Recombinant aav constructs for increased transgene expression
AssigneeWisconsin Alumni Research Foundation
InventorsKinjal Majumder, Rhiannon Abrahams
Life of this patent
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Abstract
Provided herein are constructs for producing recombinant adeno-associated virus (rAAV) vectors comprising rAAV genomes that can recombine to produce oligomerized rAAV genomes that persist longer in a cell than traditional rAAV genomes, and that comprise relatively large transgenes. The constructs comprise either a 5′ recombinant junction sequence, a 3′ recombinant junction sequence, or both, such that a recombination event will link the rAAV genomes together. Also provided are modified rAAV vectors comprising these constructs, methods for producing the vectors, and methods of using the vectors to deliver transgenes to a subject.
Claims28
A construct for producing a recombinant adeno-associated virus (rAAV) vector, the construct comprising from 5′ to 3′:
- a 5′ inverted terminal repeat (ITR), a first recombinant junction sequence, a promoter operably linked to at least a portion of a transgene, and a 3′ ITR.
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A system for expressing a transgene, the system comprising:
- a first construct for producing a first recombinant adeno-associated virus (rAAV) vector comprising, from 5′ to 3′:
- a 5′ inverted terminal repeat (ITR), a promoter operably linked to a 5′ portion of the transgene, a second recombinant junction sequence, and a 3′ ITR, wherein the second recombinant junction sequence is between the transgene and the 3′ ITR and may overlap with the 3′ ITR;
- and
- a second construct for producing a second rAAV vector comprising, from 5′ to 3′:
- the 5′ ITR, a first recombinant junction sequence, a 3′ portion of the transgene, a polyadenylation site, and the 3′ ITR.
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19-21. (canceled)
The system of claim 21, wherein the 5′ portion of the transgene and the 3′ portion of the transgene each comprise between about 200 nucleotides and about 4000 nucleotides.
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A system for expressing a transgene, the system comprising:
- a first construct for producing a first recombinant adeno-associated virus (rAAV) vector, the first construct comprising, from 5′ to 3′:
- a 5′ ITR, a promoter operably linked to a 5′ portion of the transgene, a first 3′ recombinant junction sequence, and a 3′ ITR;
- a second construct for producing a second rAAV vector, the second construct comprising, from 5′ to 3′:
- the 5′ ITR, a second 5′ recombinant junction sequence, a 3′ portion of the transgene, a polyadenylation site, and the 3′ ITR;
- and
- an intervening construct for producing an intervening rAAV vector, the intervening construct comprising, from 5′ to 3′:
- the 5′ ITR, a first 5′ recombinant junction sequence, an internal portion of the transgene, a second 3′ recombinant junction sequence, and the 3′ ITR;
- wherein the first 5′ recombinant junction sequence and the second 5′ recombination sequence are different;
- wherein the first 3′ recombinant junction sequence and the second 3′ recombinant junction sequence are different;
- wherein the first 5′ recombinant junction sequence and the first 3′ recombinant junction sequence are such that when recombined, the 5′ portion of the transgene is in frame with the internal portion of the transgene;
- and
- wherein the second 3′ recombinant junction sequence and the second 5′ recombinant junction sequence are such that when recombined, the internal portion of the transgene is in frame with the 3′ portion of the transgene.
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Dependencies are inferred from the claim text.