Compositions and methods for generating adeno-associated viral vectors with undetectable capsid gene contamination
AssigneeFred Hutchinson Cancer Research Center
InventorsArthur Dusty Miller, Christine L. Halbert, Michael J. Metzger
Life of this patent
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Abstract
In one aspect, the present invention provides an intron-modified cap expression cassette useful for generating adeno-associated virus (AAV) vector particles. In another aspect, the present invention provides a method of reducing the immune response in a mammalian subject undergoing treatment with an AAV vector.
Claims22
An intron-modified cap expression cassette useful for generating adeno-associated virus (AAV) vector particles, said expression cassette comprising:
- (a) a nucleic acid molecule comprising a nucleotide sequence encoding an AAV capsid protein operably linked to a promoter, wherein the encoded AAV capsid protein is capable of packaging AAV vectors in the presence of rep and adeno helper functions, and
- (b) at least one heterologous intron sequence operably linked to the nucleotide sequence encoding the AAV capsid protein;
- wherein the total length of the expression cassette is at least 5 kilobases.
A method of generating an expression cassette for producing adeno-associated virus (AAV) vectors, comprising inserting at least one heterologous intron sequence into a nucleic acid molecule comprising a nucleotide sequence encoding an AAV capsid protein operably linked to a promoter, wherein the total length of the expression cassette is at least 5 kilobases.
A complementation assay for measuring the presence or amount of cap gene contamination in a preparation of recombinant AAV vector virions comprising:
- (a) infecting a host cell with a test preparation of AAV vector, wherein the host cell comprises:
- (i) an indicator AAV vector with a detectable marker,
- (ii) a rep expression vector;
- and
- (iii) adenovirus helper functions;
- (b) culturing the host cell for a period of time sufficient to generate AAV vector virions from the indicator AAV vector;
- (c) contacting an indicator cell with cell lysate obtained from the host cells cultured in accordance with step (b);
- and
- (d) determining the presence or amount of indicator cells that are positive for the detectable marker, wherein a positive result indicates that the test preparation of AAV vector virions contains cap gene contamination.
A complementation assay for measuring the presence or amount of rep gene contamination in a preparation of AAV vector virions comprising:
- (a) infecting a host cell with a test preparation of AAV vector, wherein the host cell comprises:
- (i) an indicator AAV vector with a detectable marker,
- (ii) a cap expression vector;
- and
- (iii) adenovirus helper functions;
- (b) culturing the host cell for a period of time sufficient to generate AAV vector virions from the indicator AAV vector;
- (c) contacting an indicator cell with cell lysate obtained from the host cells cultured in accordance with step (b);
- and
- (d) determining the presence or amount of indicator cells that are positive for the detectable marker, wherein a positive result indicates that the test preparation of AAV vector virions contains rep gene contamination.
Dependencies are inferred from the claim text.