Titration of AAV-2 particles via a novel capsid ELISA: packaging of genomes can limit production of recombinant AAV-2.
We demonstrate the rapid and reliable quantification of physical AAV-2 (adeno-associated virus type 2) particles via a novel ELISA based on a monoclonal antibody which selectively recognizes assembled AAV-2 capsids. Titration of a variety of recombinant AAV-2 (rAAV) preparations revealed that at least 80+percent of all particles were empty, compared with a maximum of 50percent in wild-type AAV-2 stocks, indicating that the recombinant genomes were less efficiently encapsidated. This finding was confirmed upon titration of CsCl gradient fractions from recombinant and wild-type AAV-2 stocks. ELISA-based measurement of capsid numbers revealed a large number of physical particles with low densities corresponding to empty capsids in the recombinant, but not in the wild-type AAV-2 preparations. Moreover, additional expression of VP proteins during rAAV production was found to result in an excessive capsid formation, whilst yielding only minor increases in DNA-containing or transducing rAAV particles. We conclude that encapsidation of viral genomes rather than capsid assembly can be limiting for rAAV production, provided that a critical level of VP expression is maintained. The feasibility of quantifying AAV-2 capsid numbers via the ELISA allows determination of physical to DNA-containing or infectious particle ratios. These are important parameters which should help to optimize and standardize the production and application of recombinant AAV-2.
Grimm
D
D
Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany.
Kern
A
A
Pawlita
M
M
Ferrari
F
F
Samulski
R
R
Kleinschmidt
J
J
D000911:Antibodies, Monoclonal; D002213:Capsid / Q000276:immunology; D000229:Dependovirus / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans
D000911:Antibodies, Monoclonal