An episomal DNA vector platform for the persistent genetic modification of pluripotent stem cells and their differentiated progeny.

Alicia Roig-Merino,Matthias Bozza,Julia D Peterson,Richard P Harbottle,Michael D Milsom,Louise Bullen,Tristan R Mckay,Sina Stäble,Marleen Büchler-Schäff,Manuela Urban,Karin Müller-Decker,Franciscus Van Der Hoeven

doi:10.1016/j.stemcr.2021.11.011

Abstract

SummaryThe genetic modification of stem cells (SCs) is typically achieved using integrating vectors, whose potential integrative genotoxicity and propensity for epigenetic silencing during differentiation limit their application. The genetic modification of cells should provide sustainable levels of transgene expression, without compromising the viability of a cell or its progeny. We developed nonviral, nonintegrating, and autonomously replicating minimally sized DNA nanovectors to persistently genetically modify SCs and their differentiated progeny without causing any molecular or genetic damage. These DNA vectors are capable of efficiently modifying murine and human pluripotent SCs with minimal impact and without differentiation-mediated transgene silencing or vector loss. We demonstrate that these vectors remain episomal and provide robust and sustained transgene expression during self-renewal and targeted differentiation of SCs both in vitro and in vivo through embryogenesis and differentiation into adult tissues, without damaging their phenotypic characteristics.

Highlights

Pluripotent stem cells (PSCs) are an invaluable source of cells for regenerative therapies due to their capacity for proliferation, self-renewal, and their potential for multi-lineage differentiation (He et al, 2009; Schwanke et al, 2014)
PSMAR and nSMARt CAG:GFP-2A-Puro-SMAR (nSMAR) generate highly expressing stable SC lines while remaining episomal Refined SMAR vectors are based on pEPI-CMV-UCOE (Hagedorn et al, 2013) (Figure 1A)
The SMAR element was retained and the CMV promoter replaced with the CAG (Fregien and Davidson, 1986; Miyazaki et al, 1989) to provide robust transgene expression, their composition reorganized by directly coupling the selection marker to the expression cassette and SMAR motif (Bozza et al, 2020)

Summary

Introduction

Pluripotent stem cells (PSCs) are an invaluable source of cells for regenerative therapies due to their capacity for proliferation, self-renewal, and their potential for multi-lineage differentiation (He et al, 2009; Schwanke et al, 2014). Induced PSCs (iPSCs) can be derived from somatic cells (Takahashi and Yamanaka, 2006) and isolated using minimally invasive techniques. This limits concerns regarding the use of embryonic SCs (ESCs) but the risk of immune rejection as an autologous therapy. SCs are notoriously difficult to modify genetically; they are typically refractory to transfection, their extensive proliferation leads to vector dilution, and the dramatic changes in the cellular milieu following differentiation can lead to transgene silencing. A variety of methods are used to persistently genetically modify and derive SCs (Table 1), but most rely on integrating lentiviral vectors. Problems associated with random insertion into untranscribed regulatory regions (50UTR) and consequent dysregulation of neighboring genes (Cattoglio et al, 2007) (Kotterman et al, 2015) affect the use of lentiviruses in SCs (Herbst et al, 2012)

Results

Discussion

Conclusion