Recombinant viral vector system

DETAILED DESCRIPTION The present invention relates to the in vitro synthesis of a novel 165 basepair fragment of DNA which contains AAV ITR sequences and which can be synthesized in vitro and used to engineer expression vectors and/or vectors useful for genetic therapy.
This 165 bp DNA sequence, herein referred to as the "double-D sequence," is in a novel configuration not found to exist in wild type AAV.
The invention is based, in part, on the ability of the double-D sequence to provide sufficient information, in cis, for converting a circular duplex DNA molecule into a linear replicating molecule with covalently closed ends.
The replicating DNA molecule may be either encapsidated into mature AAV virions, or integrated into the host genome.
A particularly significant feature of the circular duplex DNA molecule is that the process of conversion into a linear molecule, and the replication and integration into the host genome is completely effected through the double-D sequences, thus ensuring that the heterologous gene sequences of interest remain intact.
The discovery that the majority of the AAV genome is not needed, in cis, for replication, packaging and/or integration allows one to insert larger fragments of DNA into the recombinant vectors.
In addition, the discovery that the 165 double-D sequence is sufficient for targeting vector DNA to chromosome 19 allows the insertion of any sized DNA fragment into the recombinant vectors, thereby removing any size restraints.
Nucleotide sequences which may be used in accordance with the invention include derivatives and analogs of the double-D sequences disclosed herein that are functionally equivalent in that they retain their ability to provide information, in cis, for replication encapsidation, integration and rescue of recombinant DNA.
In particular, double-D derivatives may contain additions, substitutions or deletions of the double-D nucleotide sequences while retaining their biological function.
The invention provides an in vivo system for replication and packaging of recombinant DNA into mature virions