US 5,658,776 A · Generation of high titers of recombinant AAV vectors

Abstract

Adeno-associated virus (AAV) vectors may have utility for gene therapy but heretofore a significant obstacle has been the inability to generate sufficient quantifies of such recombinant vectors in amounts that would be clinically useful for human gene therapy application. Stable, helper-free AAV packaging cell lines have been elusive, mainly due to the activities of Rep protein, which down-regulates its own expression and reverses cellular immortalization. This invention provides packaging systems and processes for packaging AAV vectors that efficiently circumvent these problems by replacing the AAV p5 promoter with a heterologous promoter and that allow for substantially increased packaging efficiency.

Claims15

Dependent claims

A process for the generation of high titers of AAV recombinant vectors comprising:

(a) providing cells containing at least one intact copy of a stably integrated recombinant AAV vector, wherein the AAV vector comprises AAV inverted terminal repeat (ITR) regions and a transcription promoter operably linked to a target polynucleotide, and wherein the expression of the rep gene is limiting in said cells;
(b) providing an AAV packaging plasmid that allows expression of the product of the rep gene, wherein in the plasmid the rep gene is operably linked to a heterologous promoter, and wherein the packaging plasmid lacks overlapping homology with AAV sequences in the vector in the cell provided in (a);
(c) inserting the AAV packaging plasmid into the cell provided in and incubating the cell under conditions that allow replication and packaging of AAV;
and
(d) isolating recombinant AAV vectors produced in step (c).

2 more elements

Dependencies are inferred from the claim text.

Generation of high titers of recombinant AAV vectors

Life of this patent

Generation of high titers of recombinant AAV vectors

Life of this patent

Abstract

Claims15