Rapid, simple, and versatile manufacturing of recombinant adeno-associated viral vectors at scale.
Adeno-associated viral (AAV) manufacturing at scale continues to hinder the application of AAV technology to gene therapy studies. Although scalable systems based on AAV-adenovirus, AAV-herpesvirus, and AAV-baculovirus hybrids hold promise for clinical applications, they require time-consuming generation of reagents and are not highly suited to intermediate-scale preclinical studies in large animals, in which several combinations of serotype and genome may need to be tested. We observed that during production of many AAV serotypes, large amounts of vector are found in the culture supernatant, a relatively pure source of vector in comparison with cell-derived material. Here we describe a high-yielding, recombinant AAV production process based on polyethylenimine (PEI)-mediated transfection of HEK293 cells and iodixanol gradient centrifugation of concentrated culture supernatant. The entire process can be completed in 1 week and the steps involved are universal for a number of different AAV serotypes. Process conditions have been optimized such that final purified yields are routinely greater than 1 x 10(14) genome copies per run, with capsid protein purity exceeding 90%. Initial experiments with vectors produced by the new process demonstrate equivalent or better transduction both in vitro and in vivo when compared with small-scale, CsCl gradient-purified vectors. In addition, the iodixanol gradient purification process described effectively separates infectious particles from empty capsids, a desirable property for reducing toxicity and unwanted immune responses during preclinical studies.
Lock
Martin
M
Gene Therapy Program, Department of Pathology and Laboratory Medicine, Division of Transfusion Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Alvira
Mauricio
M
Vandenberghe
Luk H
LH
Samanta
Arabinda
A
Toelen
Jaan
J
Debyser
Zeger
Z
Wilson
James M
JM
D000256:Adenoviridae / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics; D036022:Capsid Proteins / Q000235:genetics; D002499:Centrifugation, Density Gradient; D000229:Dependovirus / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification; D015316:Genetic Therapy; D005822:Genetic Vectors; D057809:HEK293 Cells; D006378:Helper Viruses / Q000235:genetics; D006564:Herpesviridae / Q000235:genetics; D006801:Humans; D011094:Polyethyleneimine; D014162:Transfection; D019076:Transgenes; D014283:Triiodobenzoic Acids; D014776:Virus Cultivation
D036022:Capsid Proteins; D014283:Triiodobenzoic Acids; D011094:Polyethyleneimine; C044834:iodixanol