An Evolved Adeno-associated Viral Variant Enhances Gene Delivery and Gene Targeting in Neural Stem Cells

Abstract

Gene delivery to, and gene targeting in, stem cells would be a highly enabling technology for basic science and biomedical application. Adeno-associated viral (AAV) vectors have demonstrated the capacity for efficient delivery to numerous cells, but their application to stem cells has been limited by low transduction efficiency. Due to their considerable advantages, however, engineering AAV delivery systems to enhance gene delivery to stem cells may have an impact in stem cell biology and therapy. Therefore, using several diverse AAV capsid libraries-including randomly mutagenized, DNA shuffled, and random peptide insertion variants-we applied directed evolution to create a "designer" AAV vector with enhanced delivery efficiency for neural stem cells (NSCs). A novel AAV variant, carrying an insertion of a selected peptide sequence on the surface of the threefold spike within the heparin-binding site, emerged from this evolution. Importantly, this evolved AAV variant mediated efficient gene delivery to rat, mouse, and human NSCs, as well as efficient gene targeting within adult NSCs, and it is thus promising for applications ranging from basic stem cell biology to clinical translation.

The Nontoxic Cell Cycle Modulator Indirubin Augments Transduction of Adeno-Associated Viral Vectors and Zinc-Finger Nuclease-Mediated Gene Targeting

“…In particular, incubation of cells with indirubin-3¢-monoxime, a well-tolerated CDK inhibitor with minimal toxicity in normal cells, augmented the genetargeting frequency up to 10-fold to correct a mutated reporter gene in up to 34% of infected human cells. Given the recent success in AAV-mediated gene targeting in other multipotent (Chamberlain et al, 2004;Jang et al, 2011) or pluripotent stem cells (Mitsui et al, 2009;Khan et al, 2010;Asuri et al, 2012), respectively, as well as in vivo (Papadopoulos et al, 1997;Paulk et al, 2012), these findings present a promising strategy to enhance AAV/ZFN-mediated genome engineering for potential clinical applications.…”

Directed evolution of novel adeno-associated viruses for therapeutic gene delivery

“…Selections using an error-prone AAV2 library, an AAV2 with random peptide insert library and an AAV2 pairwise shuffled library on NSCs (from the adult hippocampus) yielded an AAV2 variant containing a peptide insertion that mediated 50-fold increased transduction of rat NSCs, as well as increased transduction of murine NSCs, human fetal NSCs and human embryonic stem cell (hESC)-derived neural progenitor cells. 40 Presumably as a result of the increased transduction, the variant also exhibited a fivefold increased rate of targeted gene correction compared with natural serotypes in NSCs. 40 The most successful report of gene targeting to human pluripotent stem cells using wild-type AAV showed correct targeting of 1.3% of all colony-forming units, which corresponds to an overall gene targeting frequency for the originally infected hESCs of approximately 0.03%.…”

“…40 Presumably as a result of the increased transduction, the variant also exhibited a fivefold increased rate of targeted gene correction compared with natural serotypes in NSCs. 40 The most successful report of gene targeting to human pluripotent stem cells using wild-type AAV showed correct targeting of 1.3% of all colony-forming units, which corresponds to an overall gene targeting frequency for the originally infected hESCs of approximately 0.03%. 41 To build upon this result, Asuri et al 24 applied directed evolution to create an AAV variant capable of enhanced gene delivery and gene targeting in hESCs and human induced pluripotent stem cells.…”

“…Directed evolution was first applied to stem cells by Jang et al 40 to isolate a variant capable of efficient transduction of neural stem cells (NSCs). Selections using an error-prone AAV2 library, an AAV2 with random peptide insert library and an AAV2 pairwise shuffled library on NSCs (from the adult hippocampus) yielded an AAV2 variant containing a peptide insertion that mediated 50-fold increased transduction of rat NSCs, as well as increased transduction of murine NSCs, human fetal NSCs and human embryonic stem cell (hESC)-derived neural progenitor cells.…”

Advances in homology directed genetic engineering of human pluripotent and adult stem cells

“…Other successful examples include the isolation of AAV variants with altered receptor binding and cell tropism in vitro and in vivo, and enhanced gene delivery [34,35] . Using this approach, researchers have recently created a novel AAV variant -AAV r3.45 that mediates efficient gene delivery to both murine (infection efficiency > 40%) and human (infection efficiency > 30%) neural stem cells (NSCs) when compared to naturally occurring AAV variants (infection efficiency < 1%) [36] . More importantly, this increase in gene delivery efficiency was accompanied by up to 10-fold enhancement in the ability to repair singlebase pair mutations in rat NSCs, relative to naturally occurring AAV.…”