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

转基因猪的培育使异种移植的实现更近了一步。然而，产生表达多个转基因的转基因猪既耗时又昂贵。腺相关病毒(AAV)是一种高效、安全的基因转移载体系统。AAV的特点是免疫原性低，并允许基因长期表达。我们的研究小组在AAV的高滴度生产和纯化方面经验丰富。靶向策略已经被开发出来，以提高针对不同类型的细胞(如内皮细胞)的基因转移的特异性。一种新开发的自我互补的AAV结构允许非常快速和高水平的转基因表达。8个AAV血清型显示出不同的组织和物种特异性，已被表征。在此背景下，将构建优化的AAV载体，以实现高效的猪组织基因转移。这将包括在体外对猪细胞进行不同AAV血清型的测试，以及开发专门为有效地将基因转移到猪内皮细胞和心肌细胞而量身定做的靶向AAV载体。此外，还将生产编码免疫调节因子CTLA4-Ig和IL-10的自身互补AAV，并将其用于同种异体小鼠心脏移植模型。优化后的AAV载体将最终用于活体猪心脏的基因转移。这项技术将允许通过引入额外的免疫调节基因来进一步修改来自转基因动物的器官。

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.