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

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.

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.

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.