Recombinant adeno-associated virus particle purification comprising an affinity purification step

Abstract

The invention describes a method for obtaining purified recombinant Adeno-Associated Virus particles (rAAV), comprising the steps of: a) performing a depth filtration of a starting material previously obtained from cells producing rAAV particles, the said starting material being selected in a group comprising a cell lysate and a culture supernatant, whereby a rAAV-containing clarified composition is provided; b) submitting the rAAV-containing clarified composition to an affinity purification step, whereby a first rAAV enriched composition is provided; c) submitting the first rAAV enriched composition at least once to: c1) a step of anion-exchange chromatography on a chromatographic support wherein elution is performed by using a salt gradient, preferably a linear salt gradient, and wherein the rAAV-containing fraction is collected, whereby a second rAAV enriched composition is provided; or c2) a step of density gradient centrifugation, wherein the rAAV-containing fraction is collected, whereby a second rAAV enriched composition is provided; d) submitting the second rAAV enriched composition to a step of tangential flow filtration, whereby purified recombinant Adeno-Associated Virus particles (rAAV) are provided.

1 - 16 . (canceled) 
     
     
         17 . A method for obtaining purified recombinant Adeno-Associated Virus (rAAV) particles, comprising the steps of:
 i. performing a depth filtration of a starting material previously obtained from host cells producing rAAV particles, said starting material comprising a cell lysate and/or a cell culture supernatant, whereby a rAAV-containing clarified composition is provided;   ii. submitting the rAAV-containing clarified composition to an immunoaffinity purification step, whereby a first rAAV enriched composition is provided;   iii. submitting the first rAAV enriched composition at least once to:
 c1) a step of anion-exchange chromatography on a chromatographic support wherein elution is performed by using a salt gradient, and wherein the rAAV-containing fraction is collected, whereby a second rAAV enriched composition is provided; or 
 c2) a step of density gradient centrifugation, wherein the rAAV-containing fraction is collected, whereby a second rAAV enriched composition is provided; 
   iv. submitting the second rAAV enriched composition to a step of tangential flow filtration, whereby purified rAAV particles are provided.   
     
     
         18 . The method according to  claim 17 , wherein step b) is performed by using a chromatography support onto which antibodies or fragments thereof directed to the said rAAV particles, are immobilized. 
     
     
         19 . The method according to  claim 18 , wherein the antibodies are monoclonal. 
     
     
         20 . The method according to  claim 18 , wherein the antibodies are camelid antibodies. 
     
     
         21 . The method according to  claim 17 , wherein step b) is performed using an AVB Sepharose™ chromatography (GE Healthcare) support. 
     
     
         22 . The method according to  claim 17 , wherein the at least one chromatographic support at step c1) is a monolithic chromatographic support. 
     
     
         23 . The method according to  claim 17 , wherein elution is performed by using a linear salt gradient. 
     
     
         24 . The method according to  claim 17 , wherein step d) is performed by using a filter membrane having a molecular weight cut-off value ranging from 20 kDa to 150 kDa. 
     
     
         25 . The method according to  claim 17 , wherein the rAAV particles have an AAV capsid belonging to a serotype selected in a group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV8, AAV9, AAV10, and rhesus macaque-derived serotypes, and mixtures thereof. 
     
     
         26 . The method according to  claim 25 , wherein the rhesus macaque-derived serotype is AAVrh10. 
     
     
         27 . The method according to  claim 17 , wherein the rAAV particles have an AAV capsid belonging to the AAV5 serotype, and wherein the rAAV particle contains DNA comprising an expression cassette encoding human PDE6-beta. 
     
     
         28 . The method according to  claim 17 , wherein the rAAV particles have an AAV capsid belonging to the AAV4 serotype, and wherein the rAAV particle contains DNA comprising an expression cassette encoding human RPE65. 
     
     
         29 . The method according to  claim 17 , wherein the purified rAAV particles are suitable for use for gene therapy. 
     
     
         30 . The method according to  claim 17 , wherein the purified rAAV particles have a purity of at least 90%. 
     
     
         31 . The method according to  claim 17 , wherein the purified rAAV particles have an amount of residual host cell DNA equal or inferior to 50 ng per dose of 10 12  vector genomes (vg). 
     
     
         32 . A rAAV particle obtained by the method according to  claim 17 . 
     
     
         33 . A composition comprising the rAAV particle according to  claim 32 . 
     
     
         34 . The composition according to  claim 33 , comprising rAAV particles with a purity of at least 90% and/or an amount of residual host cell DNA equal or inferior to 50 ng per dose of 10 12  vector genomes (vg). 
     
     
         35 . A host cell transfected with an rAAV particle according to  claim 32 . 
     
     
         36 . A method of treating a disease in a subject in need thereof by gene therapy, comprising administering to said subject the rAAV particle according to  claim 32  comprising a heterologous nucleic acid encoding a gene product, or a composition comprising the rAAV particle.