissue gene delivery, however, remains a significant challenge, where the barrier effect of the vascular endothelium is a major bottleneck limiting the efficient entry of viral particles into skeletal muscle tissue. This limitation results in gene therapy of muscle diseases often relying on viral vector input at ultra-high doses (typically >1 x 10 14 vg/kg) to achieve a considerable therapeutic effect. Of note, in recent years some clinical trials of systemic AAV high dose administration have reported serious adverse events and dose-related toxic responses, such as liver injury, immune response enhancement, even death, etc., highlighting the urgent need to develop efficient, low dose delivery AAV vectors in the field of gene therapy for muscle diseases. For this reason, researchers have attempted to engineer AAV capsid structures by molecular engineering means to enhance their targeting to muscle tissue. Cited document 1 reports a novel class of variants myoAAV engineered based on AAV9 (AAV 9-MyoAAV a), exhibiting excellent skeletal muscle transduction in mice and non-human primates. The capsid introduces RGD (Arg-Gly-Asp) motif exposed on the surface, which can enhance the binding with integrin in muscle tissue, thereby improving muscle affinity. However, there is still room for further optimization of such capsids in terms of transduction efficiency, tissue specificity, trans-species conversion efficiency, etc. Citation document Citation document 1Tabebordbar M,Lagerborg KA,Stanton A,et al.Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species.Cell.2021;184(19):4919-4938.e22. Disclosure of Invention Problems to be solved by the invention To further break through the above limitations, the present invention inserts a targeting peptide consisting of "3 random amino acids+rgd/s+4 random amino acids" into AAV9 serotype capsid proteins based on the binding properties of RGD sequences to integrins. Through systematic screening and modification, a variety of novel AAV capsid variants were successfully obtained. These variants showed significantly improved cardiac and skeletal muscle transduction efficiency in mice over the existing AAV9-MyoAAV a vector. The capsid developed by the invention provides a more efficient delivery tool for gene therapy of muscle related diseases, and lays a new technical foundation for realizing efficient and accurate in-vivo gene delivery. Solution for solving the problem [1] An adeno-associated viral capsid protein comprising a targeting peptide comprising the sequence X 1-X2-X3-R-G-X6-X7-X8-X9-X10, wherein, X 6 is D or S; X 1 is A, E, G, K, L, R, S, T or V; x 2 is A, D, E, G, L, N, P, Q, T or V; X 3 is A, E, F, G, K, L, P, Q, S, T or V; x 7 is D, H, K, L, M, P, Q, R, T or V; X 8 is A, G, M, Q, R, S, T or V; x 9 is A, D, G, K, L, M, P, Q, R, S, T or V; X 10 is A, G, I, K, L, M, P, Q, R, S, T or V; optionally, the targeting peptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs 1 to 28, And/or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs 1 to 28. [2] The adeno-associated viral capsid protein according to [1], wherein the targeting peptide comprises sequence X 1-X2-X3-R-G-D-X7-X8-X9-X10, wherein, X 1 is A, G, L, S or V; X 2 is A, E, G, P, Q, T or V; x 3 is A, F, G, L, P, Q, S or T; X 7 is L, R, T or V; x 8 is A, G, Q, R, S or V; x 9 is D, G, L, M, R, S, T or V; x 10 is G, K, L, M, Q, R, S or T; Optionally, the targeting peptide comprises the amino acid sequence shown in any one of SEQ ID NOs 1,3, 5, 8, 11, 12, 15-17, 25, 27-28; Optionally, adeno-associated viruses having capsid proteins comprising the targeting peptide have increased transduction efficiency for muscle and/or heart compared to adeno-associated viruses employing capsid proteins not comprising the targeting peptide. [3] The adeno-associated viral capsid protein according to [1], wherein the targeting peptide comprises sequence X 1-X2-X3-R-G-D-X7-X8-X9-X10, wherein, X 1 is A, G or S; X 2 is A, G or Q; X 3 is A, G, L or T; X 7 is L or V; X 8 is A, G, Q or S; x 9 is D, G, L or T; X 10 is L, Q, R or S; Optionally, the targeting peptide comprises an amino acid sequence shown in any one of SEQ ID NOs 3, 5, 11, 12, 16; optionally, adeno-associated viruses having capsid proteins comprising the target