Hematopoietic stem cell gene therapy of murine protoporphyria by methylguanine-DNA-methyltransferase-mediated in vivo drug selection

Abstract

Erythropoietic protoporphyria (EPP) is an inherited defect of the ferrochelatase (FECH) gene characterized by the accumulation of toxic protoporphyrin in the liver and bone marrow resulting in severe skin photosensitivity. We previously described successful gene therapy of an animal model of the disease with erythroid-specific lentiviral vectors in the absence of preselection of corrected cells. However, the high-level of gene transfer obtained in mice is not translatable to large animal models and humans if there is no selective advantage for genetically modified hematopoietic stem cells (HSCs) in vivo. We used bicistronic SIN-lentiviral vectors coexpressing EGFP or FECH and the G156A-mutated O 6 -methylguanine-DNA-methyltransferase (MGMT) gene, which allowed efficient in vivo selection of transduced HSCs after O 6 -benzylguanine and BCNU treatment. We demonstrate for the first time that the correction and in vivo expansion of deficient transduced HSC population can be obtained by this dual gene therapy, resulting in a progressive increase of normal RBCs in EPP mice and a complete correction of skin photosensitivity. Finally, we developed a novel bipromoter SIN-lentiviral vector with a constitutive expression of MGMT gene to allow the selection of HSCs and with an erythroid-specific expression of the FECH therapeutic gene.

Living Donor Liver Transplantation for Pediatric Patients with Inheritable Metabolic Disorders

“…However, all of these reported therapies were symptomatic treatments and the evidence of their efficacy seemed to be anecdotal. To achieve a satisfactory outcome in the treatment for NLOD, a breakthrough of some sort will be needed, such as development of a gene therapy (24)(25)(26) to eradicate the intrinsic underlying disorders. At this time, however, LT combined with these reported symptomatic therapies is the sole therapeutic procedure for NLOD patients with severe manifestations.…”

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

“…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).…”

Live and let die: In vivo selection of gene-modified hematopoietic stem cells via MGMT-mediated chemoprotection

“…In murine studies, multiple groups have demonstrated that MGMT transduced HSC are able to reconstitute secondary recipients after a chemoselective regimen has been applied [ 35,37,38,60,65,118 ]. No instances of abnormal hematopoiesis or increased rates of hematological malignancy have been reported.…”