| Autor: |
Verghese SC; 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., Goloviznina NA, Skinner AM, Lipps HJ, Kurre P |
| Abstrakt: |
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. |
