Episomal Anchorage Maintains Non-Integrating Lentiviral Vectors in Dividing Cells

Abstract 4224

Insertional mutagenesis has been a major setback in retroviral stem cell gene therapy. Vector integration events can induce alternative splicing, aberrant transcripts and read-through transcription of endogenous genes. Non-integrating vectors provide a potentially safer approach in some tissue targets. However, due to the lack of nuclear retention, the vector genome is rapidly lost during cell division and differentiation. Genomic Scaffold/Matrix Associated Regions (S/MARs) efficiently anchor the DNA to nuclear matrix proteins to generate chromosome domains in the nucleus and have been successfully used for non-viral approaches to genetic modification. Here we incorporated human ß-interferon derived S/MAR sequence between GFP reporter reading frame and 3'LTR of a non-integrating 3^rd generation lentiviral transfer vector, in an effort to ‘anchor’ the vector (termed ‘aLV’) to the host cell genome in dividing cells. Our approach exploits two key properties of lentiviral life cycle, i.e. nuclear translocation of the viral genomic DNA and circularization to generate episomal genomes comprised of 1-LTR or 2-LTR circles. Integrating vector (iLV) and aLV (integrase-defective gag packaging plasmid) were VSV-G pseudotyped and concentrated, yielding stocks with titers of 2.3 x10⁸ and 3×10⁶ TU/ml, respectively. Transduction of 293T cells with aLV showed dose-dependent transduction and GFP expression similar to iLV. Transduced 293T cells were clonally expanded and maintained in vitro up to 10 weeks. A subset of 22 aLV clones and 9 iLV clones were followed over time and analyzed for transgene expression and molecular persistence of LTR circles. After >70 rounds of cell division, GFP fluorescence intensity was consistently lower, but persisted in aLV-293T clones at MFI 406.5±34.9 versus iLV at MFI 786.3±58.5. As predicted, 1-LTR and 2-LTR vector episomes were detected by PCR in genomic DNA from aLV transduced cells at the 10-week time point. Additional sequencing confirmed the presence of episomal junctions indicating the persistence of completely circularized episomes. With evidence of molecular persistence and GFP expression of S/MAR-anchored, episomal vector genomes in rapidly dividing cell lines, we next tested the performance of aLV in lineage-depleted murine hematopoietic stem and progenitor cells (mHSPCs). Overnight transduction of lin- progenitors at matched MOI yielded 96.3%±22 GFP-positive clonogenic methylcellulose colonies after iLV exposure versus 39.4%±21 after aLV exposure. Transduced mHSPCs were also transplanted into sublethally irradiated C57B/6 mice and peripheral blood leukocytes were analyzed for gene expression. Here GFP expression in the donor compartment averaged 62% for iLV compared with 10% for aLV. We are evaluating leukocyte donor subset GFP expression and will undertake secondary transplantation to formally demonstrate transduction in the stem cell compartment. In summary, lentivector mediated nuclear localization followed by beta-interferon S/MAR anchorage of the vector genome leads to episomal transgene expression in dividing target cells. Our studies suggest that S/MAR elements can serve as molecular anchors for non-integrating lentiviral episomes to provide sustained gene expression through successive rounds of cell division and progenitor differentiation in vitro and in vivo. We propose further study of aLV as a candidate vector for gene delivery to HSPCs while avoiding proviral integration and its potentially deleterious consequences.