Successful Gene Therapy of Murine Paroxysmal Nocturnal Hemoglobinuria (PNH) Using Mgmt-Mediated In Vivo Selection of Genetically Corrected, Drug-Resistant Hematopoietic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 173-173
Author(s):  
François Moreau-Gaudry ◽  
Bing Han ◽  
Emmanuel Richard ◽  
Ike E. Pantazopoulos ◽  
Hubert de Verneuil ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Murine Model ◽  
Blood Cells ◽  
Alkylating Agents ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Selective Growth Advantage ◽  
Selection Of

Abstract PNH is an acquired disorder of the hematopoietic stem cell (HSC) caused by a somatic mutation in the PIGA gene. Blood cells carrying the mutation are deficient in all glycosyl phosphatidylinositol-anchored proteins (GPI-APs). Long-term restoration of GPI-AP has been obtained in vitro using lentiviral vectors stably expressing PIGA in human HSCs. However, gene therapy for PNH might be problematic because of the lack of a growth advantage of phenotypically corrected cells. We hypothesized that providing corrected cells with a selective growth advantage might overcome this possible limitation. We chose to test this in a murine model of PNH that has GPI-AP deficient blood cells because of a somatic Piga gene mutation targeted to the HSCs (LF mice). We designed a novel bicistronic lentiviral vector (MMIP) expressing the human PIGA cDNA and the human alkylating drug resistance mutant O6-methylguanine DNA methyltransferase (MGMT G156A) under the control of the MND promoter. MGMT G156A confers protection against alkylating agents. Eight LF donor mice were injected with 5-FU. Bone marrow (BM) was harvested 5 days later for MMIP transduction performed twice at MOI 100. Transduced and mock transduced BM cells were injected into 15 lethally irradiated C57Bl/6 recipient mice. One month after BM transplantation (BMT) 8 MMIP-transduced mice were treated with the alkylating agents BCNU and O6-benzylguanine (BG) (5mg/kg and 30mg/kg). BCNU /BC treatment was repeated twice, each one month apart. Six months after BMT the percentage of donor engraftment was 75.1 ± 25% for granulocytes (G) and 88.4 ± 26% for lymphocytes (L) in the control group (n=3). Donor chimerism in the MMIP-transduced group not treated with BCNU/BG was 84.6 ± 25 and 87.8 ± 5 % (n=4). The highest donor chimerism was obtained in mice treated with BCNU/BG, 98.9 ± 0.4 and 98.6 ± 0.5 % (n=8). Next we determined the proportion of donor blood cells with a restored expression of GPI-linked proteins. Only a small proportion of GPI-AP expressing cells was found in mice receiving the mock transduced BM six months after BMT, 22±37% GPI-AP expressing RBCs; 3.1± 2% GPI-AP expressing G, and 0.5 ± 0.2% GPI-AP expressing L. In contrast, mice receiving the MMIP-transduced BM cells but not treated with BCNU/BG showed a higher level of GPI-AP+ cells: 87.8±16% for RBCs, 70.2 ±23% for G and 56.3 ± 13% for L (n=4). However, mice receiving the MMIP-transduced BM and BG/BCNU treatment, achieved an almost complete restoration of GPI-AP on peripheral blood cells: 98±3% of RBCs, 96.4 ±4.7% of G and 89.6 ± 13% of L (n=8). Findings in BM, in methylcellulose progenitor assays, and in secondary spleen colony assays confirmed that restoration of GPI-AP and BCNU/BG selection had occurred at the HSC level. This is the first report of long-term restoration of GPI-APs expression in vivo using a murine model for PNH. Our results demonstrate that BCNU/BG treatment is well tolerated (100% survival) leading to a strong in vivo selection of HSCs with restored expression GPI-AP. The selective growth advantage of converted HSCs after dual gene therapy followed by BCNU/BG treatment allows us to achieve and possibly maintain a high level of hematopoiesis with restored GPI-AP expression. The selection for the corrected HSCs might allow them to overcome a possible growth disadvantage in the competition between PNH and normal HSCs.

In Vivo Selection Of Genetically Manipulated Hematopoietic Stem Cells For Platelet Gene Therapy Of Hemophilia A

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2329-2329
Author(s):  
Qizhen Shi ◽  
Jocelyn A. Schroeder ◽  
David A. Wilcox ◽  
Robert R. Montgomery ◽  
Yingyu Chen
Keyword(s):  
Gene Therapy ◽  
Hemophilia A ◽  
Genetically Modified ◽  
Conditioning Regimen ◽  
Selective Treatment ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Fviii Activity ◽  
Selection Of

Abstract Our previous studies have demonstrated that targeting FVIII expression to platelets (2bF8) by lentiviral (LV) gene delivery to hematopoietic stem cells (HSCs) corrects bleeding diathesis in hemophilia A mice with or without inhibitors. Although the bleeding diathesis is improved in transduced recipients, the transduction efficiency using our current 2bF8 LV, is only about 10%, resulting a median level of platelet-FVIII (Plt-F8) of 1.5 mU/108 platelets even thought a myeloablative conditioning regimen was employed. It has been shown in clinical trials that efficient stem cell gene transfer and myeloablation is not required when there is a powerful selective advantage to the genetically modified cells. We hypothesize that incorporating a drug-resistance gene into the 2bF8 LV construct will allow for in vivo selection of 2bF8 LV-transduced cells which will result in the increase of therapeutic levels of Plt-F8 for hemophilia A gene therapy and reduce the potential for genotoxicity. To address our hypothesis, we constructed a new lentiviral vector, pWPT-2bF8/MGMT, which harbors dual genes, the 2bF8 gene and a drug-resistance gene, the MGMTP140K cassette. To explore the feasibility of the MGMT-based in vivo selection system, HSCs from FVIIInull mice were transduced with 2bF8/MGMT LV at an MOI (multiplicity of infectious) of 1, which is 1/10 of the MOI used for our regular 2bF8 LV transduction, and transplanted into littermates pre-conditioned with a non-myeloablative regimen, 660 cGy total body irradiation (TBI). We chose a low MOI because one of the goals of using the MGMT selection system is to reduce the potential for genotoxicity. After bone marrow (BM) reconstitution, the recipients were treated with O6-benzylguanine (BG) followed by 1, 3-bis-2 chloroethyl-1-nitrosourea (BCNU) monthly for 3 or 4 times. As determined by a chromogenic assay on platelet lysates, functional Plt-F8 expression in recipients was only 0.22 ± 0.15 mU/108 platelets before the drug treatment, but remarkably increased to 4.33 ± 5.48 mU/108 platelets (n = 16) after BG/BCNU drug-selective treatments. The levels of Plt-F8 in the untreated transduced control group remained low over the study period. FVIII activity was not detected in the plasma in any of the recipients even with Plt-F8 as high as 22 mU/108 platelets. The average copy number of 2bF8/MGMT proviral DNA per cell was determined by quantitative real-time PCR. 2bF8 proviral DNA was barely detectable (0.01 ± 0.02 copies/cell) in recipients before drug-selective treatment, but it increased to 0.42 ± 0.15 copies/cell after BG/BCNU treatments, confirming that 2bF8/MGMT genetically modified cells were effectively enriched in vivo after drug-selective treatment. When the tail clip survival test was used to assess phenotypic correction of the FVIIInull coagulation defect, 15 of 16 treated animals survived the tail clip challenge; in contrast, none of the untransduced FVIIInull control mice survived. When ROTEM analysis was used to determine the whole blood clotting time (CT), the CT was shortened from 3043 ± 728 seconds (n = 7) to 931± 273 seconds (n = 6) (P < 0.0001) in treated transduced recipients when compared to FVIIInull mice. There was no significant difference between wild type (722 ± 270 seconds, n = 7) and treated recipients. To ensure sustained Plt-F8 expression in BG/BCNU treated transduced recipients, some primary recipients were sacrificed 9 months after transplantation and BM mononuclear cells were transplanted into secondary recipients. Platelet lysate FVIII activity assays showed that the levels of Plt-F8 in secondary recipients were similar to those in primary recipients, confirming that long-term repopulating HSCs were successfully genetically modified by 2bF8/MGMT LV. When a low intensity pre-conditioning regimen of 440 cGy TBI was used, the levels of Plt-F8 increased from 0.06 ± 0.12 mU/108 platelets to 1.86 ± 2.06 mU/108 platelets after BG/BCNU drug-selective treatment. It is notable that no anti-FVIII inhibitory antibodies were detected in the treated recipients even after rhFVIII challenge, indicating that immune tolerance was induced in the treated animals. In contrast, all FVIIInull mice under the same challenge developed various levels of inhibitors. Taken together, we have established a powerful in vivo selective system that allows us to enrich 2bF8 LV-transduced cells and to enhance platelet-FVIII expression for hemophilia A gene therapy. Disclosures: No relevant conflicts of interest to declare.

Transient in vivo selection of transduced peripheral blood cells using antifolate drug selection in rhesus macaques that received transplants with hematopoietic stem cells expressing dihydrofolate reductase vectors

Blood ◽  
2004 ◽  
Vol 103 (3) ◽  
pp. 796-803 ◽  
Author(s):  
Derek A. Persons ◽  
James A. Allay ◽  
Aylin Bonifacino ◽  
Taihe Lu ◽  
Brian Agricola ◽  
...  
Keyword(s):  
Stem Cells ◽  
Drug Resistance ◽  
Drug Treatment ◽  
Blood Cells ◽  
Rhesus Macaques ◽  
Peripheral Blood Cells ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Selection Of

Abstract One of the main obstacles for effective human gene therapy for hematopoietic disorders remains the achievement of an adequate number of genetically corrected blood cells. One approach to this goal is to incorporate drug resistance genes into vectors to enable in vivo selection of hematopoietic stem cells (HSCs). Although a number of drug resistance vectors enable HSC selection in murine systems, little is known about these systems in large animal models. To address this issue, we transplanted cells transduced with dihydrofolate resistance vectors into 6 rhesus macaques and studied whether selection of vector-expressing cells occurred following drug treatment with trimetrexate and nitrobenzylmercaptopurineriboside-phosphate. In some of the 10 administered drug treatment courses, substantial increases in the levels of transduced peripheral blood cells were noted; however, numbers returned to baseline levels within 17 days. Attempts to induce stem cell cycling with stem cell factor and granulocyte-colony stimulating factor prior to drug treatment did not lead to sustained enrichment for transduced cells. These data highlight an important species-specific difference between murine and nonhuman primate models for assessing in vivo HSC selection strategies and emphasize the importance of using drugs capable of inducing selective pressure at the level of HSCs.

In Vivo Selection and Long-Term Engraftment Of Hematopoietic Stem Cells Generated Via Vascular Niche Induction Of Nonhuman Primate Induced Pluripotent Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 466-466
Author(s):  
Jennifer L Gori ◽  
Jason M Butler ◽  
Devikha Chandrasekaran ◽  
Brian C Beard ◽  
Daniel J Nolan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Nonhuman Primate ◽  
Fold Increase ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Selection Of

Clinical use of human pluripotent stem cell (PSC)-hematopoietic stem cells (HSCs) is impeded by low engraftment potential. This block suggests that additional vascular derived angiocrine signals and hematopoietic cues must be provided to produce authentic HSCs. In addition, gene modification of induced (i)PSCs with a chemotherapy resistance transgene would provide a selective mechanism to stabilize or increase engraftment of HSCs. We therefore hypothesized that modifying iPSCs to express the O6-benzylguanine (O6BG)-resistant P140K variant of methylguanine methyltransferase (MGMT), would support in vivo selection of early-engrafted iPSC-HSCs. We further postulated that Akt-activated human endothelial cells afforded by transduction of the E4ORF1 gene (E4ORF1+ECs) through angiocrine upregulation of Notch and IGF ligands would provide the necessary signals under xenobiotic-free conditions to promote definitive hematopoiesis. This vascular induction platform could drive the emergence of true HSCs. We focused on pigtail macaque (Mn)iPSCs, as a scalable, clinically relevant nonhuman primate model. MniPSCs modified to express P140K had 15-fold higher MGMT levels compared to levels in human peripheral blood mononuclear cells. P140K-MniPSCs differentiated into chemoresistant CD34+ hematopoietic progenitors (50% CD34+) with a predominant long-term (LT)-HSC-like phenotype (CD34+CD38-Thy1+CD45RA-CD49f+). Hematopoietic progenitors maintained colony forming potential after O6BG and bis-chloroethylnitrosourea (BCNU) treatment. HSCs expanded on E4ORF1+ECs maintained colony forming potential, in contrast to cells cultured with cytokines alone, with a 22-fold increase in CD34+ cell content and 10-fold increase in LT-HSC-like cells. Importantly, MniPSC-HSCs expanded with the E4ORF1+ECs had long-term engraftment in NSG mice at levels comparable to Mn bone marrow HSC engrafted mice. O6BG/BCNU treatment increased engraftment to 35% CD45+ cells the blood of mice transplanted with E4ORF1+EC expanded P140K-MniPSC-HSCs, which was maintained 16 weeks post transplantation. Primate CD45+ cell levels in the blood after selection were significantly higher for this cohort compared to mice transplanted with P140K-MniPSC-HSCs expanded in the “cytokines alone” condition (18% vs. 3% CD45+, P<0.05). On average, 15% CD34+ and 37% CD45+ cells were detected in the bone marrow of mice transplanted with E4ORF1+EC-expanded P140K-MniPSC HSCs, which is significantly higher than levels detected in the other cohorts (Table 1). CD45+ cells in the marrow were predominantly myeloid but lymphoid subsets were also present (10-25% CD3+ cells). Remarkably, the level of gene marking in CFCs and number of gene marked CFCs from mouse bone marrow was substantially higher for mice transplanted with E4ORF1+EC expanded compared to cytokine expanded P140K-MniPSC-HSCs (Table 1). Finally, to confirm engraftment of authentic HSCs, secondary transplants were established. Although engraftment was achieved in all secondary transplanted cohorts, the level of nonhuman primate cells detected was significantly higher in animals transplanted with E4ORF1+EC expanded P140K-MniPSC-HSCs. Significantly more lymphocytes (CD45+CD3+ and CD45+CD56+) and monocytes (CD45+CD14+) were detected in the blood of these secondary transplant recipients. These findings confirm generation of bona fide HSCs derived from nonhuman primate iPSCs and demonstrate that O6BG/BCNU chemotherapy supports in vivo selection of P140K-MniPSC-HSCs generated by co-culture with the E4ORF1+EC vascular platform. Our studies mark a significant advance toward clinical translation of PSC-based blood therapeutics and the development of a nonhuman primate preclinical model. Table 1 CD34+ and CD45+ engraftment and gene marking in the bone marrow of mice transplanted with nonhuman primate HPSCs from MniPSCs and bone marrow. HSCs E4ORF1+ECs O6BG/BCNU Mean %CD34+ Mean %CD45+ % gene marking in CFCs (lentivirus+) total lentivirus+ CFCs per 105 cells GFP-MniPSC + - 3 16 9 ± 2 13 ± 2 P140K-MniPSC + - 4 19 12 ± 5 17 ± 7 P140K-MniPSC - + 0.4 24 3 ± 2 2 ± 1 P140K-MniPSC + + 15 37 27 ± 24 111 ± 96 Mn BM CD34+ - - 2 21 0 0 Disclosures: Nolan: Angiocrine Bioscience: Employment. Ginsberg:Angiocrine Bioscience: Employment. Rafii:Angiocrine Bioscience: Founder Other.

Transduction of Macaque Hematopoietic Repopulating Cells with Lenti and Foamy Retroviral Vectors with MGMT Selection Cassettes To Evaluate AIDS Gene Therapy Strategies.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3273-3273
Author(s):  
Grant D. Trobridge ◽  
Brian C. Beard ◽  
David Dickerson ◽  
Christina Gooch ◽  
Philip Olsen ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Peripheral Blood ◽  
Retroviral Vectors ◽  
Hiv Replication ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Therapy Strategies ◽  
Anti Hiv

Abstract AIDS remains a significant health problem worldwide despite the advent of highly active antiretroviral therapy (HAART). Although substantial efforts have been made to develop a vaccine there is still no cure and alternative strategies are needed to treat HIV infection and to control its spread. Our goal is to evaluate lenti and foamy retroviral vectors that inhibit HIV replication by RNAi in a non-human primate SHIV model to develop a hematopoietic stem cell (HSC) gene therapy for AIDS. SHIV is a chimeric virus comprised of an SIV genome that contains the tat, rev and env genes of HIV and infects both T lymphocytes and macrophages. Infection of non-human primates with SHIV results in significant decreases in CD4+ T cells as early as 4 weeks post infection, and is currently the best large animal model available to test gene therapy strategies for AIDS. However inefficient gene delivery to hematopoietic stem cells has limited progress for AIDS gene therapy. We have developed both lenti and foamy retroviral vectors that contain methylguanine-DNA-methyltransferase (MGMT) expression cassettes to allow for in vivo selection, and have transduced macaque (M. nemestrina) long term repopulating cells with both vector systems. Following transplantation we observed rapid engraftment and levels of gene marking in the peripheral blood that should allow us to in vivo select both lenti and foamy-marked hematopoietic repopulating cells. In one animal transplanted with a lentiviral vector we obtained marking at 265 days post-transplant of over 30% in peripheral blood granulocytes and 20% in peripheral blood lymphocytes prior to in vivo selection. Anti-SHIV/HIV transgene cassettes targeting tat and rev that allow for potent inhibition of SHIV and HIV replication in vitro have been incorporated into both lenti and foamy vectors and we have transduced macaque long term repopulating cells with lenti vectors containing an anti-HIV cassette. We are currently developing protocols for efficient in vivo selection and future studies will investigate the ability of macaque hematopoietic repopulating cells transduced with lenti and foamy MGMT anti-HIV vectors to inhibit SHIV infection ex vivo and in vivo.

MGMT (P140K) Allows for Efficient and Sustained In Vivo Selection in Non-Human Primates.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3270-3270
Author(s):  
Brian C. Beard ◽  
Kate Beebe ◽  
Julia Piasecki ◽  
Christina Gooch ◽  
David Dickerson ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Negative Selection ◽  
Chemotherapy Treatment ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Elevated Liver Enzymes ◽  
Low Levels ◽  
Selection Of

Abstract In vivo selection strategies that convey a survival advantage to genetically modified cells carrying mutant forms of methylguanine methyltransferase (MGMT-P140K) have the potential to improve autologous and allogeneic stem cell gene therapy and transplantation. For some applications such as genetic diseases or anti-HIV strategies, in vivo selection may be required to increase initially low levels of gene-modified cells while for malignant diseases hematopoietic stem cell (HSC) chemo-protection may be necessary during chemotherapy dose escalation. Thus we have explored the use of gammaretrovirally expressed MGMT(P140K) mutant in three baboons. Animals received CD34−enriched cells transduced with a GALV-pseudotyped retroviral vector expressing a bicistronic message containing P140K and GFP. Two of the animals were part of a competitive repopulation assay in which one half of the cells were gene-modified with a GALV-pseudotyped vector expressing only YFP. After stable engraftment all three baboons were treated with various regimens of O6-benzylguanine (O6BG) and temozolomide (TMZ) or BCNU. Following treatment with O6BG/TMZ the selection was transient for ‘protected’ cells gene-modified with MGMT(P140K)-GFP, and the expected negative selection of ‘unprotected’ gene-modified cells (YFP transgene alone) was subtle. Conversely, positive selection of MGMT(P140K)-GFP gene-modified cells and negative selection of YFP gene-modified cells was dramatic and sustained following treatment with only a single dose of O6BG/BCNU. The increase in gene-marking (up to ~85%) is stable following selection out to 22 months. Importantly, selection of hematopoietic cells was polyclonal and no evidence of insertional mutagenesis has been detected. Aside from transient elevated liver enzymes following O6BG/BCNU treatment no additional extra-hematopoietic toxicity has been observed. We suspect that the delivery/absorption of TMZ in non-human primates is a contributing factor to transient selection because in animals with low levels (<1%) of MGMT(P140K) gene-modified cells no pronounced or sustained drop in white blood cell or platelet counts was observed following O6BG/TMZ. This is the case even up to TMZ dose levels of 700 mg/m2 that is above dose limiting toxicity in humans. In summary, MGMT selection is efficient and well tolerated in monkeys and we believe that these large animal studies closely reflect a clinical setting and will help to further improve clinical HSC gene therapy. Figure 1. Efficient in vivo selection and chemo-protection in non-human primates. (A) Representative gene-marking data in a baboon following chemotherapy treatment with either O6BG (120 mg/m2) and TMZ (600–1400 mg/m2) (solid arrows) or O6BG (120 mg/m2) and BCNU (40 mg/m2) (dashed arrows). The data is plotted as FACS+ MGMT-GFP granulocytes (closed circles) and FACS+ YFP granulocytes (open circles). (B) Absolute neutrophil counts following initial conditioning and subsequent chemotherapy treatment with O6BG/TMZ (solid arrows) or O6BG/BCNU (dashed arrow). Figure 1. Efficient in vivo selection and chemo-protection in non-human primates. (A) Representative gene-marking data in a baboon following chemotherapy treatment with either O6BG (120 mg/m2) and TMZ (600–1400 mg/m2) (solid arrows) or O6BG (120 mg/m2) and BCNU (40 mg/m2) (dashed arrows). The data is plotted as FACS+ MGMT-GFP granulocytes (closed circles) and FACS+ YFP granulocytes (open circles). (B) Absolute neutrophil counts following initial conditioning and subsequent chemotherapy treatment with O6BG/TMZ (solid arrows) or O6BG/BCNU (dashed arrow).

Efficient Gene Marking with a Clinical Gammaretrovirus Vector Expressing MGMTP140K in Baboons and Macaques

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3535-3535
Author(s):  
Brian C Beard ◽  
Megan L Welsh ◽  
Grant D Trobridge ◽  
Hans-Peter Kiem
Keyword(s):  
Gene Therapy ◽  
Alkylating Agents ◽  
Autologous Transplantation ◽  
Large Animal ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Efficient Gene ◽  
Elevated Liver Enzymes ◽  
Hematopoietic Toxicity

Abstract Using gene therapy to protect hematopoietic stem cells (HSC) from alkylating agents used in the treatment of malignant disease is an attractive strategy to alleviate prolonged neutropenia and thrombocytopenia that is dose-limiting. Both adult and pediatric patients with glioblastomas urgently need improved therapeutic strategies since even with aggressive treatment the median survival after diagnosis is approximately 12 months. Chemotherapy with the nitrosourea BCNU, the methylating agents procarbazine or temozolomide, and other alkylating agents is effective and can prolong survival but the therapeutic benefit is attenuated due to hematopoietic toxicity which limits dose-escalation of these drugs. We have previously demonstrated in the dog and non-human primate model efficient gene marking, in vivo selection and chemoprotection from temozolomide and BCNU following transplantation with MGMTP140K gene-modified cells. Thus, we wanted to explore the efficacy of autologous transplantation in macaques and baboons using cells gene-modified with an MLV-based gammaretrovirus vector developed for a pending clinical trial. This retroviral vector contains a myeloproliferative sarcoma virus LTR, negative control region deleted, dl587rev primer binding site (MND) vector backbone, which expresses MGMTP140K from the 5′ LTR promoter, and is pseudotyped with the gibbon ape leukemia virus (GALV) envelope produced from Phoenix-GALV packaging cells (PhGALV-MND.GRS.P140K c38). Following transduction the gene marking in pre-infusion colony forming units (CFUs) was 74.3% and 69.8% in the macaque and baboon respectively as determined by CFU-PCR. Intracellular MGMT-staining of cultured baboon cells 4 (76.4%) and 11 (89.9%) days after transduction confirmed high gene transfer levels. Both animals recovered neutrophil and platelet levels within expected time frames relative to historical controls and the average provirus copy number determined by real-time PCR approximately one month after transplantation was 0.14 and 0.72 in the macaque and baboon respectively. Retrovirus integration site analysis in the macaque 90 days after transplantation, and before chemotherapy, confirmed polyclonal hematopoietic reconstitution. There was no indication of progression to a pre-leukemic state. The macaque has been treated twice with O6-benzylguanine (O6BG) and BCNU and the gene marking has stably increased approximately 2.5-fold. Aside from transient elevated liver enzymes following O6BG/BCNU treatment no additional extra-hematopoietic toxicity has been observed. In summary, we have been able to achieve efficient polyclonal gene marking with MGMTP140K gene-modified cells using a vector designed for clinical application in both macaques and baboons and have preliminary evidence of in vivo selection in the macaque. We believe that these large animal studies closely reflect a clinical setting and will help to further improve clinical HSC gene therapy.

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

Blood ◽  
2007 ◽  
Vol 110 (6) ◽  
pp. 1779-1787 ◽  
Author(s):  
Claudia R. Ball ◽  
Ingo H. Pilz ◽  
Manfred Schmidt ◽  
Sylvia Fessler ◽  
David A. Williams ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Cell Clones ◽  
Selection For ◽  
The Impact ◽  
Reduced Intensity ◽  
Selection Of

AbstractEfficient in vivo selection increases survival of gene-corrected hematopoietic stem cells (HSCs) and protects hematopoiesis, even if initial gene transfer efficiency is low. Moreover, selection of a limited number of transduced HSCs lowers the number of cell clones at risk of gene activation by insertional mutagenesis. However, a limited clonal repertoire greatly increases the proliferation stress of each individual clone. Therefore, understanding the impact of in vivo selection on proliferation and lineage differentiation of stem-cell clones is essential for its clinical use. We established minimal cell and drug dosage requirements for selection of P140K mutant O6-methylguanine-DNA-methyltransferase (MGMT P140K)–expressing HSCs and monitored their differentiation potential and clonality under long-term selective stress. Up to 17 administrations of O6-benzylguanine (O6-BG) and 1,3-bis(2-chloroethyl)-1-nitroso-urea (BCNU) did not impair long-term differentiation and proliferation of MGMT P140K–expressing stem-cell clones in mice that underwent serial transplantation and did not lead to clonal exhaustion. Interestingly, not all gene-modified hematopoietic repopulating cell clones were efficiently selectable. Our studies demonstrate that the normal function of murine hematopoietic stem and progenitor cells is not compromised by reduced-intensity long-term in vivo selection, thus underscoring the potential value of MGMT P140K selection for clinical gene therapy.

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