Methods for generating high titer helper-free preparations of released recombinant AAV vectors
First Claim
1. A method of isolating a population of rAAV particles, comprising the steps of:
- (a) chromatographing an AAV producer cell lysate containing rAAV particles on a positively-charged anion exchange resin;
or chromatographing an AAV producer cell lysate containing rAAV particles on a negatively-charged cation exchange resin, and collecting a fraction containing rAAV particles; and
(b) chromatographing the fraction of step (a) on exchange resin opposite in charge to that used in step (a) and collecting a fraction containing rAAV particles;
whereby a purified population of rAAV particles is generated.
0 Assignments
0 Petitions
Accused Products
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.
100 Citations
81 Claims
-
1. A method of isolating a population of rAAV particles, comprising the steps of:
-
(a) chromatographing an AAV producer cell lysate containing rAAV particles on a positively-charged anion exchange resin;
or chromatographing an AAV producer cell lysate containing rAAV particles on a negatively-charged cation exchange resin, and collecting a fraction containing rAAV particles; and
(b) chromatographing the fraction of step (a) on exchange resin opposite in charge to that used in step (a) and collecting a fraction containing rAAV particles;
whereby a purified population of rAAV particles is generated. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 70, 71, 72, 73, 74, 75)
-
-
14. A method of isolating a population of rAAV particles, comprising the steps of:
-
(a) chromatographing an AAV producer cell culture supernatant which contains rAAV particles on a positively-charged anion exchange resin or chromatographing an AAV producer cell culture supernatant containing rAAV particles on a negatively-charged cation exchange resin, and collecting a fraction containing rAAV particles; and
(b) chromatographing the fraction of step (a) on exchange resin opposite in charge to that used in step (a) and collecting a fraction containing rAAV particles;
whereby a purified population of rAAV particles is generated. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 76, 77, 78, 79, 80, 81)
-
-
26. 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;
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) a helper virus for AAV;
b) lysing the producer cell after the incubation of step a) to produce an AAV producer cell lysate;
c) chromatographing the AAV producer cell lysate of step b) on at least one positively-charged anion exchange resin; and
d) purifing the chromatographic fractions containing rAAV particles of step c) by cation exchange chromatography to generate a purified population of rAAV vector particles. - View Dependent Claims (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66)
-
-
67. 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 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) a helper virus for AAV;
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, wherein said purifying step comprises chromatographing the AAV producer cell lysate of step b) on at least one positively-charged anion exchange resin followed by purifying on either a cation exchange resin or by tangential flow filtration to generate a purified population of rAAV vector particles. - View Dependent Claims (68, 69)
-
Specification