S/MAR sequence confers long-term mitotic stability on non-integrating lentiviral vector episomes without selection.
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.
Department of Pediatrics, Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, OR 97239, USA, Department of Surgery/Surgical Oncology, Oregon Health & Science University, Portland, OR 97239, USA, Center for Biomedical Education and Research, Institute of Cell Biology, University of Witten/Herdecke, Witten 58453, Germany, Oregon Stem Cell Center, Oregon Health & Science University, Portland, OR 97239, USA and Department of Cell & Developmental Biology Oregon Health & Science University, Portland, OR 97239, USA.
Santhosh Chakkaramakkil
SC
Verghese
Natalya A
NA
Goloviznina
Amy M
AM
Skinner
Hans J
HJ
Lipps
Peter
P
Kurre
D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D016899:Interferon-beta / Q000235:genetics; D016086:Lentivirus / Q000235:genetics*; D045170:Matrix Attachment Regions; D051379:Mice; D008938:Mitosis / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D014161:Transduction, Genetic; D019076:Transgenes
D016899:Interferon-beta