Differential Secondary Reconstitution ofIn Vivo-Selected Human SCID-Repopulating Cells in NOD/SCID versus NOD/SCID/γ chain^nullMice

Abstract

Humanized bone-marrow xenograft models that can monitor the long-term impact of gene-therapy strategies will help facilitate evaluation of clinical utility. The ability of the murine bone-marrow microenvironment in NOD/SCID versus NOD/SCID/γ chainnull mice to support long-term engraftment of MGMTP140K-transduced human-hematopoietic cells following alkylator-mediated in vivo selection was investigated. Mice were transplanted with MGMTP140K-transduced CD34+ cells and transduced cells selected in vivo. At 4 months after transplantation, levels of human-cell engraftment, and MGMTP140K-transduced cells in the bone marrow of NOD/SCID versus NSG mice varied slightly in vehicle- and drug-treated mice. In secondary transplants, although equal numbers of MGMTP140K-transduced human cells were transplanted, engraftment was significantly higher in NOD/SCID/γ chainnull mice compared to NOD/SCID mice at 2 months after transplantation. These data indicate that reconstitution of NOD/SCID/γ chainnull mice with human-hematopoietic cells represents a more promising model in which to test for genotoxicity and efficacy of strategies that focus on manipulation of long-term repopulating cells of human origin.

Efficiency and Safety of O6-Methylguanine DNA Methyltransferase (MGMTP140K)-MediatedIn VivoSelection in a Humanized Mouse Model

“…Recapitulating earlier studies by other groups in murine as well as xenotransplant models (Reese et al, 1999;Ragg et al, 2000;Sawai et al, 2001;Jansen et al, 2002;Pollok et al, 2003;Zielske et al, 2003;Cai et al, 2006Cai et al, , 2010Milsom et al, 2008;Giordano et al, 2011), our study documents robust and stable chemoselection of transgenic human cells over extended periods in a model employing the xenotransplantation of MGMT-transduced CB-derived CD34 integration pattern. An overall oligoclonal repertoire was detected in all experimental groups at the end of the 24-28-week observation period without enrichment of clones with integrations in or near-specific gene classes.…”

“…An overall oligoclonal repertoire was detected in all experimental groups at the end of the 24-28-week observation period without enrichment of clones with integrations in or near-specific gene classes. In the past, the majority of studies on MGMT-mediated in vivo selection, including the recent clinical trial, primarily used gammaretroviral vectors (Pollok et al, 2003;Zielske et al, 2003;Cai et al, 2006Cai et al, , 2008Cai et al, , 2010Adair et al, 2012). As these vectors have a preference to integrate near the TSS of genes and carry a substantial risk of gene activation, we here evaluated a SIN lentiviral vector in order to reduce this risk.…”

“…Transgenic overexpression of the O 6 -benzylguanine (BG)-resistant MGMT P140K point mutant has been demonstrated to allow for efficient enrichment of transduced hematopoietic cells by the combined application of the BG and BCNU or TMZ. These studies have been conducted in murine models (Reese et al, 1999;Ragg et al, 2000;Sawai et al, 2001;Jansen et al, 2002;Davis et al, 2003;Milsom et al, 2008;Giordano et al, 2011), including models of b-thalassemia (Persons et al, 2003;Zhao et al, 2009) and erythropoietic protoporphyria (Richard et al, 2004), humanized mice (Pollok et al, 2003;Zielske et al, 2003;Cai et al, 2006Cai et al, , 2008Cai et al, , 2010, as well as large animal models (Neff et al, 2003(Neff et al, , 2005Beard et al, 2009Beard et al, , 2010Larochelle et al, 2009;Gori et al, 2012;Trobridge et al, 2012). MGMT P140K (henceforth referred to as MGMT) also has been utilized to select for primary lung epithelium (Reese et al, 2008) and muscle cells (Lee et al, 2009) in vivo or to enrich for transgenic and protected lymphocytes in the context of novel gene therapy approaches for AIDS (Trobridge et al, 2009;Kiem et al, 2010).…”

“…Nevertheless, a number of questions remain, as some of these studies were compromised by relatively short observation periods (Pollok et al, 2003;Zielske et al, 2003;Cai et al, 2006) or insufficient depth of clonal analysis (Neff et al, 2003(Neff et al, , 2005Beard et al, 2009Beard et al, , 2010Larochelle et al, 2009). Furthermore, the majority of these studies utilized gammaretroviral vectors (Pollok et al, 2003;Zielske et al, 2003;Cai et al, 2006Cai et al, , 2008Cai et al, , 2010Adair et al, 2012), which inherently carry a relatively high risk for insertional mutagenesis (Modlich et al, 2009). Therefore, to improve the safety of MGMT-mediated myeloprotection and in vivo chemoselection strategies, we here analyzed MGMT expression from a third-generation SIN lentiviral vector employing the truncated elongation factor 1a (i.e., elongation factor-1a short version; EFS) promoter for transgene expression.…”

Therapeutic Modulation of DNA Damage and Repair Mechanisms in Blood Cells

“…Haiyan Wang, Shanbao Cai, Eva Tonsing-Carter and Karen E. Pollok (2011). Therapeutic Modulation of DNA Damage and Repair Mechanisms in Blood Cells, DNA Repair and Human Health, Dr. Sonya Vengrova (Ed.…”