Stable differentiation and clonality of murine long-term hematopoiesis after extended reduced-intensity selection for MGMT P140K transgene expression

Ball, Claudia R.; Pilz, Ingo H.; Schmidt, Manfred; Fessler, Sylvia; Williams, David A.; Kalle, Christof von; Glimm, Hanno

doi:10.1182/blood-2006-11-053710

Cited by 14 publications

(19 citation statements)

References 55 publications

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“…Others have observed successful in vivo selection using nontherapeutic, single-gene ␥-retroviral or foamy viral marking vectors and low MOI for HSC transduction to result in initial low levels of transduced cells. 35,36 Chang et al, using a lentiviral vector encoding both human factor IX and MGMT driven by the cellular PGK promoter, showed that in vivo selection could be used to increase factor IX levels from very low levels to those that would be curative. 37 However, in this study, the VCN reached 2.3 after therapeutic selection, consistent with some clones having multiple vector copies.…”

Section: Discussionmentioning

confidence: 99%

“…Others have also observed that more efficient in vivo selection occurs when there is an initial threshold number of vector-expressing cells. 35 Thus, it is difficult to conclude whether the EF1␣-MGMT-wPRE-containing vector is superior to the MSCV-MGMT-based vector. However, given recent genotoxicity studies from our and other groups, the cellular EF1␣ promoter offers an enhanced safety profile.…”

Section: Discussionmentioning

confidence: 99%

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Amelioration of murine β-thalassemia through drug selection of hematopoietic stem cells transduced with a lentiviral vector encoding both γ-globin and the MGMT drug-resistance gene

Zhao¹,

Pestina²,

Nasimuzzaman³

et al. 2009

Blood

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Correction of murine models of ␤-thalassemia has been achieved through high-level globin lentiviral vector gene transfer into mouse hematopoietic stem cells (HSCs). However, transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells. We therefore developed a double gene lentiviral vector encoding both human ␥-globin under the transcriptional control of erythroid regulatory elements and methylguanine methyltransferase (MGMT), driven by a constitutive cellu-

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Amelioration of murine β-thalassemia through drug selection of hematopoietic stem cells transduced with a lentiviral vector encoding both γ-globin and the MGMT drug-resistance gene

Zhao¹,

Pestina²,

Nasimuzzaman³

et al. 2009

Blood

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“…23,53,54 Previous work in a murine model of MGMTP140K demonstrated that clones survived serial transplantation and multiple rounds of chemotherapy, but the RIS profile was not studied due to a relatively limited number of identified integration sites. 55 Therefore, we wanted to determine whether in vivo selection using MGMTP140K caused a disproportionate bias for RIS in or near proto-oncogene TSS as an indication of a preleukemic state. Although numerous RIS were identified less than 500 K from proto-oncogene TSS, including genes such as MDS1/Evi1 and LMO2, there was no overall selection for these sites after multiple rounds of chemotherapy, indicating that selection is dependent upon the protection elicited by the overexpression of MGMTP140K during chemotherapy and not due to outgrowth of potentially dangerous clones with an intrinsic growth advantage.…”

Section: Discussionmentioning

confidence: 99%

Long-term polyclonal and multilineage engraftment of methylguanine methyltransferase P140K gene-modified dog hematopoietic cells in primary and secondary recipients

Beard

Sud

Keyser

et al. 2009

Blood

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Overexpression of methylguanine methyltransferase P140K (MGMTP140K) has been successfully used for in vivo selection and chemoprotection in mouse and large animal studies, and has promise for autologous and allogeneic gene therapy. We examined the long-term safety of MGMTP140K selection in a clinically relevant dog model. Based on the association of provirus integration and protooncogene activation leading to leukemia in the X-linked immunodeficiency trial, we focused our analysis on the distribution of retrovirus integration sites (RIS) relative to proto-oncogene transcription start sites (TSS). We analyzed RIS near protooncogene TSS before (n ‫؍‬ 157) and after (n ‫؍‬ 129) chemotherapy in dogs that received MGMTP140K gene-modified cells and identified no overall increase of RIS near proto-oncogene TSS after chemotherapy. We also wanted to determine whether in vivo selected cells retained fundamental characteristics of hematopoietic stem cells. To that end, we performed secondary transplantation of IntroductionHematopoietic stem cell (HSC) gene therapy has advanced to the point at which engraftment of gene-modified cells in large animal models has reached clinically therapeutic levels (Ͼ 10%-15%) for a variety of diseases (for review, see Neff et al 1 ). In addition, several significant clinical gene therapy trials have clearly demonstrated the potential of retrovirus gene-modified cells as an effective cure for the genetic diseases of X-linked severe combined immunodeficiency (SCID-X1), 2 adenosine deaminase-deficient severe combined immunodeficiency, 3 and X-linked chronic granulomatous disease (X-CGD). 4 These large animal and clinical studies are aided by either the use of ablative radiation or an inherent growth/survival advantage of the gene-modified cells after transplantation. For many other single-gene genetic diseases (ie, ␤-thalassemias and pyruvate kinase deficiency), acquired diseases (ie, glioblastoma and acute myeloid leukemia), or infectious diseases (ie, AIDS) in which retrovirus gene therapy is appropriate, a potential limiting curative factor is gene-marking levels below therapeutic thresholds due to the inability to use ablative conditioning and no substantial growth advantage/ in vivo selection of gene-modified cells (for reviews, see Neff et al 1 and Trobridge et al 5 ). This requires alternate in vivo selection strategies after transplantation to increase the percentage of gene-modified cells to therapeutic levels. To realize the full clinical potential of retrovirus gene therapy, safe in vivo selection strategies using drug resistance gene therapy will be critical to propel these and other promising gene transfer applications.The basic premise of drug resistance gene therapy is to stably deliver a transgene that confers resistance to a cytotoxic drug via several different mechanisms (for reviews, see Neff et al, 1 Trobridge et al, 5 and Sorrentino 6 ). Several selectable markers have been extensively evaluated because of their ability to protect cells from cytotoxic agents, including d...

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“…A number of drug-resistance enzymes, and particularly drug-resistant human dihydrofolate reductase (hDHFR) Allay et al, 1998;Gori et al, 2007) and O 6 -methylguanine-DNA-methyltransferase (MGMT) variants (Fontes et al, 2006;Kramer et al, 2006;Ball et al, 2007) have been tested for application as selective markers for mammalian cells. Transduction of a drug-resistance gene in hematopoietic stem cells (HSC) offers the possibility to enrich the transduced cells by administration of the corresponding myelo-suppressive chemotherapy agent prior to or following engraftment.…”

Section: Introductionmentioning

confidence: 99%

Selectively weakened binding of methotrexate by human dihydrofolate reductase allows rapid ex vivo selection of mammalian cells

Volpato

Mayotte

Fossati

et al. 2011

J of Molecular Recognition

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Ex vivo selection of transduced hematopoietic stem cells (HSC) with drug-resistance genes offers the possibility to enrich transduced cells prior to engraftment, toward increased reconstitution in transplant recipients. We evaluated the potential of highly methotrexate (MTX)-resistant variants of human dihydrofolate reductase (hDHFR) for this application. Two subsets of hDHFR variants with reduced affinity for MTX that had been previously identified in a bacterial system were considered: those with substitutions at positions 31, 34, and/or 35, and those with substitutions at position 115. The variants were characterized for their resistance to pemetrexed (PMTX), an antifolate that is related to MTX. We observed a strong correlation between decreased binding to both antifolates, although the identity of specific sequence variations modulated the correlation. We chose a subset of hDHFR variants for tests of ex vivo MTX resistance, taking into consideration their residual specific activity and their decrease in affinity for the related antifolates. Murine myeloid progenitors and other differentiated hematopoietic cells were transduced and exposed to MTX in a nucleotide-free medium. Bone marrow (BM) cells including 15% cells infected with F31R/Q35E were enriched to 98% transduced cells within 6 days of ex vivo selection. hDHFR variant F31R/Q35E allowed a strong ex vivo enrichment upon a short exposure to MTX relative to a less resistant variant of hDHFR, L22Y. We have thus demonstrated that bacterial selection of highly antifolate-resistant hDHFR variants can provide selectable markers for rapid ex vivo enrichment of hematopoietic cells.

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Stable differentiation and clonality of murine long-term hematopoiesis after extended reduced-intensity selection for MGMT P140K transgene expression

Cited by 14 publications

References 55 publications

Amelioration of murine β-thalassemia through drug selection of hematopoietic stem cells transduced with a lentiviral vector encoding both γ-globin and the MGMT drug-resistance gene

Amelioration of murine β-thalassemia through drug selection of hematopoietic stem cells transduced with a lentiviral vector encoding both γ-globin and the MGMT drug-resistance gene

Long-term polyclonal and multilineage engraftment of methylguanine methyltransferase P140K gene-modified dog hematopoietic cells in primary and secondary recipients

Selectively weakened binding of methotrexate by human dihydrofolate reductase allows rapid ex vivo selection of mammalian cells

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