Development of Novel Cell Surface CD34-Targeted Recombinant Adenoassociated Virus Vectors for Gene Therapy

Recombinant adenoassociated virus (rAAV) type 2 vectors have been used to transduce a wide variety of cell types, including hematopoietic progenitor cells. For in vivo gene transfer, it is desirable to have an rAAV vector that specifically transduces selected target cells. As a first step toward generating an rAAV vector capable of targeting delivery in vivo, we have engineered a chimeric protein combining the AAV capsid protein and the variable region of a single-chain antibody against human CD34 molecules, a cell surface marker for hematopoietic stem/progenitor cells. Inclusion of the chimeric CD34 single-chain antibody–AAV capsid proteins within an rAAV virion significantly increased the preferential infectivity of rAAV for the CD34+ human myoleukemia cell line KG-1, which is normally refractory to rAAV transduction. Antibodies against the single-chain antibody and the CD34 protein blocked this transduction. This chimeric vector represents a significant improvement in the host range of rAAV and the first step toward specific gene delivery by rAAV vectors to cells of choice, in this case, hematopoietic progenitor cells, for the treatment of human disease. We have constructed rAAV type 2 vectors encoding modified capsid proteins that allow for cell-type specific targeting to cells that express the CD34 protein. Specifically, a fusion protein was constructed, consisting of the N terminus of the AAV virion protein, VP2, and a single-chain antibody directed against CD34. Vector particles packaged in the presence of the VP2–antibody fusion protein are bound specifically to CD34-expressing KG-1 cells. Moreover, while KG-1 cells were resistant to transduction by unmodified rAAV vectors, the modified vector particles were able to transduce these cells. Antibodies against either the CD34 molecule or the single-chain antibody blocked the modified rAAV virion trasduction of KG-1 cells. Although improvements in titer of these cell type-specific targetable vectors will be necessary before implementation in in vivo gene therapy, this work demonstrates that the tropism of the rAAV particle can be altered by addition of chimeric capsid fusion proteins during viral particle formation.