Raav expression systems for genetic modification of specific capsid proteins
AssigneeUniversity of Florida Research Foundation, Inc.
InventorsKenneth H. Warrington, Shaun R. Opie, Nicholas Muzyczka
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Abstract
Disclosed are improved recombinant adeno-associated viral (rAAV) vectors having mutations in one or more capsid proteins. Exemplary vectors are provided that have altered affinity for heparin or heparin sulfate, as well as vectors, expression systems, and rAAV virions that lack functional VP2 protein expression, but are nevertheless, fully virulent. Also provided by the invention are rAAV vector-based compositions, virus particles, host cells, and pharmaceutical formulations that comprise them useful in the expression of selected therapeutic proteins, polypeptides, peptides, antisense oligonucleotides and/or ribozymes in selected mammals, including organs, tissues, and human host cells.
Claims28
A composition, comprising:
- (a) a first expression vector comprising a coding sequence of a first AAV capsid protein under control of an expression control sequence;
- and
- (b) a second expression vector comprising coding sequences for a second AAV capsid protein under control of an expression control sequence and a third AAV capsid protein under control of an expression control sequence;
- wherein the first and the second expression vectors are not on the same nucleic acid segment and wherein the first, the second, and the third capsid proteins are distinct capsid proteins selected from the group consisting of an AAV2 Vp1 protein, an AAV2 Vp2 protein and an AAV2 Vp3 protein, and further wherein the composition is modified 1) by a mutation in the coding sequence for the Vp2 protein such that the coding sequence for Vp2 does not express a functional AAV Vp2 capsid protein and/or 2) at least one mutation such that binding to HPSG is altered, impaired, or prevented wherein the at least one mutation is an arginine-to-alanine mutation or an arginine-to-lysine mutation at an amino acid residue R487, R585, or R588, wherein binding by the mutated capsid protein.
An infectious recombinant adeno-associated virus virion comprising:
- (a) a first expression vector comprising a coding sequence of a first AAV capsid protein under control of an expression control sequence;
- and
- (b) a second expression vector comprising a coding sequence of a second AAV capsid protein under control of an expression control sequence and a third AAV capsid protein under control of an expression control sequence;
- wherein the first and the second expression vectors are not on the same nucleic acid segment and wherein the first, the second, and the third capsid proteins are distinct capsid proteins selected from the group consisting of an AAV2 Vp1 protein, an AAV2 Vp2 protein and an AAV2 Vp3 protein, and further wherein the composition is modified 1) by a mutation in the coding sequence for the Vp2 protein such that the coding sequence for Vp2 does not express a functional AAV Vp2 capsid protein and/or 2) at least one mutation such that binding to HPSG is altered, impaired, or prevented wherein the at least one mutation is an arginine-to-alanine mutation or an arginine-to-lysine mutation at an amino acid residue corresponding to R487, R585, or R588, of an AAV2 capsid protein.
Dependencies are inferred from the claim text.