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Lentiviral MGMT(P140K)-mediated in vivo selection employing a ubiquitous chromatin opening element (A2UCOE) linked to a cellular promoter.

Abstract
Notwithstanding recent successes, insertional mutagenesis as well as silencing and variegation of transgene expression still represent considerable obstacles to hematopoietic gene therapy. This also applies to O(6)-methylguanine DNA methyltransferase (MGMT)-mediated myeloprotection, a concept recently proven clinically effective in the context of glioblastoma therapy. To improve on this situation we here evaluate a SIN-lentiviral vector expressing the MGMT(P140K)-cDNA from a combined A2UCOE/PGK-promoter. In a murine in vivo chemoselection model the A2UCOE.PGK.MGMT construct allowed for significant myeloprotection as well as robust and stable selection of transgenic hematopoietic cells. In contrast, only transient enrichment and severe myelotoxicity was observed for a PGK.MGMT control vector. Selection of A2UCOE.PGK.MGMT-transduced myeloid and lymphoid mature and progenitor cells was demonstrated in the peripheral blood, bone marrow, spleen, and thymus. Unlike the PGK and SFFV promoters used as controls, the A2UCOE.PGK promoter allowed for sustained vector copy number-related transgene expression throughout the experiment indicating an increased resistance to silencing, which was further confirmed by CpG methylation studies of the PGK promoter. Thus, our data support a potential role of the A2UCOE.PGK.MGMT-vector in future MGMT-based myeloprotection and chemoselection strategies, and underlines the suitability of the A2UCOE element to stabilize lentiviral transgene expression in hematopoietic gene therapy.
AuthorsRuhi Phaltane, Nico Lachmann, Sebastian Brennig, Mania Ackermann, Ute Modlich, Thomas Moritz
JournalBiomaterials (Biomaterials) Vol. 35 Issue 25 Pg. 7204-13 (Aug 2014) ISSN: 1878-5905 [Electronic] Netherlands
PMID24875758 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
CopyrightCopyright © 2014 Elsevier Ltd. All rights reserved.
Chemical References
  • Antineoplastic Agents
  • Chromatin
  • O(6)-benzylguanine
  • Guanine
  • O(6)-Methylguanine-DNA Methyltransferase
  • Phosphoglycerate Kinase
Topics
  • Animals
  • Antineoplastic Agents (pharmacology)
  • Chromatin (chemistry, genetics)
  • Cloning, Molecular
  • DNA Copy Number Variations
  • Female
  • Gene Expression
  • Genetic Therapy
  • Genetic Vectors
  • Guanine (analogs & derivatives, pharmacology)
  • Hematopoietic Stem Cells (metabolism)
  • Lentivirus (genetics)
  • Mice
  • Mice, Inbred C57BL
  • O(6)-Methylguanine-DNA Methyltransferase (chemistry, genetics)
  • Phosphoglycerate Kinase (chemistry, genetics)
  • Promoter Regions, Genetic
  • Sequence Analysis, DNA
  • Transgenes

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