Methods of manufacturing microdystrophin gene therapy constructs
AssigneeREGENXBIO Inc.
InventorsChunping Qiao, Devin McDougald, Ye Liu, Olivier Danos
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Abstract
Provided are gene constructs and nucleic acids that encode a microdystrophin protein for use in gene therapy and methods of producing recombinant AAV particles containing the nucleic acids. The microdystrophin gene constructs and expression cassettes were engineered for improved therapy with respect to efficacy, potency and safety to the subject when expressed by a viral vector in muscle cells and/or CNS cells.
Claims23
A method of producing recombinant adeno-associated viruses (AAVs) comprising:
- (a) culturing a host cell containing:
- (i) an artificial genome comprising a nucleotide sequence encoding a microdystrophin protein comprising the amino acid sequence of SEQ ID NO:
- 79, or the reverse complement of the nucleotide sequence;
- (ii) a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and capsid protein operably linked to expression control elements that drive expression of the AAV rep and capsid proteins in the host cell in culture and supply the rep and cap proteins in trans;
- and
- (iii) sufficient adenovirus helper genes to permit replication and packaging of the artificial genome by the AAV capsid proteins;
- and
- (b) recovering recombinant AAV encapsidating the artificial genome from the cell culture.
A method of producing recombinant adeno-associated viruses (AAVs) comprising:
- (a) culturing a host cell containing:
- (i) an artificial genome comprising a nucleotide sequence encoding a microdystrophin protein comprising the amino acid sequence of SEQ ID NO:
- 1, or the reverse complement of the nucleotide sequence;
- (ii) a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and capsid protein operably linked to expression control elements that drive expression of the AAV rep and capsid proteins in the host cell in culture and supply the rep and cap proteins in trans;
- and
- (iii) sufficient adenovirus helper genes to permit replication and packaging of the artificial genome by the AAV capsid proteins;
- and
- (b) recovering recombinant AAV encapsidating the artificial genome from the cell culture.
A method of producing recombinant adeno-associated viruses (AAVs) comprising:
- (a) culturing a host cell containing:
- (i) an artificial genome comprising the nucleotide sequence of SEQ ID NO:
- 53;
- (ii) a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and capsid protein operably linked to expression control elements that drive expression of the AAV rep and capsid proteins in the host cell in culture and supply the rep and cap proteins in trans;
- and
- (iii) sufficient adenovirus helper genes to permit replication and packaging of the artificial genome by the AAV capsid proteins;
- and
- (b) recovering recombinant AAV encapsidating the artificial genome from the cell culture.
Dependencies are inferred from the claim text.