Selection of hematopoietic stem cells with a combination of galactose-bound vinyl polymer and soybean agglutinin, a galactose-specific lectin

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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1030. A Rhesus Macaque Transplant Model for Drug Selection of Hematopoietic Stem Cells Transduced with MGMT Vectors

Molecular Therapy ◽

10.1016/j.ymthe.2005.07.577 ◽

2005 ◽

Vol 11 ◽

pp. S398

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Rhesus Macaque ◽

Drug Selection ◽

Hematopoietic Stem ◽

Transplant Model ◽

Selection Of

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Differentiation and proliferation of hematopoietic stem cells

Blood ◽

10.1182/blood.v81.11.2844.2844 ◽

1993 ◽

Vol 81 (11) ◽

pp. 2844-2853 ◽

Cited By ~ 907

Author(s):

M Ogawa

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cell Potential ◽

Hematopoietic Stem ◽

Gm Csf ◽

Complex Process ◽

Specific Factors ◽

Differentiation And Proliferation ◽

Kinetics Of ◽

Selection Of

Abstract Available evidence indicates that qualitative changes in hematopoietic stem cells and progenitors, such as the decision of stem cells to self- renew or differentiate, or selection of lineage potentials by the multipotential progenitors during differentiation (commitment), are intrinsic properties of the progenitors and are stochastic in nature. In-contrast, proliferative kinetics of the progenitors, namely survival and expansion of the progenitors, appear to be controlled by a number of interacting cytokines. While proliferation and maturation of committed progenitors is controlled by late-acting lineage-specific factors such as Ep, M-CSF, G-CSF, and IL-5, progenitors at earlier stages of development are controlled by a group of several overlapping cytokines. IL-3, GM-CSF, and IL-4 regulate proliferation of multipotential progenitors only after they exit from G0 and begin active cell proliferation. Triggering of cycling by dormant primitive progenitors and maintenance of B-cell potential of the primitive progenitors appears to require interactions of early acting cytokines including IL-6, G-CSF, IL-11, IL-12, LIF, and SF. Currently, this simple model fits our understanding of the interactions of growth factors with hematopoietic progenitors. Naturally the model risks oversimplification of a very complex process. However, because the model is testable, it will hopefully challenge investigators to design new experiments to examine its validity.

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Stable Polyclonal Murine Long-Term Hematopoiesis without Clonal Exhaustion of MGMT P140K Expressing Murine Hematopoietic Stem Cells after Extended Reduced Intensity Selection.

Blood ◽

10.1182/blood.v108.11.3271.3271 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3271-3271

Author(s):

Claudia R. Ball ◽

Manfred Schmidt ◽

Ingo H. Pilz ◽

Fessler Sylvia ◽

David A. Williams ◽

...

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Retroviral Vector ◽

Hematopoietic Stem ◽

Reduced Intensity ◽

Selection Of

Abstract Gene therapy is a promising approach for the therapy of hereditary diseases, but after the occurrence of adverse side effects in a SCID-X1 gene therapy trial increased biological safety has become a major goal of gene therapy. A reduction of the number of transplanted cells could help achieve this goal by reducing the statistical likelihood of insertional mutagenesis simply by simply reducing the number of transplanted cells carrying potentially untoward insertion sites. As we have previously shown, incorporation of the selectable marker gene MGMT P140K into a retroviral vector allows a reduced intensity and toxicity in vivo selection of low numbers of genetically modified hematopoietic cells by chemotherapy with O6-benzylguanine (O6BG) and nitrosourea drugs such as 1,3-bis-2 chloroethyl-1-nitrosourea (BCNU). However, it is still not known whether extended selection over longer periods of time influences the long-term proliferation and differentiation capacity of murine haematopoietic stem cells. To address this question, serial transplantations of murine MGMT-P140K-expressing hematopoiesis combined with repeated administrations of O6-BG and BCNU were performed. After ex vivo gene transfer of a MGMT/IRES/eGFP-encoding retroviral vector, bone marrow cells were transplanted into syngeneic C57 BL/6J mice and serially transplanted. First, 2nd and 3rd generation recipient mice were subsequently treated every four weeks in order to amplify treatment effects on the long-term clonal behaviour of modified hematopoietic stem cells. Lineage contribution of transduced hematopoiesis was monitored by FACS over a total of 17 rounds of selection and clonality was monitored by LAM-PCR over a total of 16 rounds of selection. In primary mice, the percentage of transduced blood cells increased from 4.7 ± 0.8 % to 36.4 ± 9.8 % (n=12) and in secondary mice from 29.9 ± 7.2 % to 65.1 ± 8.7 % (n=18) after selection without inducing persistent peripheral blood cytopenia. Lineage analysis showed an unchanged multilineage differentiation potential in the transduced compared to control cells in 1st and 2nd generation animals. LAM PCR analysis of peripheral blood revealed stable oligo- to polyclonal hematopoiesis in 1st, 2nd and 3rd generation mice. Evidence of predominant clones or clonal exhaustion was not observed despite of up to 16 rounds of BCNU/O6-BG treatment. Interestingly, pairs of secondary transplanted mice which had received bone marrow cells from identical donors showed very similar clonal composition, engraftment kinetics under selection and lineage contribution of the transduced hematopoiesis. This is molecular proof that extensive self-renewal of transplantable stem cells had occurred in the primary mice resulting in a net symmetric refilling of the stem cell compartment. In summary, we demonstrate that even extended selection of MGMT-P140K-expressing hematopoietic stem cells by repetitive chemotherapy does not affect differentiation or proliferation potential and does not result in clonal exhaustion. Our results have important implications for the clinical use of MGMT selection strategies intending to employ amplification of a limited number of genetically modified clones in clinical gene therapy.

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