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

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.

A chimeric HS4 insulator–scaffold attachment region enhances transgene expression in transfected Chinese hamster ovary cells

“…Matrix attachment regions can increase expression levels of the transgene in stably transfected CHO cells [6][7][8][9][10][11][12]. However, the characteristics and mechanism of MARs function have not been elucidated, and further studies are needed to develop improved methods for transgene expression.…”

“…Scaffold/matrix attachment regions (S/MARs) can block transgene silencing [6][7][8] and increase transgene expression levels and stability in host cells [9][10][11][12][13][14][15]. In addition, S/MARs can also reduce variations in transgene expression among different cells to some extent, and the rate of transgene genomic integration can be increased [11].…”

The IS2 Element Improves Transcription Efficiency of Integration-Deficient Lentiviral Vector Episomes

“…Up to now, most attempts to improve IDLVs have focused on improving the stability of DNA episomal circles either through transient cell cycle arrest 50 or by the inclusion of SAR elements alone 48 , 49 , 50 or combined with replication origin ( http://www.vivebiotech.com/technology ). However, efficient stable transgene expression in highly dividing cells using IDLVs can be difficult to achieve.…”

“…Several research groups have achieved varying levels of success in their attempt to improve IDLV expression by inserting different fragments of the β-interferon SARs element into IDLVs. 48 , 49 , 50 However, the use of SARs elements to improve IDLV transcription efficiency has only been studied in relation to immunoglobulin κ (Igκ) SARs sequences. Grandchamp et al.…”

Effect of promoter, promoter mutation and enhancer on transgene expression mediated by episomal vectors in transfected HEK293, Chang liver and primary cells

“…Gene therapy involves the delivery of a therapeutic gene into a patient's cells as a drug to treat diseases. Vector systems play the key role in driving the expression of transgenes in gene therapy [1,2]. Episomal vectors can replicate in synchrony with each cell cycle division within the host genome for sustained, nonviral and nonintegrating transgene expression in vitro and in vivo [3][4][5], which does not cause insertional mutagenesis in gene therapy [6].…”