973. Efficient Transduction of Bone Marrow-Derived Mesenchymal Stem Cells for Cancer Gene Therapy Using a Tropism-Modified Adeno-Associated Virus (AAV) Vector

Abstract

Top of pageAbstract Exogenously administered bone marrow-derived mesenchymal stem cells (MSCs) will preferentially engraft at tumor sites and contribute to the population of stromal fibroblasts. This has allowed the development of therapeutic strategies based on the local production of biological agents by gene-manipulated MSCs following transplantation. Adeno-associated virus (AAV) - based vectors are attractive for the introduction of therapeutic genes into MSCs due to their ability to promote stable gene expression in the absence of integration. While there are currently at least 8 naturally occurring serotypes of AAV, the AAV2 serotype has been associated with most gene therapy studies to date. Previously, our laboratory has generated a novel recombinant AAV vector based on AAV2 that contains an “RGD” polypeptide sequence (4C-RGD) inserted into the AAV capsid protein(s) following VP1 amino acid 588, “rAAV2(RGD)”. As with many of the available non-viral transfection methods, we found that AAV serotypes 1, 2, and 5 poorly infect human MSCs, even at very high multiplicities of infection (MOI). In contrast, rAAV2(RGD) is able to transduce greater than 80% of human MSCs in vitro. This represents a nearly three-order of magnitude enhancement in gene transfer efficiency compared to unmodified AAV2-based vectors. One potential obstacle to AAV-mediated gene therapy is the prolonged delay in transgene expression following AAV infection in vivo. Our ex vivo gene therapy approach circumvents this delay as demonstrated by rAAV2(RGD)-mediated transgene expression in as little as 72 hours post-infection in infected MSCs. Importantly, rapid onset of transgene expression was achieved in the absence of adenoviral help and in conjunction with increasing MOIs of rAAV2(RGD). Characterization of AAV-transduced MSCs for tri-lineage differentiation potential (adipogenic, chondrogenic, and osteogenic), duration of transgene expression, and maintenance of transgene expression levels in vitro following rAAV2(RGD) infection is ongoing. With the demonstration that human bone marrow-derived MSCs engraft into tumor microenvironments, AAV-transduced MSCs could provide an innovative delivery strategy for cancer gene therapies based on the local production of biological agents in the tumor microenvironment. Since such a treatment strategy will require safe and efficient ways of delivering and expressing therapeutic genes in human MSCs, the use of RGD-modified AAV vectors may provide the ideal platform to accomplish genetic manipulation of these cells.