Directed evolution of adeno-associated virus for enhanced gene delivery and gene targeting in human pluripotent stem cells.

Efficient approaches for the precise genetic engineering of human pluripotent stem cells (hPSCs) can enhance both basic and applied stem cell research. Adeno- associated virus (AAV) vectors are of particular interest for their capacity to mediate efficient gene delivery to and gene targeting in various cells. However, natural AAV serotypes offer only modest transduction of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), which limits their utility for efficiently manipulating the hPSC genome. Directed evolution is a powerful means to generate viral vectors with novel capabilities, and we have applied this approach to create a novel AAV variant with high gene delivery efficiencies (~50%) to hPSCs, which are importantly accompanied by a considerable increase in gene-targeting frequencies, up to 0.12%. While this level is likely sufficient for numerous applications, we also show that the gene-targeting efficiency mediated by an evolved AAV variant can be further enhanced (>1%) in the presence of targeted double- stranded breaks (DSBs) generated by the co-delivery of artificial zinc finger nucleases (ZFNs). Thus, this study demonstrates that under appropriate selective pressures, AAV vectors can be created to mediate efficient gene targeting in hPSCs, alone or in the presence of ZFN- mediated double-stranded DNA breaks.

Enantioselective separation on chiral Au nanoparticles.

The surfaces of chemically synthesized Au nanoparticles have been modified with d- or l-cysteine to render them chiral and enantioselective for adsorption of chiral molecules. Their enantioselective interaction with chiral compounds has been probed by optical rotation measurements during exposure to enantiomerically pure and racemic propylene oxide. The ability of optical rotation to detect enantiospecific adsorption arises from the fact that the specific rotation of polarized light by (R)- and (S)-propylene oxide is enhanced by interaction with Au nanoparticles. This effect is related to previous observations of enhanced circular dichroism by Au nanoparticles modified by chiral adsorbates. More importantly, chiral Au nanoparticles modified with either d- or l-cysteine selectively adsorb one enantiomer of propylene oxide from a solution of racemic propylene oxide, thus leaving an enantiomeric excess in the solution phase. Au nanoparticles modified with l-cysteine (d-cysteine) selectively adsorb the (R)-propylene oxide ((S)-propylene oxide). A simple model has been developed that allows extraction of the enantiospecific equilibrium constants for (R)- and (S)-propylene oxide adsorption on the chiral Au nanoparticles.

Enhanced gene targeting of adult and pluripotent stem cells using evolved adeno-associated virus.

Efficient approaches for the precise genetic engineering of stem cells can enhance both basic and applied stem cell research. Adeno-associated virus (AAV) vectors have demonstrated high-efficiency gene delivery and gene targeting to numerous cell types, and AAV vectors developed specifically for gene delivery to stem cells have further increased gene targeting frequency compared to plasmid construct techniques. This chapter details the production and purification techniques necessary to generate adeno-associated viral vectors for use in high-efficiency gene targeting of adult or pluripotent stem cell applications. Culture conditions used to achieve high gene targeting frequencies in rat neural stem cells and human pluripotent stem cells are also described.