S/MAR sequence confers long-term mitotic stability on non-integrating lentiviral vector episomes without selection

Abstract

Insertional oncogene activation and aberrant splicing have proved to be major setbacks for retroviral stem cell gene therapy. Integrase-deficient human immunodeficiency virus-1-derived vectors provide a potentially safer approach, but their circular genomes are rapidly lost during cell division. Here we describe a novel lentiviral vector (LV) that incorporates human ß-interferon scaffold/matrix-associated region sequences to provide an origin of replication for long-term mitotic maintenance of the episomal LTR circles. The resulting ‘anchoring’ non-integrating lentiviral vector (aniLV) achieved initial transduction rates comparable with integrating vector followed by progressive establishment of long-term episomal expression in a subset of cells. Analysis of aniLV-transduced single cell-derived clones maintained without selective pressure for >100 rounds of cell division showed sustained transgene expression from episomes and provided molecular evidence for long-term episome maintenance. To evaluate aniLV performance in primary cells, we transduced lineage-depleted murine hematopoietic progenitor cells, observing GFP expression in clonogenic progenitor colonies and peripheral blood leukocyte chimerism following transplantation into conditioned hosts. In aggregate, our studies suggest that scaffold/matrix-associated region elements can serve as molecular anchors for non-integrating lentivector episomes, providing sustained gene expression through successive rounds of cell division and progenitor differentiation in vitro and in vivo.

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

“…In the present study, the minimal S/MAR sequence in the SNIL vector enabled the sustained expression of eGFP for 61 days, and this expression was detected in approximately 40% of the cells. Verghese et al reported the performance of the aniLV vector, a NIL vector containing the original 2.1-kb S/MAR sequence (Verghese et al, 2014). The authors used the original long S/MAR sequence and detected persistent GFP expression for 56 days, although GFP expression was detected only in 10% of the aniLV stably transduced cells.…”

Characteristic element of matrix attachment region mediates vector attachment and enhances nerve growth factor expression in Chinese hamster ovary cells

“…The use of attached vectors eliminates the potential risks of integrated vectors, such as the generation of mutations (Koirala et al, 2014). Attached vectors expressing the MAR element have attracted a lot of attention (Argyros et al, 2012;Mizutani et al, 2013;Verghese et al, 2014) because they are not integrated into the host (mammalian cell) genome. They bind to the matrix during the mitotic phase, and their stable attachment can prompt a highly effective, continuous, and safe expression.…”

Retrovirus-based vectors for transient and permanent cell modification

“…LV episomes equipped with a scaffold/matrix attachment region (S/MAR) element derived from human interferon b, which served as an anchoring element, were mitotically stable even without selection pressure. However, although above background levels, in vivo expression after transplantation of transduced hematopoietic progenitors in mice was low [51]. Alternatively, insertion of the simian virus 40 (SV40) origin of replication (oriT) could provide a means to achieve SV40 large T-dependent LV episome replication.…”