US 6,995,006 B2 · Methods for generating high titer helper-free preparations of released recombinant AAV vectors

Abstract

This invention provides methods and compositions for producing high titer, substantially purified preparations of recombinant adeno-associated virus (AAV) that can be used as vectors for gene delivery. At the onset of vector production, AAV producer cells of this invention typically comprise one or more AAV packaging genes, an AAV vector comprising a heterologous (i.e. non-AAV) transgene of interest, and a helper virus such as an adenovirus. The AAV vector preparations produced are generally replication incompetent but are capable of mediating delivery of a transgene of interest (such as a therapeutic gene) to any of a wide variety of tissues and cells. The AAV vector preparations produced according to this invention are also substantially free of helper virus as well as helper viral and cellular proteins and other contaminants. The invention described herein provides methods of producing rAAV particles by culturing producer cells under conditions, such as temperature and pH, that promote release of virus. Also provided is a quantitative, high-throughput assay useful in the assessment of viral infectivity and replication, as well as in the screening of agent that affect viral infectivity and/or replication.

Claims53

Dependent claims

A method of generating a population of rAAV particles comprising culturing a producer cell under a stress condition, said producer cell comprising (i) one or more AAV packaging genes, wherein each said AAV packaging gene encodes an AAV replication or encapsidation protein;

(ii) a recombinant AAV (rAAV) vector that comprises a heterologous non-AAV polynucleotide flanked by at least one AAV inverted terminal repeat (ITR);
and (iii) helper virus function for AAV, whereby about two-fold or more rAAV particles are produced compared to a producer cell not grown under said stress condition.

A method of generating a population of recombinant adeno-associated virus (rAAV) particles, comprising the steps of:

a) incubating an AAV producer cell under conditions that are permissive for replication of AAV and which comprise inducing a stress in the AAV producer cell;
wherein said AAV producer cell comprises:
(i) one or more AAV packaging genes, wherein each said AAV packaging gene encodes an AAV replication or encapsidation protein;
(ii) a recombinant AAV (rAAV) pro-vector that comprises a heterologous non-AAV polynucleotide flanked by at least one AAV inverted terminal repeat (ITR);
and
(iii) a helper virus for AAV or a polynucleotide sequence of said helper virus that encodes at least one helper virus function;
whereby about two-fold or more rAAV particles are produced compared to a producer cell not grown under said stress condition;
b) lysing the producer cell after the incubation of step a) to produce an AAV producer cell lysate;
and
c) purifying the AAV producer cell lysate to generate a population of recombinant adeno-associated virus (rAAV) particles.

7 more elements

Dependencies are inferred from the claim text.

Facts

Application

10/020,482Filed 30 Oct 2001 · division

Priority

5 Sep 1997

Published

US 2002/0160501 A131 Oct 2002

Granted

7 Feb 2006

Term ends

Not estimatedThe source records don’t support an estimate.

Classification26 codes

C07D 207/32
Having two double bonds between ring members or between ring members and non-ring members: with only hydrogen atoms, hydrocarbon or substituted…
A61K 48/0091
Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy:…
C07C 2/32
With hydrides or organic compounds: as complexes, e.g. acetyl-acetonates
C07D 207/34
Having two double bonds between ring members or between ring members and non-ring members: with hetero atoms or with carbon atoms having three…

Family

Application60/084,193Us provisional application · filed 5 Sep 1997

PCT/US98/18600Parent of this continuation-in-part · filed 4 Sep 1998

Application60/071,733Us provisional application · filed 16 Nov 1998

Application60/123,685Us provisional application · filed 10 Mar 1999

Application09/526,333Parent of this division · filed 15 Mar 2000

US 2002/0160501 A1Pre-grant publication · published 31 Oct 2002

US 6,566,118 B1Parent of this division

Application09/142,474Parent of this continuation-in-part

Application09/526,333Parent of this continuation-in-part

US 6,995,006 B2This patent

From the US filing history. Foreign family members aren’t listed.

Methods for generating high titer helper-free preparations of released recombinant AAV vectors

Life of this patent

Methods for generating high titer helper-free preparations of released recombinant AAV vectors

Life of this patent

Abstract

Claims53