IN-VITRO PACKAGING OF ADENOASSOCIATED VIRUS-DNA

We have developed an in vitro procedure for packaging of recombinant a deno-associated virus (AAV). By using AAV replicative-form DNA as the substrate, it is possible to synthesize an infectious AAV particle in vitro that can be used to transfer a marker gene to mammalian cells, T he packaging procedure requires the presence of both the AAV Rep and c apsid proteins. Two kinds of in vitro products can be formed which fac ilitate DNA transfer. Both are resistant to heat and have a density in cesium chloride gradients that is indistinguishable from that of the in vivo-synthesized wild-type virus. This indicates that the particles formed have the appropriate protein-to-DNA ratio and a structure that shares the heat resistance of mature AAV particles. The two types of particles can be distinguished by their sensitivity to chloroform and DNase I treatment. The chloroform-resistant product is, by several cri teria, an authentic AAV particle, In addition to having the correct de nsity and being resistant to treatment with chloroform, DNase I, and h eat, this particle is efficiently synthesized only if the AAV genome c ontains intact terminal repeats, which are known to be required for AA V packaging, It is also precipitated by a monoclonal antibody that rec ognizes mature virus particles but not bound by an antibody that recog nizes monomeric or denatured capsid proteins, The chloroform-resistant species is not made when aphidicolin is present in the reaction mixtu re, suggesting that active DNA replication is required for in vitro pa ckaging, In contrast, the chloroform-sensitive product has several fea tures that suggest it is an incompletely assembled virus particle, It is sensitive to DNase I, does not require the presence of AAV terminal repeats, and is capable of transferring DNA that is theoretically too large to package. Sucrose gradient centrifugation of the in vitro-syn thesized products reveals that the particles have sedimentation values between 60S and 110S, which is consistent with partially assembled an d mature AAV particles. The in vitro packaging procedure should be use ful for studying the mechanism by which a human icosahedral DNA virus particle is assembled, and it may be useful for producing recombinant AAV for gene therapy. The chloroform-sensitive particle may also be us eful for transferring DNA that is too large to be packaged in mature r ecombinant AAV.