Viral, nonviral and peptide-mediated intra-articular transfer of genes and proteins

We have been examining the ability of different vectors to transfer therapeutic genes directly to joints following intra-articular injection. To date, we have examined the efficiency of intra-articular gene transfer to the rabbit knee mediated by adenoviral, adenoassociated virus (AAV), retrovirus and herpes simplex virus vectors as well as by over 100 different nonviral formulations. Moreover, we have examined the ability to repeat dose with the different types of viral vectors. A summary of our experiments to identify a clinically useful vector for intra-articular gene transfer will be presented. In an attempt to improve intra-articular gene transfer, we also have screened an M13 peptide phage display library for peptides able to facilitate internalization into synovial cells. We have identified a class of cationic peptides (PTD), similar to the cationic protein transduction domains found in Antennapedia (Antp) and HIV TAT peptide, as well as a class of more neutral and hydrophobic peptides (HAP). To determine the ability of the PTD and HAP peptides to facilitate internalization, the peptides were biotinylated and coupled to avidin-β-gal or streptavidin-Cy3. Although internalization mediated by the HAP peptides in culture and in vivo was not as efficient as the cationic peptides, they appeared to be more cell type specific. Several of the HAP and PTD peptides were fused to an antimicrobial peptide, KLAK, generating novel apoptotic peptides. These fusion peptides were able to impair rabbit and human synovial cell viability through disruption of mitochondria and induction of apoptosis. Intra-articular injection of these apoptotic peptides into arthritic rabbit joints resulted in extensive synovial cell apoptosis as well as reduction in leukocytic infiltration. An update of our progress using the different internalizing peptides for delivery of therapeutic proteins and genes will be presented.