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 (ade no-associated virus type 2) particles via a novel ELISA based on a monoclon al antibody which selectively recognizes assembled AAV-2 capsids. Titration of a variety of recombinant AAV-2 (rAAV) preparations revealed that at lea st 80% of aff particles were empty, compared with a maximum of 50% in wild- type AAV-2 stocks, indicating that the recombinant genomes were less effici ently encapsidated. This finding was confirmed upon titration of CsCl gradi ent fractions from recombinant and wild-type AAV-2 stocks. ELISA-based meas urement of capsid numbers revealed large number of physical particles with low densities corresponding to empty capsids in the combinant, but not in t he wild-type AAV-2 preparations. Moreover, additional expression of VP prot eins during rAAV production was found to result in an excessive capsid form ation, whilst yielding only minor increases in DNA-containing or transducin g rAAV particles. We conclude that encapsidation of viral genomes rather th an capsid assembly can be limiting fbr rAAV production, provided that a cri tical level of VP expression is maintained The feasibility of quantifying A AV-2 capsid numbers via ther ELISA allows determination of physical to DNA- containing or infectious particle ratios These are important parameters whi ch should help to optimize and standardize the production and application o f recombinant AAV-2.