Transcriptionally-activated AAV inverted terminal repeats (ITRs) for use with recombinant AAV vectors
AssigneeTargeted Genetics Corporation
InventorAndrew L. Feldhaus
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Abstract
This invention provides transcriptionally-activated AAV ITRs (inverted terminal repeats) which are small and transcriptionally active and uses thereof to optimize the expression of relatively large transgenes packaged in recombinant AAV vectors.
Claims35
A polynucleotide comprising a transcriptionally-activated Adeno-associated virus (AAV) inverted terminal repeat (ITR), wherein the transcriptionally-activated ITR is less than about 400 bp in length and comprises a heterologous transcriptionally active element, and wherein the transcriptionally-activated ITR exhibits at least about a two-fold increase in transcriptional activity relative to a wild-type ITR under conditions permissive for transcription.
A polynucleotide comprising, in order:
- a first ITR which is a transcriptionally-activated ITR, wherein the transcriptionally-activated ITR is less than about 400 bp in length and comprises a transcriptionally active element, and wherein the transcriptionally-activated ITR exhibits at least a two-fold increase in transcriptional activity relative to a wild-type ITR under conditions permissive for transcription;
- and
- a second ITR selected from the group consisting of a wild-type ITR, a transcriptionally-activated ITR, a D sequence, a trs, or a portion of a wild-type ITR.
A polynucleotide according to any of the previous claims further comprising a gene operably linked to the transcriptionally-activated ITR.
An AAV viral particle comprising a polynucleotide of any of the previous claims.
A method of packaging a recombinant AAV vector, comprising the steps of:
- a) providing a mammalian cell;
- b) introducing a recombinant AAV vector, said vector comprising:
- a first ITR which is a transcriptionally-activated ITR, wherein the transcriptionally-activated ITR is less than about 400 bp in length and comprises a transcriptionally active element, and wherein the transcriptionally-activated ITR exhibits at least a two-fold increase in transcriptional activity relative to a wild-type ITR under conditions permissive for transcription;
- and a second ITR selected from the group consisting of a wild-type ITR, a transcriptionally-activated ITR, a D sequence, a trs, or a portion of a wild-type ITR;
- c) providing Rep and Cap proteins within the cell;
- d) providing helper virus or helper virus functions;
- and
- e) incubating the cell under conditions suitable for replication and packaging of the AAV vector.
Dependencies are inferred from the claim text.