Non-integrating lentiviral vectors based on the minimal S/MAR sequence retain transgene expression in dividing cells.

Zhen Xu,Peng Xu,Yajun Wang,Wenli Mu,Lingling Zhang,Guang Liu,Jing Lu,Feng Chen,Xuemin Xie,Depei Liu

doi:10.1007/s11427-016-0067-0

Abstract

Safe and efficient gene transfer systems are the basis of gene therapy applications. Non-integrating lentiviral (NIL) vectors are among the most promising candidates for gene transfer tools, because they exhibit high transfer efficiency in both dividing and non-dividing cells and do not present a risk of insertional mutagenesis. However, non-integrating lentiviral vectors cannot introduce stable exogenous gene expression to dividing cells, thereby limiting their application. Here, we report the design of a non-integrating lentiviral vector that contains the minimal scaffold/matrix attachment region (S/MAR) sequence (SNIL), and this SNIL vector is able to retain episomal transgene expression in dividing cells. Using SNIL vectors, we detected the expression of the eGFP gene for 61 days in SNIL-transduced stable CHO cells, either with selection or not. In the NIL group without the S/MAR sequence, however, the transduced cells died under selection for the transient expression of NIL vectors. Furthermore, Southern blot assays demonstrated that the SNIL vectors were retained extrachromosomally in the CHO cells. In conclusion, the minimal S/MAR sequence retained the non-integrating lentiviral vectors in dividing cells, which indicates that SNIL vectors have the potential for use as a gene transfer tool.

Highlights

Gene therapy involves introducing a therapeutic gene/RNA into a target cell/tissue to restore deficient protein function caused by a genetic disorder (Kootstra and Verma, 2003; Verma and Weitzman, 2005)
Minimal scaffold/matrix attachment region (S/MAR)-containing non-integrating lentiviral vectors and non-S/MAR controls were packaged from the S/MAR-based non-integrating lentiviral (SNIL) and Non-integrating lentiviral (NIL) plasmids, respectively, using the integrasedefective packaging plasmid pLV-HELP-NIL and pseudotyped using the vesicular stomatitis virus glycoprotein (VSV-G) envelope protein
The 48-h p24 titer of the S/MAR-containing lentivirus was 77.905 ng mL 1, which is approximately 9.738×108 LP mL 1 (LP: lentiviral particles; 1 ng p24=1.25×107 lentiviral particles as indicated by the lentivirus titer kit manual). These results suggest that the minimal S/MAR sequence does not disrupt transgene expression and viral packaging

Summary

Introduction

Gene therapy involves introducing a therapeutic gene/RNA into a target cell/tissue to restore deficient protein function caused by a genetic disorder (Kootstra and Verma, 2003; Verma and Weitzman, 2005). Many factors influence this process, the success of gene therapy relies considerably on the selection of an appropriate gene delivery system. An ideal gene delivery system should be capable of efficient gene transfer in various cell types without producing a pathogenic effect (Kootstra and Verma, 2003). Human immunodeficiency virus (HIV)-based lentiviral vectors have been accepted as efficient gene transfer tools because they have a relatively large packaging capacity, broad cell tropism, specific cell-type targeting, weak immunogenesis and are produced (Abordo-Adesida et al, 2005; Cronin et al, 2005; Kafri et al, 1997). Similar to other integrating systems, the integrating feature of lentiviral vectors can cause unbalanced gene expression and gene silencing and might even present risks of insertional mutagenesis, which can lead to serious effects, such as malignant transformation (Kohn et al, 2003)

Methods

Results

Conclusion