Recombinant adeno-associated virus-mediated transgene delivery into porcine organs to allow immunological modifications of xenografts

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The breeding of transgenic pigs brings the realization of xenotransplantation closer. However, the generation of transgenic pigs expressing multiple transgenes is time-consuming and expensive. Adeno-associated virus (AAV) can serve as an efficient and safe vector system for gene transfer into donor organs. AAV is characterized by low immunogenicity and allows long-term gene expression. Our research group is experienced in high titer production and purification of AAV. Targeting strategies have been developed that enhance the specificity of gene transfer towards distinct types of cells such as endothelial cells. A newly developed self-complementary AAV construct allows very rapid and elevated transgene expression. Eight AAV serotypes, which show different tissue and species specificities, have been characterized. On this background AAV vectors will be constructed that are optimized towards efficient gene transfer into porcine tissues. This will involve the testing of different AAV serotypes on porcine cells in vitro and the development of a targeting AAV vector specially tailored for an efficient gene transfer into porcine endothelial and heart muscle cells. Furthermore, a self complementary AAV coding the immunomodulatory factors CTLA4-Ig and interleukin-10 will be produced and used in a allogenic mouse heart transplantation model. The optimized AAV vector will finally be used for gene transfer into porcine hearts in vivo. This technique will allow further modifications of organs derived from transgenic animals by introducing additional immunomodulatory genes.