(453e) Directed Evolution of Adeno-Associated Virus for Enhanced Gene Delivery to Pluripotent and Adult Neural Stem Cells

Stem cells are characterized by their ability to self-renew and to generate one or more mature cell types. Both pluripotent stem cells (PSCs) and neural stem cells (NSCs) present attractive targets in gene therapy and/or cell replacement therapies to treat nervous system injury or disease. Unfortunately, most gene delivery vectors are not capable of targeted gene delivery to stem cells. Adeno-associated virus (AAV) is a promising gene delivery vector that has been used in nearly 100 clinical trials to date. We have applied directed evolution, a high-throughput molecular engineering approach, to create AAV variants capable of efficient and specific transduction of human pluripotent stem cells or adult neural stem cells. Here, we report the in vitro and in vivo infection properties of our novel AAV variants and demonstrate the variants’ utility for genetic engineering of stem cells.

Following a directed evolution approach consisting of one round of evolution and three selection steps on human embryonic stem cells, a variant of AAV2 was isolated and found to mediate increased transduction of several human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC) lines. In addition, the variant was capable of gene-targeting frequencies of up to 0.1%, approximately 10-fold higher than the targeting frequencies in pluripotent stem cells obtained using naturally occurring AAV serotypes. Furthermore, using zinc finger nucleases in conjunction with AAV-mediated gene targeting, this variant achieved gene-targeting frequencies of 1.2%, a >10-fold increase in the already efficient AAV-mediated gene targeting in hESCs. This capability can enable the use of hPSCs for in vitro studies of disease mechanisms, developmental processes, and drug discovery and toxicity.

Furthermore, following a directed evolution approach consisting of three rounds of evolution and ten selection steps on adult rat NSCs, a variant containing a peptide insertion was isolated and found to mediate a 50-fold increase in the transduction of rat NSCs. The variant also exhibited increased transduction of murine NSCs, human fetal NSCs, and human embryonic stem cell-derived neural progenitor cells in vitro. Moreover, the variant was capable of gene-targeting frequencies of up to 0.16%, approximately 5-fold higher than the targeting frequencies in rat NSCs obtained using naturally occurring AAV serotypes. To build upon the in vitro success of this variant, the in vivo infection properties of this novel AAV variant were analyzed. It was capable of efficient and selective transduction of rat and mouse neural stem cells in the adult hippocampus compared to several natural AAV serotypes. Delivery of a constitutively-active β-catenin transgene demonstrated that variant r3.45 can be used to study mechanisms through which key regulatory genes are acting in NSCs and that r3.45 can be used to deliver transgenes that can induce changes in the NSC population in the hippocampus.