In vitro packaging of adeno-associated virus DNA

DETAILED DESCRIPTION It is an object of the present invention to provide a method for packaging a DNA substrate in an in vitro reaction to produce adeno-associated virus (AAV) particles capable of transduction of mammalian cells.
It is a further object of the present invention to provide a method for producing cell-free extracts that are useful for in vitro packaging of DNA substrates into AAV particles that are capable of transduction.
In vitro packaged AAV particles and methods for their use in transduction of mammalian cells are also provided.
These and other objects of the present invention as will hereinafter become readily apparent are accomplished by providing a method for packaging a DNA substrate into an AAV capsid in vitro to produce a viral particle or a recombinant viral particle that is capable of transferring the packaged DNA substrate to a recipient mammalian cell resulting in expression or function of the substrate DNA or some part of the substrate DNA in the recipient cell.
In particular, the method of the present invention comprises (a) transfecting a mammalian cell culture permissive for AAV replication with a dAAV vector containing AAV capsid and Rep gene coding sequences; (b) infecting the cell culture with a helper virus; (c) preparing an extract from the transfected cell culture; (d) combining said extract with a DNA substrate; and, (e) incubating said extract under conditions to promote packaging of the substrate DNA.
In more particular embodiments, the method of the present invention comprises additionally the step of heating the packaged extract at elevated temperatures and optionally the step of extracting the packaged extract with chloroform.
In other embodiments the present invention provides in vitro packaged AAV particles capable of transduction of recipient mammalian cells.
In one such embodiment, the substrate DNA packaged into the AAV particles is not restricted by size and sequence constraints typical of previous rAAV vectors made by in vivo packaging methods