Non-integrating gamma-retroviral vectors as a versatile tool for transient zinc-finger nuclease delivery

Abstract

Designer nucleases, like zinc-finger nucleases (ZFNs), represent valuable tools for targeted genome editing. Here, we took advantage of the gamma-retroviral life cycle and produced vectors to transfer ZFNs in the form of protein, mRNA and episomal DNA. Transfer efficacy and ZFN activity were assessed in quantitative proof-of-concept experiments in a human cell line and in mouse embryonic stem cells. We demonstrate that retrovirus-mediated protein transfer (RPT), retrovirus-mediated mRNA transfer (RMT), and retrovirus-mediated episome transfer (RET) represent powerful methodologies for transient protein delivery or protein expression. Furthermore, we describe complementary strategies to augment ZFN activity after gamma-retroviral transduction, including serial transduction, proteasome inhibition, and hypothermia. Depending on vector dose and target cell type, gene disruption frequencies of up to 15% were achieved with RPT and RMT, and >50% gene knockout after RET. In summary, non-integrating gamma-retroviral vectors represent a versatile tool to transiently deliver ZFNs to human and mouse cells.

Retrovirus-based vectors for transient and permanent cell modification

“…This optimized system efficiently delivered FLP recombinase and Sleeping Beauty (SB) transposase activity to target cells and was also successful in expressing biologically relevant amounts of ZFN [56]. Similar to the concept of repeated RET, repetitive transduction with RMT vectors increased the low ZFN activity obtained with a single administration [56].…”

“…Inclusion of a posttranscriptional regulatory element (PRE) to improve RNA stability and expression levels [73], as well as codon optimization of the transgene along with introduction of an optimized Kozak consensus sequence for translation initiation further enhanced RMT potency [72]. This optimized system efficiently delivered FLP recombinase and Sleeping Beauty (SB) transposase activity to target cells and was also successful in expressing biologically relevant amounts of ZFN [56]. Similar to the concept of repeated RET, repetitive transduction with RMT vectors increased the low ZFN activity obtained with a single administration [56].…”

“…Repeated RET was also successfully used to elevate targeted gene knockout frequencies upon transfer of zinc finger nucleases (ZFN) to K562 and murine embryonic stem cells [56]. Thus far, no major cytotoxicities were reported upon repeated RET [3,56]. As repeated transfer can optimally be accomplished ex vivo, this strategy appears most suitable for approaches requiring restricted but prolonged transgene expression in cultured cells, for example for targeted cell fate conversion.…”

“…Demonstrating biological utility, repeated RET of the transcription factor (TF) Oct4 (fused to a VP16 domain) could substitute for Oct4 expression from iRVV in reprogramming of human fibroblasts to the induced pluripotent state [3]. Repeated RET was also successfully used to elevate targeted gene knockout frequencies upon transfer of zinc finger nucleases (ZFN) to K562 and murine embryonic stem cells [56]. Thus far, no major cytotoxicities were reported upon repeated RET [3,56].…”

Targeted transgene insertion into the CHO cell genome using Cre recombinase‐incorporating integrase‐defective retroviral vectors

“…Bobis-Wozowicz et al (2014) reported a versatile tool for ZFN protein delivery mediated by IDRV. In this case, although targeted gene knockouts in mammalian cells were possible, transgene insertion into a specific genomic locus was not reported.…”