T-cell specific in vivo gene delivery with DART-AAVs targeted to CD8.

One of the biggest challenges for in vivo gene therapy are vectors mediating highly selective gene transfer into a defined population of therapy-relevant cells. Here we present DARPin-targeted AAVs (DART-AAVs) displaying DARPins specific for human and murine CD8. Insertion of DARPins into the GH2/GH3 loop of the capsid protein 1 (VP1) of AAV2 and AAV6 resulted in high selectivity for CD8-positive T cells with unimpaired gene delivery activity. Remarkably, the capsid core structure was unaltered with protruding DARPins detectable. In complex primary cell mixtures, including donor blood or systemic injections into mice, the CD8-targeted AAVs were by far superior to unmodified AAV2 and AAV6 in terms of selectivity, target cell viability and gene transfer rates. In vivo, up to 80% of activated CD8+ T cells were hit upon a single vector injection into conditioned humanized or immunocompetent mice. While gene transfer rates decreased significantly under non-activated conditions, genomic modification selectively in CD8+ T cells was still detectable upon Cre delivery into indicator mice. In both mouse models, selectivity for CD8+ T cells was close to absolute with exceptional detargeting from liver. The CD8-AAVs described here expand strategies for immunological research and in vivo gene therapy options.

GP64-pseudotyped lentiviral vectors target liver endothelial cells and correct hemophilia A mice.

Lentiviral vectors (LV) are efficient vehicles for in vivo gene delivery to the liver. LV integration into the chromatin of target cells ensures their transmission upon proliferation, thus allowing potentially life-long gene therapy following a single administration, even to young individuals. The glycoprotein of the vesicular stomatitis virus (VSV.G) is widely used to pseudotype LV, as it confers broad tropism and high stability. The baculovirus-derived GP64 envelope protein has been proposed as an alternative for in vivo liver-directed gene therapy. Here, we perform a detailed comparison of VSV.G- and GP64-pseudotyped LV in vitro and in vivo. We report that VSV.G-LV transduced hepatocytes better than GP64-LV, however the latter showed improved transduction of liver sinusoidal endothelial cells (LSEC). Combining GP64-pseudotyping with the high surface content of the phagocytosis inhibitor CD47 further enhanced LSEC transduction. Coagulation factor VIII (FVIII), the gene mutated in hemophilia A, is naturally expressed by LSEC, thus we exploited GP64-LV to deliver a FVIII transgene under the control of the endogenous FVIII promoter and achieved therapeutic amounts of FVIII and correction of hemophilia A mice.

Secreted Particle Information Transfer (SPIT) - A Cellular Platform for In Vivo Genetic Engineering.

A multitude of tools now exist that allow us to precisely manipulate the human genome in a myriad of different ways. However, successful delivery of these tools to the cells of human patients remains a major barrier to their clinical implementation. Here we introduce a new cellular approach for in vivo genetic engineering, Secreted Particle Information Transfer (SPIT) that utilizes human cells as delivery vectors for in vivo genetic engineering. We demonstrate the application of SPIT for cell-cell delivery of Cre recombinase and CRISPR-Cas9 enzymes, we show that genetic logic can be incorporated into SPIT and present the first demonstration of human cells as a delivery platform for in vivo genetic engineering in immunocompetent mice. We successfully applied SPIT to genetically modify multiple organs and tissue stem cells in vivo including the liver, spleen, intestines, peripheral blood, and bone marrow. We anticipate that by harnessing the large packaging capacity of a human cell's nucleus, the ability of human cells to engraft into patients' long term and the capacity of human cells for complex genetic programming, that SPIT will become a paradigm shifting approach for in vivo genetic engineering.