Lentiviral MGMT(P140K)-mediated in vivo selection employing a ubiquitous chromatin opening element (A2UCOE) linked to a cellular promoter.
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.
['Phaltane|Ruhi|R|', 'Lachmann|Nico|N|', 'Brennig|Sebastian|S|', 'Ackermann|Mania|M|', 'Modlich|Ute|U|', 'Moritz|Thomas|T|']
D000818:Animals D000970:Antineoplastic Agents D002843:Chromatin D003001:Cloning, Molecular D056915:DNA Copy Number Variations D005260:Female D015870:Gene Expression D015316:Genetic Therapy D005822:Genetic Vectors D006147:Guanine D006412:Hematopoietic Stem Cells D016086:Lentivirus D051379:Mice D008810:Mice, Inbred C57BL D019853:O(6)-Methylguanine-DNA Methyltransferase D010735:Phosphoglycerate Kinase D011401:Promoter Regions, Genetic D017422:Sequence Analysis, DNA D019076:Transgenes
Gene therapy In vivo selection Lentiviral vector MGMT UCOE