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.

1 - 7 . (canceled) 
     
     
         8 . 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; and (b) chromatographing an AAV producer cell lysate containing rAAV particles on a negatively-charged cation exchange resin, whereby a purified population of rAAV particles is generated. 
     
     
         9 . The method of  claim 8 , wherein step a is performed before step b. 
     
     
         10 . The method of  claim 8 , wherein step b is performed before step a. 
     
     
         11 . The method of  claim 10 , further comprising step (c) of chromatographing the lysate containing rAAV particles on a negatively-charged cation exchange resin, said step performed after steps (a) and (b). 
     
     
         12 . The method of  claim 11 , wherein heparin sulfate is used for step (c). 
     
     
         13 . The method of  claim 8 , further comprising the step of subjecting the producer cells to tangential flow filtration. 
     
     
         14 . The method of  claim 8 , wherein the lysate is subjected to tangential flow filtration. 
     
     
         15 . The method of  claim 14 , wherein tangential flow filtration is performed prior to chromatography. 
     
     
         16 . The method of  claim 14 , wherein tangential flow filtration is performed after chromatography. 
     
     
         17 . The method of  claim 8 , wherein said anion exchange resin is an N-charged amino or imino resin. 
     
     
         18 . The method of  claim 17 , wherein said anion exchange resin is selected from the group consisting of a POROS 50 PI resin, a diethylaminoethyl (DEAE) resin, a trimethylaminoethyl (TMAE) resin, a quaternary amine resin and a polyethylenimine (PEI) resin. 
     
     
         19 . The method of  claim 8 , wherein said cation exchange resin is a sulfo-, phospho- or carboxy-based cationic resin. 
     
     
         20 . The method of  claim 19 , wherein said cation exchange resin is selected from the group consisting of an HS resin, an SP resin, and a carboxymethyl (CM) resin. 
     
     
         21 . The method of  claim 8 , wherein the producer cell is cultured under suspension conditions. 
     
     
         22 . A method of isolating a population of rAAV particles, comprising the steps of: (a) chromatographing AAV producer cell culture supernatant which contains rAAV particles on a positively-charged anion exchange resin; and (b) chromatographing the AAV producer cell culture supernatant containing rAAV particles on a negatively-charged cation exchange resin, whereby a purified population of rAAV particles is generated. 
     
     
         23 . The method of  claim 22 , wherein step a is performed before step b. 
     
     
         24 . The method of  claim 22 , wherein step b is performed before step a. 
     
     
         25 . The method of  claim 24 , further comprising step (c) of chromatographing the lysate containing rAAV particles on a negatively-charged cation exchange resin, said step performed after steps (a) and (b). 
     
     
         26 . The method of  claim 25  wherein heparin sulfate is used for step (c). 
     
     
         27 . The method of  claim 22 , further comprising the step of subjecting the culture supernatant to tangential flow filtration. 
     
     
         28 . The method of  claim 27 , wherein tangential flow filtration is performed prior to chromatography. 
     
     
         29 . The method of  claim 27 , wherein tangential flow filtration is performed after chromatography. 
     
     
         30 . The method of  claim 22 , wherein said anion exchange resin is an N-charged amino or imino resin. 
     
     
         31 . The method of  claim 20 , wherein said anion exchange resin is selected from the group consisting of a POROS 50 PI resin, a diethylaminoethyl (DEAE) resin, a trimethylaminoethyl (TMAE) resin, a quaternary amine resin and a polyethylenimine (PEI) resin. 
     
     
         32 . The method of  claim 22 , wherein said cation exchange resin is a sulfo-, phospho- or carboxy-based cationic resin. 
     
     
         33 . The method of  claim 32 , wherein said cation exchange resin is selected from the group consisting of an HS resin, an SP resin, and a carboxymethyl (CM) resin. 
     
     
         34 . The method of  claim 22 , wherein the producer cell is cultured under suspension conditions. 
     
     
         35 . A method of isolating rAAV particles comprising the steps of (a) chromatographing an AAV producer cell lysate containing rAAV particles on a positively charged anion exchange resin; and (b) subjecting the product of step a to tangential flow filtration to generate a purified population of rAAV. 
     
     
         36 . The method of  claim 35 , wherein step-a is performed before step b. 
     
     
         37 . The method of  claim 35 , wherein step b is performed before step a. 
     
     
         38 . The method of  claim 35 , wherein said anion exchange resin is an N-charged amino or imino resin. 
     
     
         39 . The method of  claim 39 , wherein said anion exchange resin is selected from the group consisting of a POROS 50 PI resin, a diethylaminoethyl (DEAE) resin, a trimethylaminoethyl (TMAE) resin, a quaternary amine resin and a polyethylenimine (PEI) resin. 
     
     
         40 . The method of  claim 35 , wherein the producer cell is cultured under suspension conditions. 
     
     
         41 . A method of isolating 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; and (b) subjecting the product of step a to tangential flow filtration to generate a purified population of rAAV. 
     
     
         42 . The method of  claim 41 , wherein step a is performed before step b. 
     
     
         43 . The method of  claim 41 , wherein step b is performed before step a. 
     
     
         44 . The method of  claim 41 , wherein said anion exchange resin is an N-charged amino or imino resin. 
     
     
         45 . The method of  claim 44 , wherein said anion exchange resin is selected from the group consisting of a POROS 50 PI resin, a diethylaminoethyl (DEAE) resin, a trimethylaminoethyl (TMAE) resin, a quaternary amine resin and a polyethylenimine (PEI) resin. 
     
     
         46 . The method of  claim 41 , wherein the producer cell is cultured under suspension conditions. 
     
     
         47 . 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. 
     
     
         48 . The method of  claim 47 , wherein the producer cell is attachment dependent. 
     
     
         49 . The method of  claim 47 , wherein the producer cell is grown in suspension. 
     
     
         50 . A method of generating a population of recombinant adeno-associated virus (rAAV) particles, comprising the steps of:
 a) providing an AAV producer cell that 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; 
   b) incubating the producer cell provided in step a) under conditions that are permissive for replication of AAV;   c) lysing the producer cell after the incubation of step b) to produce an AAV producer cell lysate;   d) chromatographing the AAV producer cell lysate of step c) on at least one positively-charged anion exchange resin; and   e) purifying the chromatographic fractions containing rAAV particles of step d) by cation exchange chromatography or tangential flow filtration to generate a purified population of rAAV vector particles.   
     
     
         51 . A method of generating a population, of rAAV particles according to  claim 50 , wherein said purifying step e) comprises subjecting the fractions to cation exchange chromatography. 
     
     
         52 . A method of generating a population of rAAV particles according to  claim 50 , wherein said purifying step e) comprises subjecting the fractions to tangential flow filtration. 
     
     
         53 . A method of generating a population of rAAV particles according to  claim 50 , wherein said rAAV pro-vector comprises a heterologous non-AAV polynucleotide flanked by two AAV inverted terminal repeats (ITRs). 
     
     
         54 . A method of generating a population of rAAV particles according to  claim 50 , wherein said AAV producer cell comprises at least one AAV packaging gene that is stably integrated into the genome of said AAV producer cell. 
     
     
         55 . A method of generating a population of rAAV particles according to  claim 50 , wherein said providing of the producer cell in step a) comprises introducing the helper virus into the producer cell already introduced with the AAV packaging gene(s) and the rAAV pro-vector. 
     
     
         56 . A method of generating a population of rAAV particles according to  claim 50 , wherein the providing of the producer cell in step a) comprises introducing the rAAV pro-vector and the helper virus simultaneously or sequentially into the producer cell already introduced with the AAV packaging gene(s). 
     
     
         57 . A method of generating a population of rAAV particles according to  claim 50 , wherein the providing of the producer cell in step a) comprises introducing the AAV packaging gene(s) and the rAAV pro-vector simultaneously or sequentially into the host cell already introduced with the helper virus. 
     
     
         58 . A method of generating a population of rAAV particles according to  claim 50 , wherein said AAV producer cell comprises an AAV rep gene and an AAV cap gene. 
     
     
         59 . A method of generating a population of rAAV particles according to  claim 50 , wherein said AAV rep gene and AAV cap gene are stably integrated into the genome of said AAV producer cell. 
     
     
         60 . A method of generating a population of rAAV particles according to  claim 50 , wherein the providing of the producer cell in step a) comprises introducing into the producer cell at least one AAV split packaging gene. 
     
     
         61 . A Method of generating a population of rAAV particles according to  claim 50 , wherein said helper virus is an adenovirus. 
     
     
         62 . A method of generating a population of rAAV particles according to  claim 50 , wherein said helper virus is a temperature-sensitive helper virus and said step of incubating the producer cell is conducted at a temperature that is permissive for replication of AAV but non-permissive for replication of the temperature-sensitive helper virus. 
     
     
         63 . A method of generating a population of rAAV particles according to  claim 50 , wherein said helper virus is a temperature-sensitive adenovirus. 
     
     
         64 . A method of generating a population of rAAV particles according to  claim 50 , wherein said helper virus is adenovirus Ad-ts149. 
     
     
         65 . A method of generating a population of rAAV particles according to  claim 50 , wherein said AAV producer cell lysate is also affinity purified on a resin having a ligand that is specific for one or more surface molecules present on AAV. 
     
     
         66 . A method of generating a population of rAAV particles according to  claim 65 , wherein the affinity purification is conducted after ion-exchange chromatography. 
     
     
         67 . A method of generating a population of rAAV particles according to  claim 65 , wherein said ligand is an antibody that is specific for a surface molecule present on AAV. 
     
     
         68 . A method of generating a population of rAAV particles according to  claim 50 , wherein the AAV producer cells of step b) are concentrated prior to lysis. 
     
     
         69 . A method of generating a population of rAAV particles according to  claim 68 , wherein the AAV producer cells of step b) are concentrated by centrifugation or by tangential flow filtration prior to lysis. 
     
     
         70 . A method of generating a population of rAAV particles according to  claim 50 , wherein said step of lysing the AAV producer cell is conducted by subjecting the cells to a lytic technique selected from the group consisting of microfluidization, sonication, and freeze-thawing 
     
     
         71 . A method of generating a population of rAAV particles according to  claim 70 , wherein said step of lysing the AAV producer cell is conducted by subjecting the cells to microfluidization. 
     
     
         72 . A method of generating a population of rAAV particles according to  claim 50 , wherein the AAV producer cell lysate of step c) is treated with a nuclease prior to chromatography. 
     
     
         73 . A method of generating a population of rAAV particles according to  claim 72 , wherein said nuclease is Benzonase. 
     
     
         74 . A method of generating a population of rAAV particles according to  claim 50 , wherein the AAV producer cell lysate of step c) is clarified prior to chromatography. 
     
     
         75 . A method of generating a population of rAAV particles according to  claim 74 , wherein the AAV producer cell lysate of step c) is clarified by filtration or centrifugation prior to chromatography. 
     
     
         76 . A method of generating a population of rAAV particles according to  claim 50 , wherein the AAV producer cells are concentrated prior to lysis, resuspended in a buffer comprising saline at an ionic strength at least that of a 50 mM NaCl solution, lysed, and then clarified by filtration prior to chromatography. 
     
     
         77 . A method of generating a population of rAAV particles according to  claim 51 , wherein chromatographic fractions containing rAAV particles are concentrated by filtration or centrifugation after elution from the chromatographic resin. 
     
     
         78 . A method of generating a population of rAAV particles according to  claim 51 , wherein chromatographic fractions containing rAAV particles are concentrated by tangential flow filtration 
     
     
         79 . A method of generating a population of rAAV particles according to  claim 50 , wherein said anion exchange resin is an N-charged amino or amino resin. 
     
     
         80 . A method of generating a population of rAAV particles according to  claim 50 , wherein said anion exchange resin is selected from the group consisting of a POROS 50 PI resin, a diethylaminoethyl (DEAE) resin, a trimethylaminoethyl (TMAE) resin, a quaternary amine resin and a polyethylenimine (PEI) resin. 
     
     
         81 . A method of generating a population of rAAV particles according to  claim 51 , wherein said cation exchange resin is a sulfo-, phospho- or carboxy-based cationic, resin. 
     
     
         82 . A method of generating a population of rAAV particles according to  claim 51 , wherein said cation exchange resin is selected from the group consisting of an HS resin, an SP resin, and a carboxymethyl (CM) resin. 
     
     
         83 . A method of generating a population of rAAV particles according to  claim 50 , wherein the producer cell of step a) is an attachment-dependent mammalian cell line. 
     
     
         84 . A method of generating a population of rAAV particles according to  claim 50 , wherein said step b) of incubating the producer cell provided in step a) conducted in a vessel selected from the group consisting of a tissue culture flask, a roller bottle, a spinner flask, a tank reactor, a fermentor, and a bioreactor. 
     
     
         85 . A method of generating a population of rAAV particles according to  claim 50 , wherein said step b) of incubating the producer cell provided in step a) is conducted using a microcarrier. 
     
     
         86 . A method of generating a population of rAAV particles according to  claim 50 , wherein said vessel is a hollow-fiber, packed-bed or fluidized-bed bioreactor. 
     
     
         87 . A method of generating a population of rAAV particles according to  claim 50 , wherein the producer cell of step a) is a suspension-adapted mammalian cell line. 
     
     
         88 . A method of generating a population of rAAV particles according to  claim 50 , wherein said step b) of incubating the producer cell provided in step a) is conducted in a vessel selected from the group consisting of a spinner flask, a tank reactor and an air lift fermentor. 
     
     
         89 . A method of generating a population of rAAV particles according to  claim 50 , wherein said step b) of incubating the producer cell provided in step a) is performed in rAAV medium essentially as shown in Table 2. 
     
     
         90 . A method of generating a population of rAAV particles according to  claim 50 , wherein the producer cells are 293 N3s cells or HeLa S3 cells. 
     
     
         91 . A method of generating a population of rAAV particles according to  claim 50 , wherein step b) is conducted for at least 5 days. 
     
     
         92 . A method of generating a population of rAAV particles according to  claim 50 , wherein step b) of incubating the producer cell is conducted in a multi-liter bioreactor and wherein at least about 10 9  replicative units of rAAV per liter of bioreactor volume are isolated after step e). 
     
     
         93 . A method of generating a population of recombinant adeno-associated virus (rAAV) particles, comprising the steps of:
 a) providing an AAV producer cell that 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; 
   b) subjecting the producer cell provided in step a) to a sub-lethal stress; and   c) incubating the stressed producer cell of step b) under conditions that are permissive for replication of AAV.   
     
     
         94 . A method of generating a population of rAAV particles according to  claim 93 , wherein said sub-lethal stress is selected from the group consisting of a nutritional stress, an osmotic stress, a pH stress, a temperature stress, an aerobic stress, a mechanical stress, a radiational stress and a toxic stress. 
     
     
         95 . A method of generating a population of rAAV particles according to  claim 93 , wherein said sub-lethal stress is a nutritional stress. 
     
     
         96 . A method of generating a population of rAAV particles according to  claim 93 , wherein said sub-lethal stress is an osmotic stress. 
     
     
         97 . A method of generating a population of rAAV particles according to  claim 93 , wherein said sub-lethal stress is a pH stress. 
     
     
         98 . A method of generating a population of rAAV particles according to  claim 97 , wherein said pH stress comprises raising the pH to above pH 7.2. 
     
     
         99 . A method of generating a population of rAAV particles according to  claim 97 , wherein said pH stress comprises elevating the pH to at least 7.4, and wherein the majority of the AAV particles produced are released into the supernatant. 
     
     
         100 . A method of generating a population of rAAV particles according to  claim 97 , wherein said pH stress comprises elevating the pH to about 8.0. 
     
     
         101 . A method of generating a population of rAAV particles according to  claim 93 , wherein said sub-lethal stress is a temperature stress. 
     
     
         102 . A method of generating a population of rAAV particles according to  claim 93 , wherein said sub-lethal stress is an aerobic stress. 
     
     
         103 . A method of generating a population of rAAV particles according to  claim 93 , wherein said sub-lethal stress is a mechanical stress. 
     
     
         104 . A method of generating a population of rAAV particles according to  claim 93 , wherein said sub-lethal stress is a radiational stress. 
     
     
         105 . A method of generating a population of rAAV particles according to  claim 93 , wherein said sub-lethal stress is a toxic stress. 
     
     
         106 . A method of generating a population of rAAV particles according to  claim 95 , wherein said nutritional stress is imposed by culturing the producer cells in a medium that is deficient in one or more amino acids. 
     
     
         107 . A method of generating a population of rAAV particles according to  claim 95 , wherein said nutritional stress is imposed by culturing the producer cells in a medium that is deficient in aspartic acid. 
     
     
         108 . A method of generating a population of rAAV particles according to  claim 95 , wherein said nutritional stress is imposed by culturing the producer cells in a medium that is deficient in glutamic acid. 
     
     
         109 . A method of generating a population of rAAV particles according to  claim 108 , wherein the deficient medium contains less than 10 μmol/L of aspartic acid. 
     
     
         110 . A method of generating a population of rAAV particles according to  claim 95 , wherein the deficient medium contains less than 2 μmol/L of glutamic acid. 
     
     
         111 . A method of generating a population of rAAV particles according to  claim 95 , wherein said nutritional stress is imposed by culturing the producer cells in a medium that is deficient in serum. 
     
     
         112 . A method of generating a population of rAAV particles according to  claim 95 , wherein the cells are subjected to said nutritional stress by introducing the cells into a nutritionally deficient medium. 
     
     
         113 . A method of generating a population of rAAV particles according to  claim 95 , wherein the cells are subjected to said nutritional stress by culturing the cells in a medium until the medium becomes nutritionally deficient. 
     
     
         114 . A method of generating a population of rAAV particles according to  claim 93 , wherein said purified population of rAAV vector particles is substantially free of replication-competent AAV and of helper virus and cellular proteins. 
     
     
         115 . A method of generating a population of rAAV particles according to  claim 93 , in which elution from the chromatographic resin is conducted by increasing the salt concentration and chromatographic eluants comprising rAAV particles are subsequently treated to reduce the effective salt concentration by dilution, dialysis, diafiltration or concentration. 
     
     
         116 . A method of generating a population of rAAV particles according  claim 93 , including the step of subjecting a fraction comprising AAV particles to heparin sulfate chromatography. 
     
     
         117 . A method of generating a population of recombinant adeno-associated virus (rAAV) particles, comprising the steps of:
 a) providing an AAV producer cell that 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; 
   b) incubating the producer cell provided in step a) under conditions that are permissive for replication of AAV and which comprise inducing a sub-lethal stress in the AAV producer cell;   c) lysing the producer cell after the incubation of step b) to produce an AAV producer cell lysate; and   d) purifying the AAV producer cell lysate to generate a population of recombinant adeno-associated virus (rAAV) particles.   
     
     
         118 . A method of generating a population of rAAV particles according to  claim 117 , wherein said purifying step d) comprises chromatographing the AAV producer cell lysate of step c) 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. 
     
     
         119 . The method of  claim 118 , wherein said purifying step d) comprises chromatographing the AAV producer cell lysate of step c) on at least one negatively charged cation exchange resin followed by purifying on an anion exchange resin. 
     
     
         120 . A method of generating a population of rAAV particles according to  claim 117 , wherein said purifying step d) comprises chromatographing the AAV producer cell lysate of step c) on a positively-charged anion exchange resin followed by tangential flow filtration to generate a purified population of rAAV vector particles. 
     
     
         121 . A method of generating a population of rAAV particles according to  claim 117 , wherein said rAAV pro-vector comprises a heterologous non-AAV polynucleotide flanked by two AAV inverted terminal repeats (ITRs). 
     
     
         122 . A method of generating a population of rAAV particles according to  claim 117 , wherein said AAV producer cell comprises at least one AAV packaging gene that is stably integrated into the genome of said AAV producer cell. 
     
     
         123 . A method of generating a population of rAAV particles according to  claim 117 , wherein said AAV producer cell comprises an AAV rep gene and an AAV cap gene. 
     
     
         124 . A method of generating a population of rAAV particles according to  claim 117 , wherein said helper virus is adenovirus. 
     
     
         125 . A method of generating a population of virus particles, comprising the step of:
 a) incubating a producer cell in a cell culture medium under conditions comprising a condition that promotes release of virus particles, whereby virus particles are released from the producer cell into the culture medium.   
     
     
         126 . The method of  claim 125 , wherein the virus is recombinant adeno-associated virus (rAAV), and wherein the 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) 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.   
     
     
         127 . The method of  claim 126 , wherein the condition that promotes release of virus particles is pH. 
     
     
         128 . The method of  claim 127 , wherein the pH is about 7.4 to about 8.0. 
     
     
         129 . The method of  claim 128 , wherein the pH is about 8.0. 
     
     
         130 . The method of  claim 126 , wherein the condition that promotes virus release is osmolality. 
     
     
         131 . The method of  claim 130 , wherein the osmolality is about 300 mOsm. 
     
     
         132 . The method of  claim 131 , wherein the pH is about 8.00. 
     
     
         133 . The method of  claim 132 , wherein the pH is maintained at about 8.00. 
     
     
         134 . The method of  claim 133 , wherein the pH is adjusted by using a sodium salt. 
     
     
         135 . The method of  claim 131 , wherein the osmolality is adjusted using an ionic salt. 
     
     
         136 . The method of  claim 135 , wherein the ionic salt is NaCl. 
     
     
         137 . The method of  claim 130 , wherein the initial osmolality of the cell culture is about 300 mOsm. 
     
     
         138 . The method of  claim 137 , wherein the pH is about 8.00. 
     
     
         139 . The method of  claim 137 , wherein the osmolality is adjusted using an ionic salt. 
     
     
         140 . The method of  claim 139 , wherein the ionic salt is NaCl. 
     
     
         141 . The method of  claim 126 , wherein the condition that promotes virus release is temperature. 
     
     
         142 . The method of  claim 141 , wherein the temperature is about 37° C. to about 40° C. 
     
     
         143 . The method of  claim 142 , wherein the temperature is about 39° C. 
     
     
         144 . The method of  claim 133 , wherein the temperature is about 39° C. 
     
     
         145 . The method of  claim 144 , wherein the osmolality is about 300 to about 350 mOsm. 
     
     
         146 . The method of  claim 137 , wherein the temperature is about 39° C. 
     
     
         147 . The method of  claim 126 , wherein the condition that promotes release of virus particles is conductivity. 
     
     
         148 . The method of  claim 147 , wherein the conductivity is at least about 10 mS. 
     
     
         149 . The method of  claim 147 , wherein the conductivity is about 10 mS. 
     
     
         150 . The method of  claim 147 , wherein the conductivity is about 15 mS. 
     
     
         151 . The method of  claim 147 , wherein the conductivity is adjusted using a sodium salt. 
     
     
         152 . The method of  151 , wherein the sodium salt is NaCl. 
     
     
         153 . The method of  claim 126 , wherein the condition that promotes release of virus particles is an agent or condition that permeabilizes the producer cell. 
     
     
         154 . The method of  claim 133 , wherein producer cells are cultured for about 48 to about 96 hours after introduction of helper virus function. 
     
     
         155 . The method of  claim 143 , wherein producer cells are cultured for about 48 to about 96 hours after introduction of helper virus function. 
     
     
         156 . The method of  claim 126 , wherein helper virus function is provided by helper-virus. 
     
     
         157 . A method of generating a population of rAAV particles according to  claim 156 , wherein said helper virus is an adenovirus. 
     
     
         158 . The method of  claim 126 , further comprising the step of (b) harvesting the viral particles from the cell culture medium, thereby obtaining a population of rAAV particles. 
     
     
         159 . The method of  claim 158 , further comprising the steps of:
 c) chromatographing the AAV producer cell culture medium on a positively-charged anion exchange resin; and   d) purifying the chromatographic fractions containing rAAV particles of step c) by cation exchange chromatography or tangential flow filtration to generate a purified population of rAAV vector particles.   
     
     
         160 . The method of  claim 159 , wherein step (d) is cation exchange chromatography. 
     
     
         161 . The method of  claim 158 , further comprising the steps of
 c) chromatographing the AAV producer cell culture medium on a negatively-charged cation exchange resin;   d) purifying the chromatographic fractions containing rAAV particles of step c) by anion exchange chromatography; and   e) purifying the chromatographic fractions containing rAAV particles of step d) by cation exchange chromatography to generate a purified population of rAAV vector particles.   
     
     
         162 . The method of  claim 161 , wherein the chromatography of step e) is performed using heparin sulfate. 
     
     
         163 . A method of generating a population of rAAV particles according to  claim 126 , wherein said rAAV vector comprises a heterologous non-AAV polynucleotide flanked by two AAV inverted terminal repeats (ITRs). 
     
     
         164 . A method of generating a population of rAAV particles according to  claim 126 , wherein said AAV producer cell comprises at least one AAV packaging gene that is stably integrated into the genome of said AAV producer cell. 
     
     
         165 . A method of generating a population of rAAV particles according to  claim 126 , wherein said AAV producer cell comprises an AAV rep gene and an AAV cap gene. 
     
     
         166 . A method of generating a population of rAAV particles according to  claim 165 , wherein said AAV rep gene and AAV cap gene are stably integrated into the genome of said AAV producer cell. 
     
     
         167 . A method of generating a population of rAAV particles according to  claim 126 , wherein the producer cell is an attachment-dependent mammalian cell line. 
     
     
         168 . A method of generating a population of rAAV particles according to  claim 126 , wherein the producer cell is a suspension-adapted mammalian cell line. 
     
     
         169 - 177 . (canceled)