Loss of expression of the Hoxa-9 homeobox gene impairs the proliferation and repopulating ability of hematopoietic stem cells

The homeobox gene Hoxa-9 is normally expressed in primitive bone marrow cells, and overexpression of Hoxa-9 markedly expands hematopoietic stem cells, suggesting a function in early hematopoiesis. We present evidence for major functional defects in Hoxa-9-/- hematopoietic stem cells. Hoxa-9-/- marrow cells have normal numbers of immunophenotypic stem cells (Lin-c-kit+flk-2-Sca-1+ [KLFS] cells). However, sublethally irradiated Hoxa-9-/- mice develop persistent pancytopenia, indicating unusual sensitivity to ionizing irradiation. In competitive transplantation assays, Hoxa-9-/- cells showed an 8-fold reduction in multilineage long-term repopulating ability, a defect not seen in marrow cells deficient for the adjacent Hoxa-10 gene. Single-cell cultures of KLFS cells showed a 4-fold reduction in large high-proliferation potential colonies. In liquid cultures, Hoxa-9-deficient Lin-Sca-1+ cells showed slowed proliferation (a 5-fold reduction in cell numbers at day 8) and delayed emergence of committed progenitors (a 5-fold decrease in colony-forming cells). Slowing of proliferation was accompanied by a delay in myeloid maturation, with a decrease in Gr-1hiMac-1hi cells at the end of the culture. Retroviral transduction with a Hoxa-9 expression vector dramatically enhanced the cytokine-driven proliferation and in vivo engraftment of Hoxa-9-/- marrow cells. Hoxa-9 appears to be specifically required for normal hematopoietic stem cell function both in vitro and in vivo.

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Serial transplantation of methotrexate-resistant bone marrow: protection of murine recipients from drug toxicity by progeny of transduced stem cells

Blood ◽

10.1182/blood.v75.2.337.bloodjournal752337 ◽

1990 ◽

Vol 75 (2) ◽

pp. 337-343 ◽

Cited By ~ 2

Author(s):

CA Corey ◽

AD DeSilva ◽

CA Holland ◽

DA Williams

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Gene Transfer ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Retroviral Vectors ◽

Hematopoietic Stem ◽

Genetic Sequences ◽

Marrow Cells

Recombinant retroviral vectors have been used to transfer a variety of genetic sequences into hematopoietic stem cells. Although transfer and expression of foreign genetic sequences into reconstituting stem cells is one approach to somatic gene therapy, few studies have shown long lasting phenotypic changes in recipient mice in vivo. In this study, we show successful transfer of a methotrexate-resistant cDNA (DHFRr) into reconstituting hematopoietic stem cells using a retroviral vector, FrDHFRr, in which the DHFR cDNA is expressed off a hybrid Friend/Moloney long term repeat. Both primary and secondary recipients transplanted with bone marrow cells infected with this recombinant retrovirus show improved survival and protection from methotrexate- induced marrow toxicity when compared with control animals. These data suggest that retroviral-mediated gene transfer of DHFRr cDNA leads to a stable change in the phenotype of hematopoietic stem cells and progeny derived from those cells in vivo after bone marrow transplantation. Gene transfer using recombinant retroviral vectors seems to be one rational approach to establishing chemotherapy-resistant bone marrow cells.

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Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.

Journal of Experimental Medicine ◽

10.1084/jem.183.4.1797 ◽

1996 ◽

Vol 183 (4) ◽

pp. 1797-1806 ◽

Cited By ~ 2081

Author(s):

M A Goodell ◽

K Brose ◽

G Paradis ◽

A S Conner ◽

R C Mulligan

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Side Population ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Quiescent Stem Cells ◽

Marrow Cells

Hematopoietic stem cells (HSC) are multipotent cells that reside in the bone marrow and replenish all adult hematopoietic lineages throughout the lifetime of the animal. While experimenting with staining of murine bone marrow cells with the vital dye, Hoechst 33342, we discovered that display of Hoechst fluorescence simultaneously at two emission wavelengths revealed a small and distinct subset of whole bone marrow cells that had phenotypic markers of multipotential HSC. These cells were shown in competitive repopulation experiments to contain the vast majority of HSC activity from murine bone marrow and to be enriched at least 1,000-fold for in vivo reconstitution activity. Further, these Hoechst-stained side population (SP) cells were shown to protect recipients from lethal irradiation at low cell doses, and to contribute to both lymphoid and myeloid lineages. The formation of the Hoechst SP profile was blocked when staining was performed in the presence of verapamil, indicating that the distinctly low staining pattern of the SP cells is due to a multidrug resistance protein (mdr) or mdr-like mediated efflux of the dye from HSC. The ability to block the Hoechst efflux activity also allowed us to use Hoechst to determine the DNA content of the SP cells. Between 1 and 3% of the HSC were shown to be in S-G2M. This also enabled the purification of the G0-G1 and S-G2M HSC had a reconstitution capacity equivalent to quiescent stem cells. These findings have implications for models of hematopoietic cell development and for the development of genetic therapies for diseases involving hematopoietic cells.

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Development of a Novel Selective Amplifier Gene for Controllable Expansion of Transduced Hematopoietic Cells

Blood ◽

10.1182/blood.v90.10.3884 ◽

1997 ◽

Vol 90 (10) ◽

pp. 3884-3892 ◽

Cited By ~ 26

Author(s):

Keiko Ito ◽

Yasuji Ueda ◽

Masaki Kokubun ◽

Masashi Urabe ◽

Toshiya Inaba ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Hematopoietic Cells ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Selective Amplifier ◽

Marrow Cells

Abstract To overcome the low efficiency of gene transfer into hematopoietic cells, we developed a novel system for selective expansion of transduced cells. To this end, we constructed a chimeric cDNA (GCRER) encoding the fusion protein between the granulocyte colony-stimulating factor receptor (G-CSFR) and the hormone-binding domain (HBD) of the estrogen receptor (ER) as a selective amplifier gene. Use of the intracellular signaling pathway of G-CSFR was considered to be appropriate, because G-CSF has the ability not only to stimulate the neutrophil production, but also to expand the hematopoietic stem/progenitor cell pool in vivo. To activate the exogenous G-CSFR signal domain selectively, the estrogen/ER-HBD system was used as a molecular switch in this study. When the GCRER gene was expressed in the interleukin-3 (IL-3)–dependent murine cell line, Ba/F3, the cells showed IL-3–independent growth in response to G-CSF or estrogen. Moreover, the Ba/F3 cells transfected with the Δ(5-195)GCRER, whose product lacks the extracellular G-CSF–binding domain, did not respond to G-CSF, but retained the ability for estrogen-dependent growth. Further, murine bone marrow cells transduced with the GCRER or Δ(5-195)GCRER gene with retroviral vectors formed a significant number of colonies in response to estrogen, as well as G-CSF, whereas estrogen did not stimulate colony formation by untransduced murine bone marrow cells. It is noteworthy that erythroid colonies were apparently formed by the bone marrow cells transduced with the GCRER gene in the presence of estrogen without the addition of erythropoietin, suggesting that the signals from the G-CSFR portion of the chimeric molecules do not preferentially induce neutrophilic differentiation, but just promote the differentiation depending on the nature of the target cells. We speculate that when the selective amplifier genes are expressed in the primitive hematopoietic stem cells, the growth signal predominates and that the population of transduced stem cells expands upon estrogen treatment, even if some of the cells enter the differentiation pathway. The present study suggests that this strategy is applicable to the in vivo selective expansion of transduced hematopoietic stem cells.

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Identification of Non-Hematopoietic Stem Cells in Mouse Bone Marrow Osteoblastic Zone.

Blood ◽

10.1182/blood.v104.11.677.677 ◽

2004 ◽

Vol 104 (11) ◽

pp. 677-677

Author(s):

Hidetaka Sato ◽

Fumio Arai ◽

Mami Shibata ◽

Atsushi Hirao ◽

Toshio Suda ◽

...

Keyword(s):

Skeletal Muscle ◽

Stem Cells ◽

Bone Marrow ◽

Endothelial Cells ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Fibroblastic Cells ◽

Marrow Cells

Abstract Bone marrow contains hematopoietic and non-hematopoietic stem cells. Although the character of hematopoietic stem cells (HSCs) in vivo has been extensively studied, our understanding of non-hematopoietic stem cells is derived only from in vitro expanded culture such as mesenchymal stem cells (MSCs). Since the number of non-hematopoietic stem cells in bone marrow is small, their characteristics, anatomical location and physiological role in vivo remain unknown. We attempted to overcome this problem by introducing a novel method. We extracted bone marrow cells from osteoblastic zone by enzymatic digestion. Bone marrow extracted cells (BMECs) contained non-hematopoietic cells (CD45−/Ter-119− cells) more than flushed out bone marrow cells (BMCs). Non-hematopoietic cells in BMECs were divided into two types, CD34+ (BME 34+) cells and CD34− (BME 34−) cells. When BME 34− cells were cultured to expand to a confluent layer in MSC culture condition, they formed fibroblastic cells, which have the ability to differentiate into osteocytes, chondrocytes and adipocytes, indicating that BME 34− cells are the in vivo origin of MSCs. On the other hand, BME CD34+ cells contained mostly non-hematopoietic SP fraction and they were Sca-1+ cells. Immunohistochemical analyses show that CD31-BME 34+ cells localized on osteoblasts and endothelial cells in bone marrow. When BME 34+ cells were co-cultured with stromal (OP9) cells, they formed a colony of myogenic cells, endothelial cells or adipogenic cells. However, different from BME 34− cells, fibroblastic cells expanded from BME 34+ cells did not differentiate to osteocytes, chondrocytes or adipocytes in MSC culture condition. When 4,000-6,000 BME 34+ cells were transplanted into irradiated mice, these cells engrafted as non-hematopoietic Vimentin+ fibroblastic-like cells in various organs such as liver, spleen, pancreas, colon, intestine, kidney, skin, brain, lung, heart, skeletal muscle, and bone marrow. In spleen and liver, transplanted cells formed clusters of Vimentin+ fibroblastic cells indicating that BME 34+ cells have ability to expand in vivo to some extent. When BME 34+ cells were directly injected into tibial muscles which were damaged by cardiotoxin, these cells gave rise to mature skeletal muscle cells and endothelial cells. In summary, these studies show distinguishable two types of non-hematopoietic stem cells in bone marrow. It is possible that mobilization and recruitment of these bone marrow-derived non-hematopoietic stem cells play a major role in the setting of wound healing, organ regeneration and tumor growth. In addition, non-hematopoietic two types of BMECs may contribute to clarify the debate on the stem cell plasticity of bone marrow cells.

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Serial transplantation of methotrexate-resistant bone marrow: protection of murine recipients from drug toxicity by progeny of transduced stem cells

Blood ◽

10.1182/blood.v75.2.337.337 ◽

1990 ◽

Vol 75 (2) ◽

pp. 337-343 ◽

Cited By ~ 99

Author(s):

CA Corey ◽

AD DeSilva ◽

CA Holland ◽

DA Williams

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Gene Transfer ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Retroviral Vectors ◽

Hematopoietic Stem ◽

Genetic Sequences ◽

Marrow Cells

Abstract Recombinant retroviral vectors have been used to transfer a variety of genetic sequences into hematopoietic stem cells. Although transfer and expression of foreign genetic sequences into reconstituting stem cells is one approach to somatic gene therapy, few studies have shown long lasting phenotypic changes in recipient mice in vivo. In this study, we show successful transfer of a methotrexate-resistant cDNA (DHFRr) into reconstituting hematopoietic stem cells using a retroviral vector, FrDHFRr, in which the DHFR cDNA is expressed off a hybrid Friend/Moloney long term repeat. Both primary and secondary recipients transplanted with bone marrow cells infected with this recombinant retrovirus show improved survival and protection from methotrexate- induced marrow toxicity when compared with control animals. These data suggest that retroviral-mediated gene transfer of DHFRr cDNA leads to a stable change in the phenotype of hematopoietic stem cells and progeny derived from those cells in vivo after bone marrow transplantation. Gene transfer using recombinant retroviral vectors seems to be one rational approach to establishing chemotherapy-resistant bone marrow cells.

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Reversible Expression of CD34 by Murine Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v94.8.2548.420k38_2548_2554 ◽

1999 ◽

Vol 94 (8) ◽

pp. 2548-2554 ◽

Cited By ~ 291

Author(s):

Takashi Sato ◽

Joseph H. Laver ◽

Makio Ogawa

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Cell Populations ◽

Hematopoietic Stem ◽

Adult Mice ◽

Cd34 Expression ◽

Marrow Cells

We used a mouse transplantation model to address the recent controversy about CD34 expression by hematopoietic stem cells. Cells from Ly-5.1 C57BL/6 mice were used as donor cells and Ly-5.2 mice were the recipients. The test cells were transplanted together with compromised marrow cells of Ly-5.2 mice. First, we confirmed that the majority of the stem cells with long-term engraftment capabilities of normal adult mice are CD34−. We then observed that, after the injection of 150 mg/kg 5-fluorouracil (5-FU), stem cells may be found in both CD34− and CD34+ cell populations. These results indicated that activated stem cells express CD34. We tested this hypothesis also by using in vitro expansion with interleukin-11 and steel factor of lineage−c-kit+ Sca-1+ CD34− bone marrow cells of normal mice. When the cells expanded for 1 week were separated into CD34− and CD34+ cell populations and tested for their engraftment capabilities, only CD34+ cells were capable of 2 to 5 months of engraftment. Finally, we tested reversion of CD34+ stem cells to CD34− state. We transplanted Ly-5.1 CD34+post–5-FU marrow cells into Ly-5.2 primary recipients and, after the marrow achieved steady state, tested the Ly-5.1 cells of the primary recipients for their engraftment capabilities in Ly-5.2 secondary recipients. The majority of the Ly-5.1 stem cells with long-term engraftment capability were in the CD34− cell fraction, indicating the reversion of CD34+ to CD34−stem cells. These observations clearly demonstrated that CD34 expression reflects the activation state of hematopoietic stem cells and that this is reversible.

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Lentiviral gene transfer regenerates hematopoietic stem cells in a mouse model for Mpl-deficient aplastic anemia

Blood ◽

10.1182/blood-2010-09-308262 ◽

2011 ◽

Vol 117 (14) ◽

pp. 3737-3747 ◽

Cited By ~ 20

Author(s):

Dirk Heckl ◽

Daniel C. Wicke ◽

Martijn H. Brugman ◽

Johann Meyer ◽

Axel Schambach ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mouse Model ◽

Hematopoietic Stem Cells ◽

Aplastic Anemia ◽

Bone Marrow Cells ◽

Specific Expression ◽

Hematopoietic Stem ◽

Egfp Reporter

AbstractThpo/Mpl signaling plays an important role in the maintenance of hematopoietic stem cells (HSCs) in addition to its role in megakaryopoiesis. Patients with inactivating mutations in Mpl develop thrombocytopenia and aplastic anemia because of progressive loss of HSCs. Yet, it is unknown whether this loss of HSCs is an irreversible process. In this study, we used the Mpl knockout (Mpl−/−) mouse model and expressed Mpl from newly developed lentiviral vectors specifically in the physiologic Mpl target populations, namely, HSCs and megakaryocytes. After validating lineage-specific expression in vivo using lentiviral eGFP reporter vectors, we performed bone marrow transplantation of transduced Mpl−/− bone marrow cells into Mpl−/− mice. We show that restoration of Mpl expression from transcriptionally targeted vectors prevents lethal adverse reactions of ectopic Mpl expression, replenishes the HSC pool, restores stem cell properties, and corrects platelet production. In some mice, megakaryocyte counts were atypically high, accompanied by bone neo-formation and marrow fibrosis. Gene-corrected Mpl−/− cells had increased long-term repopulating potential, with a marked increase in lineage−Sca1+cKit+ cells and early progenitor populations in reconstituted mice. Transcriptome analysis of lineage−Sca1+cKit+ cells in Mpl-corrected mice showed functional adjustment of genes involved in HSC self-renewal.

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Role Of Sf3b1 On Hematopoiesis

Blood ◽

10.1182/blood.v122.21.600.600 ◽

2013 ◽

Vol 122 (21) ◽

pp. 600-600

Author(s):

Manabu Matsunawa ◽

Ryo Yamamoto ◽

Masashi Sanada ◽

Aiko Sato ◽

Yusuke Shiozawa ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Transplantation ◽

Hematopoietic Stem Cells ◽

Marrow Transplantation ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Ring Sideroblasts ◽

Marrow Cells

Abstract Frequent pathway mutation involving multiple components of the RNA splicing machinery is a cardinal feature of myeloid neoplasms showing myeloid dysplasia, in which the major mutational targets include U2AF35, ZRSR2, SRSF2 and SF3B1. Among these, SF3B1 mutations were strongly associated with MDS subtypes characterized by increased ring sideroblasts, such as refractory anemia and refractory cytopenia with multiple lineage dysplasia with ring sideroblasts, suggesting the critical role of SF3B1 mutations in these MDS subtypes. However, currently, the molecular mechanism of SF3B1mutation leading to the ring sideroblasts formation and MDS remains unknown. The SF3B1 is a core component of the U2-small nuclear ribonucleoprotein (U2 snRNP), which recognizes the 3′ splice site at intron–exon junctions. It was demonstrated that Sf3b1 null mice were shown to be embryonic lethal, while Sf3b1 +/- mice exhibited various skeletal alterations that could be attributed to deregulation of Hox gene expression due to haploinsufficiency of Sf3b1. However, no detailed analysis of the functional role of Sf3b1 in hematopoietic system in these mice has been performed. So, to clarify the role of SF3B1 in hematopoiesis, we investigated the hematological phenotype of Sf3b1 +/- mice. There was no significant difference in peripheral blood counts, peripheral blood lineage distribution, bone marrow total cellularity or bone marrow lineage composition between Sf3b1 +/+ and Sf3b1 +/- mice. Morphologic abnormalities of bone marrow and increased ring sideroblasts were not observed. However, quantitative analysis of bone marrow cells from Sf3b1 +/- mice revealed a reduction of the number of hematopoietic stem cells (CD34 neg/low, cKit positive, Sca-1 positive, lineage-marker negative: CD34-KSL cells) measured by flow cytometry analysis, compared to Sf3b1 +/+ mice. Whereas examination of hematopoietic progenitor cells revealed a small decrease in KSL cell populations and megakaryocyte - erythroid progenitors (MEP) in Sf3b1 +/- mice, and common myeloid progenitors (CMP), granulocyte - monocyte progenitors (GMP) and common lymphoid progenitors (CLP) remained unchanged between Sf3b1 +/+ and Sf3b1 +/- mice. In accordance with the reduced number of hematopoietic stem cells in Sf3b1 +/- mice, the total number of colony-forming unit generated from equal number of whole bone marrow cells showed lower colony number in Sf3b1 +/- mice in vitro. Competitive whole bone marrow transplantation assay, which irradiated recipient mice were transplanted with donor whole bone marrow cells from Sf3b1 +/+ or Sf3b1 +/- mice with an equal number of competitor bone marrow cells, revealed impaired competitive whole bone marrow reconstitution capacity of Sf3b1 +/- mice in vivo. These data demonstrated Sf3b1 was required for hematopoietic stem cells maintenance. To further examine the function of hematopoietic stem cells in Sf3b1 +/- mice, we performed competitive transplantation of purified hematopoietic stem cells from Sf3b1 +/+ or Sf3b1 +/- mice into lethally irradiated mice together with competitor bone marrow cells. Sf3b1 +/- progenitors showed reduced hematopoietic stem cells reconstitution capacity compared to those from Sf3b1 +/+ mice. In serial transplantation experiments, progenitors from Sf3b1 +/- mice showed reduced repopulation ability in the primary bone marrow transplantation, which was even more pronounced after the second bone marrow transplantation. Taken together, these data demonstrate that Sf3b1 plays an important role in normal hematopoiesis by maintaining hematopoietic stem cell pool size and regulating hematopoietic stem cell function. To determine the molecular mechanism underlying the observed defect in hematopoietic stem cells of Sf3b1 +/- mice, we performed RNA-seq analysis. We will present the results of our biological assay and discuss the relation of Sf3b1 and hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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HIF-1α is a negative regulator of plasmacytoid DC development in vitro and in vivo

Blood ◽

10.1182/blood-2012-03-417022 ◽

2012 ◽

Vol 120 (15) ◽

pp. 3001-3006 ◽

Cited By ~ 19

Author(s):

Andreas Weigert ◽

Benjamin Weichand ◽

Divya Sekar ◽

Weixiao Sha ◽

Christina Hahn ◽

...

Keyword(s):

Bone Marrow ◽

Cell Function ◽

Bone Marrow Cells ◽

Negative Regulator ◽

Low Oxygen ◽

Hematopoietic Stem ◽

Lineage Differentiation ◽

Marrow Cells

Abstract Hypoxia-inducible factors (HIFs) regulate hematopoiesis in the embryo and maintain hematopoietic stem cell function in the adult. How hypoxia and HIFs contribute to hematopoietic lineage differentiation in the adult is ill defined. Here we provide evidence that HIF-1 limits differentiation of precursors into plasmacytoid dendritic cells (pDCs). Low oxygen up-regulated inhibitor of DNA binding 2 (ID2) and suppressed Flt3-L–induced differentiation of bone marrow cells to pDCs in wild-type but not HIF-1αfl/fl LysM-Cre bone marrow cells. Moreover, pDC differentiated normally in hypoxic ID2−/− bone marrow cultures. Finally, we observed elevated pDC frequencies in bone marrow, blood, and spleen of HIF-1αfl/fl LysM-Cre and ID2−/−, but not HIF-2αfl/fl LysM-Cre mice. Our data indicate that the low oxygen content in the bone marrow might limit pDC development. This might be an environmental mechanism to restrict the numbers of these potentially autoreactive cells.

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Successful treatment of murine β-thalassemia using in vivo selection of genetically modified, drug-resistant hematopoietic stem cells

Blood ◽

10.1182/blood-2003-03-0677 ◽

2003 ◽

Vol 102 (2) ◽

pp. 506-513 ◽

Cited By ~ 103

Author(s):

Derek A. Persons ◽

Esther R. Allay ◽

Nobukuni Sawai ◽

Phillip W. Hargrove ◽

Thomas P. Brent ◽

...

Keyword(s):

Stem Cells ◽

Gene Transfer ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Lentiviral Vector ◽

Drug Resistant ◽

Hematopoietic Stem ◽

In Vivo Selection ◽

Selection Of

AbstractSuccessful gene therapy of β-thalassemia will require replacement of the abnormal erythroid compartment with erythropoiesis derived from genetically corrected, autologous hematopoietic stem cells (HSCs). However, currently attainable gene transfer efficiencies into human HSCs are unlikely to yield sufficient numbers of corrected cells for a clinical benefit. Here, using a murine model of β-thalassemia, we demonstrate for the first time that selective enrichment in vivo of transplanted, drug-resistant HSCs can be used therapeutically and may therefore be a useful approach to overcome limiting gene transfer. We used an oncoretroviral vector to transfer a methylguanine methyltransferase (MGMT) drug-resistance gene into normal bone marrow cells. These cells were transplanted into β-thalassemic mice given nonmyeloablative pretransplantation conditioning with temozolomide (TMZ) and O6-benzylguanine (BG). A majority of mice receiving 2 additional courses of TMZ/BG demonstrated in vivo selection of the drug-resistant cells and amelioration of anemia, compared with untreated control animals. These results were extended using a novel γ-globin/MGMT dual gene lentiviral vector. Following drug treatment, normal mice that received transduced cells had an average 67-fold increase in γ-globin expressing red cells. These studies demonstrate that MGMT-based in vivo selection may be useful to increase genetically corrected cells to therapeutic levels in patients with β-thalassemia.

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