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

Santhosh Chakkaramakkil Verghese, Natalya A. Goloviznina, Amy Skinner, Hans J. Lipps, Peter Kurre

Research output: Contribution to journalArticle

25 Citations (Scopus)

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.

Original languageEnglish (US)
JournalNucleic Acids Research
Volume42
Issue number7
DOIs
StatePublished - 2014

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Cell Division
Plasmids
Maintenance
Integrases
Chimerism
Replication Origin
Insertional Mutagenesis
Cell Lineage
Cell- and Tissue-Based Therapy
Hematopoietic Stem Cells
Transgenes
Oncogenes
Genetic Therapy
Interferons
HIV-1
Leukocytes
Stem Cells
Clone Cells
Transplantation
Genome

ASJC Scopus subject areas

  • Genetics

Cite this

S/MAR sequence confers long-term mitotic stability on non-integrating lentiviral vector episomes without selection. / Verghese, Santhosh Chakkaramakkil; Goloviznina, Natalya A.; Skinner, Amy; Lipps, Hans J.; Kurre, Peter.

In: Nucleic Acids Research, Vol. 42, No. 7, 2014.

Research output: Contribution to journalArticle

Verghese, Santhosh Chakkaramakkil ; Goloviznina, Natalya A. ; Skinner, Amy ; Lipps, Hans J. ; Kurre, Peter. / S/MAR sequence confers long-term mitotic stability on non-integrating lentiviral vector episomes without selection. In: Nucleic Acids Research. 2014 ; Vol. 42, No. 7.
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