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CN-121974988-A - Adeno-associated virus capsid protein VP1 mutant and application thereof

CN121974988ACN 121974988 ACN121974988 ACN 121974988ACN-121974988-A

Abstract

The invention relates to an adeno-associated virus capsid protein VP1 mutant and application thereof, belonging to the biotechnology field. Compared with the adeno-associated virus capsid protein VP1 with an initial amino acid sequence shown as SEQ ID NO.2, the adeno-associated virus capsid protein VP1 mutant provided by the invention has the advantages that the 567 th threonine mutation is alanine, the 575 th glutamine mutation is proline, the 585 th arginine mutation is asparagine, the 587 th asparagine mutation is aspartic acid, histidine is inserted between the 588 th amino acid and the 589 th amino acid, and the adeno-associated virus capsid protein VP1 mutant obviously increases the production efficiency of recombinant adeno-associated virus and the transduction efficiency of the recombinant adeno-associated virus in muscle tissues, so that the adeno-associated virus capsid protein VP1 mutant has great application prospects in the establishment of muscle-associated disease models or the preparation of gene therapeutic drugs for muscle-associated diseases.

Inventors

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Assignees

  • 布林凯斯(深圳)生物技术有限公司

Dates

Publication Date
20260505
Application Date
20251224

Claims (10)

  1. 1. An adeno-associated virus capsid protein VP1 mutant, which is characterized in that, compared with adeno-associated virus capsid protein VP1 with an amino acid sequence shown in SEQ ID NO.2, the 567 th threonine mutation is alanine, the 575 th glutamine mutation is proline, the 585 th arginine mutation is asparagine, the 587 th asparagine mutation is aspartic acid, and histidine is inserted between the 588 th amino acid and the 589 th amino acid.
  2. 2. The adeno-associated virus capsid protein VP1 mutant of claim 1, wherein the amino acid sequence of the adeno-associated virus capsid protein VP1 mutant is shown in SEQ ID No. 3.
  3. 3. An adeno-associated virus capsid mutant comprising the adeno-associated virus capsid protein VP1 mutant of claim 1 or 2.
  4. 4. An adeno-associated virus mutant comprising the adeno-associated virus capsid mutant of claim 3 and a genome.
  5. 5. A recombinant adeno-associated viral vector comprising the adeno-associated viral mutant of claim 4, wherein the adeno-associated viral mutant has a gene of interest encoding a protein of interest and/or a functional RNA integrated into the genome.
  6. 6. The recombinant adeno-associated viral vector according to claim 5, wherein the adeno-associated viral mutant further comprises a promoter integrated into the genome.
  7. 7. A nucleic acid molecule encoding the adeno-associated virus capsid protein VP1 mutant of claim 1 or 2, the adeno-associated virus capsid mutant of claim 3, the adeno-associated virus mutant of claim 4 or the recombinant adeno-associated virus vector of claim 5 or 6.
  8. 8. A method of gene delivery, which is not diagnostic or therapeutic for diseases, characterized in that it uses the recombinant adeno-associated viral vector of claim 5 or 6 to deliver a gene of interest encoding a protein of interest and/or a functional RNA to a target site of muscle tissue.
  9. 9. Use of the adeno-associated viral capsid protein VP1 mutant of claim 1 or 2 or the adeno-associated viral capsid mutant of claim 3 or the adeno-associated viral mutant of claim 4 or the recombinant adeno-associated viral vector of claim 5 or 6 or the nucleic acid molecule of claim 7 for gene delivery or for modeling of a disease, wherein said use is for diagnostic and therapeutic purposes of a non-disease, wherein said gene delivery comprises delivery of a gene of interest and/or a functional RNA encoding a protein of interest to a target site of muscle tissue, wherein said disease model comprises a muscle-associated disease model.
  10. 10. Use of the adeno-associated viral capsid protein VP1 mutant of claim 1 or 2 or the adeno-associated viral capsid mutant of claim 3 or the adeno-associated viral mutant of claim 4 or the recombinant adeno-associated viral vector of claim 5 or 6 or the nucleic acid molecule of claim 7 for the preparation of a gene delivery reagent or for the preparation of a gene therapy drug, wherein the gene delivery comprises delivery of a gene of interest encoding a protein of interest and/or a functional RNA to a target site of muscle tissue, wherein the gene therapy drug comprises a gene therapy drug for a muscle-related disease.

Description

Adeno-associated virus capsid protein VP1 mutant and application thereof Technical Field The invention relates to an adeno-associated virus capsid protein VP1 mutant and application thereof, belonging to the biotechnology field. Background Adeno-associated virus (AAV) is a single stranded DNA replication defective parvovirus whose genome consists of terminal inverted repeat ITRs and middle Rep and Cap genes. Wherein ITRs play an important role in AAV replication and packaging, the Rep gene codes for nonstructural proteins involved in AAV replication, packaging and genome integration, the Cap gene codes for structural proteins VP1, VP2 and VP3, the 3 structural proteins are assembled in proportion to form a viral capsid which serves as a viral gene delivery vector, and in addition, another open reading frame nested in the Cap gene codes for assembly activator protein AAP which is involved in targeting and assembly of capsid proteins. After AAV is transduced into cells, replication can be performed with the aid of helper virus (typically adenovirus). The recombinant adeno-associated virus (rAAV) is a gene delivery virus vector modified on the basis of nonpathogenic wild AAV, has the advantages of high safety, multiple types, wide infection host range, high targeting specificity, capability of carrying exogenous genes for long-term stable expression and the like, is widely applied to the fields of treatment of related rare diseases such as ophthalmology, liver, brain, muscle, heart and the like, and becomes one of the gene treatment vectors with the highest clinical application value. To date, 9 rAAV-based gene therapy drugs are marketed in batches worldwide, including Elevidys developed and marketed by Sarepta Therapeutics for the treatment of Dunaliella muscular dystrophy in children 4-5 years. These drugs have an average selling price of over 100 tens of thousands of dollars, and such an expensive price places a heavy burden on the patient's home and society. The main reasons for high selling price of the gene medicine based on the virus vector include large dosage caused by low transduction efficiency, high production cost caused by low packaging efficiency, and the like. Therefore, enhancing the transduction capacity of the viral vector and enhancing the packaging efficiency of the viral vector contributes to reducing the amount of rAAV used, thereby further improving the safety of rAAV and reducing the production cost of rAAV. Among them, AAV serotype 2 is the most widely studied AAV, and commercially available gene drugs such as Luxturna and Upstaza are rAAV constructed based on AAV 2. However, the transduction efficiency of AAV2 on muscle cells is extremely low, and the production efficiency of AAV2 is also low, which severely limits the application of rAAV constructed based on AAV2 in the development of gene therapy drugs for muscle tissues, and therefore, the development of AAV2 mutants with higher production efficiency and transduction efficiency is necessary. Disclosure of Invention In order to solve the problems, the invention provides an adeno-associated virus capsid protein VP1 mutant, wherein compared with adeno-associated virus capsid protein VP1 with an initial amino acid sequence shown as SEQ ID NO.2, the 567 th threonine mutation is alanine, the 575 th glutamine mutation is proline, the 585 th arginine mutation is asparagine, the 587 th asparagine mutation is aspartic acid, and histidine is inserted between the 588 th amino acid and the 589 th amino acid. In one embodiment of the invention, the amino acid sequence of the adeno-associated virus capsid protein VP1 mutant is shown in SEQ ID NO. 3. In one embodiment of the invention, the nucleotide sequence of the nucleic acid molecule encoding the adeno-associated virus capsid protein VP1 mutant is shown in SEQ ID NO. 4. In one embodiment of the invention, the adeno-associated virus capsid protein VP1 mutant is an adeno-associated virus type 2 (AAV 2) capsid protein VP1 mutant. The invention also provides an adeno-associated virus capsid mutant, which comprises the adeno-associated virus capsid protein VP1 mutant. The invention also provides an adeno-associated virus mutant, which comprises the adeno-associated virus capsid mutant and a genome. The invention also provides a recombinant adeno-associated virus vector, which is characterized in that the recombinant adeno-associated virus vector comprises the adeno-associated virus mutant, and a target gene and/or functional RNA for encoding a target protein are integrated on the genome of the adeno-associated virus mutant. In one embodiment of the invention, the protein of interest comprises at least one of a marker protein or an active protein. In one embodiment of the invention, the marker protein comprises at least one of a fluorescent protein or an enzyme reaction chromogenic protein. In one embodiment of the invention, the marker protein comprises at least one of BFP、CFP、GFP、YFP、RFP、iRFP、Cer