Zinc finger nuclease-expressing baculoviral vectors mediate targeted genome integration of reprogramming factor genes to facilitate the generation of human induced pluripotent stem cells.

Integrative gene transfer using retroviruses to express reprogramming factors displays high efficiency in generating induced pluripotent stem cells (iPSCs), but the value of the method is limited because of the concern over mutagenesis associated with random insertion of transgenes. Site-specific integration into a preselected locus by engineered zinc-finger nuclease (ZFN) technology provides a potential way to overcome the problem. Here, we report the successful reprogramming of human fibroblasts into a state of pluripotency by baculoviral transduction-mediated, site-specific integration of OKSM (Oct3/4, Klf4, Sox2, and c-myc) transcription factor genes into the AAVS1 locus in human chromosome 19. Two nonintegrative baculoviral vectors were used for cotransduction, one expressing ZFNs and another as a donor vector encoding the four transcription factors. iPSC colonies were obtained at a high efficiency of 12% (the mean value of eight individual experiments). All characterized iPSC clones carried the transgenic cassette only at the ZFN-specified AAVS1 locus. We further demonstrated that when the donor cassette was flanked by heterospecific loxP sequences, the reprogramming genes in iPSCs could be replaced by another transgene using a baculoviral vector-based Cre recombinase-mediated cassette exchange system, thereby producing iPSCs free of exogenous reprogramming factors. Although the use of nonintegrating methods to generate iPSCs is rapidly becoming a standard approach, methods based on site-specific integration of reprogramming factor genes as reported here hold the potential for efficient generation of genetically amenable iPSCs suitable for future gene therapy applications.

Baculoviral transduction facilitates TALEN-mediated targeted transgene integration and Cre/LoxP cassette exchange in human-induced pluripotent stem cells.

Safety and reliability of transgene integration in human genome continue to pose challenges for stem cell-based gene therapy. Here, we report a baculovirus-transcription activator-like effector nuclease system for AAVS1 locus-directed homologous recombination in human induced pluripotent stem cells (iPSCs). This viral system, when optimized in human U87 cells, provided a targeted integration efficiency of 95.21% in incorporating a Neo-eGFP cassette and was able to mediate integration of DNA insert up to 13.5 kb. In iPSCs, targeted integration with persistent transgene expression was achieved without compromising genomic stability. The modified iPSCs continued to express stem cell pluripotency markers and maintained the ability to differentiate into three germ lineages in derived embryoid bodies. Using a baculovirus-Cre/LoxP system in the iPSCs, the Neo-eGFP cassette at the AAVS1 locus could be replaced by a Hygro-mCherry cassette, demonstrating the feasibility of cassette exchange. Moreover, as assessed by measuring γ-H2AX expression levels, genome toxicity associated with chromosomal double-strand breaks was not detectable after transduction with moderate doses of baculoviral vectors expressing transcription activator-like effector nucleases. Given high targeted integration efficiency, flexibility in transgene exchange and low genome toxicity, our baculoviral transduction-based approach offers great potential and attractive option for precise genetic manipulation in human pluripotent stem cells.

Systemic delivery of fusogenic membrane glycoprotein-expressing neural stem cells to selectively kill tumor cells.

Intravenously injected neural stem cells (NSCs) can infiltrate both primary and metastatic tumor sites; thus, they are attractive tumor-targeting vehicles for delivering anticancer agents. However, because the systemic distribution of the injected NSCs involves normal organs and might induce off-target actions leading to unintended side effects, clinical applications of this approach is impeded. Given that the vesicular stomatitis virus glycoprotein (VSV-G) can promote the formation of multinucleated syncytia to kill cells in a pH-dependent manner, we engineered a pH sensor of VSV-G and generated a novel VSV-G mutant that efficiently promotes syncytium formation at the tumor extracellular pH (pHe) but not at pH 7.4. Using transduced NSCs derived from induced pluripotent stem cells (iPSCs), the VSV-G mutant was delivered into mice with metastatic breast cancers in the lung through tail vein injection. Compared with the conventional stem cell-based gene therapy that uses the herpes simplex virus thymidine kinase (HSVtk) suicide gene, this treatment did not display toxicity to normal non-targeted organs while retaining therapeutic effects in tumor-bearing organs. Our findings demonstrate the effectiveness of a new approach for achieving tumor-selective killing effects following systemic stem cell administration. Its potential in stem cell-based gene therapy for metastatic cancer is worthy of further exploration.