PEPTIDE-MODIFIED HYBRID RECOMBINANT ADENO-ASSOCIATED VIRUS SEROTYPE BETWEEN AAV9 AND AAVrh74 WITH REDUCED LIVER TROPISM AND INCREASED MUSCLE TRANSDUCTION

A serotype and virus technology, applied in the field of muscle genetic diseases and treatment of neuromuscular genetic diseases

Inactive Publication Date: 2021-12-14
GENETHON +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] AAV2 vectors displaying the RGDLGLS (SEQ ID NO:8) peptide inserted into the AAV capsid (position R588 relative to VP1 protein numbering) efficiently transduce primary murine breast cancer cells in vitro but fail to transduce mammary glands in vivo Cancer tumor cells (Michelfelder et al., PLoS ONE, 2009, 4, e5122)

Method used

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  • PEPTIDE-MODIFIED HYBRID RECOMBINANT ADENO-ASSOCIATED VIRUS SEROTYPE BETWEEN AAV9 AND AAVrh74 WITH REDUCED LIVER TROPISM AND INCREASED MUSCLE TRANSDUCTION
  • PEPTIDE-MODIFIED HYBRID RECOMBINANT ADENO-ASSOCIATED VIRUS SEROTYPE BETWEEN AAV9 AND AAVrh74 WITH REDUCED LIVER TROPISM AND INCREASED MUSCLE TRANSDUCTION
  • PEPTIDE-MODIFIED HYBRID RECOMBINANT ADENO-ASSOCIATED VIRUS SEROTYPE BETWEEN AAV9 AND AAVrh74 WITH REDUCED LIVER TROPISM AND INCREASED MUSCLE TRANSDUCTION

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0169] Example: Peptide-modified hybrid AAV9-rh74 serotype vectors

[0170] 1. Materials and Methods

[0171] Plasmid construction of new serotypes

[0172] To construct a plasmid containing the AAV2 Rep sequence and the hybrid Cap 9-rh74, a 1029nt fragment containing the hypervariable portion of the AAV-rh74 Cap flanked by the AAV9 Cap sequence fragment and the 5' restriction site BsiWI was synthesized and 3' Eco47III (GENEWIZ). This fragment was then inserted into plasmid pAAV2-9 containing AAV2Rep and AAV9Cap using the mentioned restriction sites to replace the AAV9 Cap corresponding sequence. Peptide implantation was performed by replacing the QQNAAP hexapeptide (SEQ ID NO: 11 ) in the AAV9-rh74 capsid with the amino acid sequence GQSGRGDLGLSAQAA (SEQ ID NO: 13).

[0173] AAV production

[0174] HEK293T cells were grown in suspension in 250 mL of serum-free medium. Cells were transfected with 3 plasmids: i) a transgenic plasmid containing the AAV2 ITR flanked by an ex...

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Abstract

The invention relates to a recombinant adeno-associated virus (AAV) capsid protein, which is a peptide-modified hybrid between AAV serotype 9 (AAV9) and AAV serotype 74 (AAVrh74) capsid proteins comprising at least one copy of a peptide comprising the RGD motif, wherein said recombinant peptide-modified hybrid AAV capsid protein has a further reduced liver tropism and an increased muscle transduction compared to the recombinant hybrid AAV capsid protein not having said peptide. The invention relates also to the derived peptide-modified hybrid AAV serotype vector particles packaging a gene of interest and their use in gene therapy, in particular for treating neuromuscular genetic diseases, in particular muscular genetic diseases.

Description

technical field [0001] The present invention relates to recombinant adeno-associated virus (AAV) capsids, which are peptide-modified hybrids between AAV serotype 9 (AAV9) and AAV serotype rh74 (AAVrh74) capsid proteins, and hybrids without said peptides. Compared with AAV capsid protein, this capsid protein has further decreased hepatic tropism and increased muscle transduction. The present invention also relates to derived peptide-modified hybrid AAV serotype vector particles packaging target genes, and their use in gene therapy, especially for the treatment of neuromuscular genetic diseases, especially for muscle genetic diseases. Background technique [0002] Recombinant adeno-associated virus (rAAV) vectors are widely used for in vivo gene transfer. The rAAV vector is a non-enveloped vector composed of a capsid with a diameter of 20nm and a single-stranded DNA of 4.7kb. The genome carries two genes, rep and cap, flanked by two palindromic terminal regions called invert...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K48/00C12N15/864C07K14/015
CPCC12N2750/14122C12N2750/14143C12N2750/14171C12N15/86C07K14/005C12N2750/14145C12N7/00A61K48/00A61P35/00C12N2750/14152C12N2310/20A61P25/28C07K14/075C12N9/22C12N15/111C12N2750/14121
Inventor G·龙齐蒂P·维达尔F·明戈齐
Owner GENETHON
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