Enhanced in vivo selection of bone marrow cells by retroviral-mediated coexpression of mutant O6-methylguanine-DNA-methyltransferase and HOXB4

Milsom, Michael D.; Woolford, Lorna B; Margison, Geoffrey P.; Humphries, R. Keith; Fairbairn, Leslie J.

doi:10.1016/j.ymthe.2004.07.019

Cited by 31 publications

(29 citation statements)

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“…A complementary approach has been to identify intrinsic regulators such as transcription factors [13] and key mediators of signaling pathways [14,15] that can be manipulated to activate or promote HSC self-renewal divisions. A striking example of the latter strategy is the use of retrovirally engineered overexpression of the homeobox transcription factor HOXB4 to stimulate expansions of HSC numbers in vitro of up to 80-fold [16][17][18][19][20] with evidence of similarly enhanced HSC expansion in vivo [21][22][23][24]. Significant enhancement of HSC self-renewal has also been elicited by repeated delivery of HOXB4 protein to the cell as an exogenous supplied TAT-HOX fusion protein [25].…”

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confidence: 99%

Near-maximal expansions of hematopoietic stem cells in culture using NUP98-HOX fusions

Ohta

Sekulovic

Bakovic

et al. 2007

Experimental Hematology

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Objective-Strategies to expand hematopoietic stem cells (HSCs) ex vivo are of key interest. The objective of this study was to resolve if ability of HOXB4, previously documented to induce a significant expansion of HSCs in culture, may extend to other HOX genes and also to further analyze the HOX sequence requirements to achieve this effect.Methods-To investigate the ability of Nucleoporin98-Homeobox fusion genes to stimulate HSC self-renewal, we evaluated their presence in 10-to 20-day cultures of transduced mouse bone marrow cells. Stem cell recovery was measured by limiting-dilution assay for long-term competitive repopulating cells (CRU Assay).Results-These experiments revealed remarkable expansions of Nucleoporin98-Homeoboxtransduced HSCs (1000-fold to 10,000-fold over input) in contrast to the expected decline of HSCs in control cultures. Nevertheless, the Nucleoporin98-Homeobox-expanded HSCs displayed no proliferative senescence and retained normal lympho-myeloid differentiation activity and a controlled pool size in vivo. Analysis of proviral integration patterns showed the cells regenerated in vivo were highly polyclonal, indicating they had derived from a large proportion of the initially targeted HSCs. Importantly, these effects were preserved when all HOX sequences flanking the homeodomain were removed, thus defining the homeodomain as a key and independent element in the fusion.Conclusion-These findings create new possibilities for investigating HSCs biochemically and genetically and for achieving clinically significant expansion of human HSCs.The self-renewal function of hematopoietic stem cells (HSCs) is essential to their ability to sustain lifelong hematopoiesis and to regenerate the hematopoietic system after myeloablative treatments. This property is the basis of an increasing range of applications of HSC transplants to treat various malignant and genetic disorders [1,2]. Further improvements in the safety and therapeutic utility of HSC transplants can be readily envisaged if robust methods for largescale ex vivo expansion of HSCs were available. For example, the low absolute numbers of HSCs in most cord blood samples [3] [4] restrict the clinical utility of these products. A method for significantly expanding HSCs could not only address these insufficiencies but also broaden the exploitation of reduced conditioning regimens and associated therapeutic benefits.One approach that has allowed some HSC expansion in vitro to be achieved has focused on the identification of optimized combinations and concentrations of externally acting growth factors and related molecules [5][6][7][8][9][10][11][12]. A complementary approach has been to identify intrinsic regulators such as transcription factors [13] and key mediators of signaling pathways [14,15] that can be manipulated to activate or promote HSC self-renewal divisions. A striking example of the latter strategy is the use of retrovirally engineered overexpression of the homeobox transcription factor HOXB4 to stimulate expansions of HSC numb...

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confidence: 99%

Near-maximal expansions of hematopoietic stem cells in culture using NUP98-HOX fusions

Ohta

Sekulovic

Bakovic

et al. 2007

Experimental Hematology

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“…An example of how this may be achieved comes through the co-ordinate expression of a drug resistance gene and the homeobox transcription factor HOXB4, which has previously been shown to facilitate the in vitro and in vivo expansion of normal HSC. Retroviral co-expression of HOXB4 along with either DHFR [ 122 ] or MGMT [ 12 ] has been demonstrated to dramatically improve the recovery of transduced cells following challenge with either an anti-folate or O 6 alkylator regimen, respectively. However, an effective method of applying such a pro-proliferative strategy which minimizes the potential that expanded cells will become transformed has yet to be devised.…”

Section: Stem Cell Exhaustionmentioning

confidence: 99%

“…There is a limited amount of preliminary data which suggests that MGMT overexpression may restrict the proliferation of hematopoietic cell lines in a dose dependent manner [ 128 ] and perturb the engraftment of HSC [ 12 ] (T. Moritz, University of Duisburg/Essen Medical School, personal communication). It is difficult to envisage a mechanism which would explain these effects.…”

Section: Using a Sledgehammer To Crack A Nut?mentioning

confidence: 99%

“…Hence, despite the concern that this expansion of transduced cells may represent a "pre-leukemic event", this trial provides further evidence of the feasibility of in vivo expansion of transduced human HSC/progenitors to elicit a therapeutic effect. In this context, the transfer and expression of chemoresistance genes has been suggested as one method to effect in vivo selection in genetic diseases where no selective pressure is exerted on the stem cell or progenitor populations by the disease phenotype [ 4,[11][12][13][14].…”

Section: Introductionmentioning

confidence: 99%

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

Milsom

Williams

2007

DNA Repair

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Gene transfer into hematopoietic stem cells (HSC) provides a potential means of correcting monogenic defects and altering drug sensitivity of normal bone marrow to cytotoxic agents. These applications have significant therapeutic potential but the translation of successful murine studies into human therapies has been hindered by low gene transfer in large animals (including humans), and recent serious side effects in a human immunodeficiency trial related to insertional mutagenesis. The latter trial, along with other subsequent trials, while bringing into focus the potential risks of integrating vector systems, also clearly demonstrate the potential usefulness of in vivo selection as it relates to inefficient stem cell transduction. Developing from initial studies by our group and other investigators in which drug resistance was utilized to demonstrate the feasibility of using gene transfer to effect protection from myelotoxicity of chemotherapeutic agents, expression of mutant forms of O 6 -methyguanine-DNA-methytransferase (MGMT) coupled with the simultaneous use of pharmacologic inhibitors and chemotherapeutic agents has been shown to provide a powerful method to select HSC in vivo. While stem and progenitor cell protection and resulting selection in vivo has potential applications for the treatment of selected cancers (allowing dose escalation) andor correction of monogenic disease (allowing an iatrogenic survival advantage of transduced cells in vivo), such an in vivo selection may have untoward effects on stem cell behavior. These deleterious effects may include stem cell exhaustion; lineage skewing; accumulation of genotoxic lesions; and clonal dominance driven towards a pro-leukemic phenotype. Knowledge of the likelihood of such deleterious events occurring as well as their potential implications will be critical to future clinical applications and may also enhance our understanding of both normal stem cell behavior and the evolution of hematopoietic malignancies.

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“…13,14 An example of a gene that provides a proliferative advantage is the HoxB4 gene. Expression of this gene increases self-renewal capacity of HSCs and thereby increases the number of transduced HSCs over nontransduced HSCs.…”

Section: Gene Delivery Into Hscmentioning

confidence: 99%

Gene therapy with drug resistance genes

2005

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A major side effect of cancer chemotherapy is myelosuppression. Expression of drug-resistance genes in hematopoietic stem cells (HSC) using gene transfer methodologies holds the promise of overcoming marrow toxicity in cancer chemotherapy. Adequate protection of marrow cells in cancer patients from myelotoxicity in this way would permit the use of escalating doses of chemotherapy for eradicating residual disease. A second use of drug-resistance genes is for coexpression with a therapeutic gene in HSCs to provide a selection advantage to gene-modified cells. In this review, we discuss several drug resistance genes, which are well suited for in vivo selection as well as other newer candidate genes with potential for use in this manner.

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Enhanced in vivo selection of bone marrow cells by retroviral-mediated coexpression of mutant O6-methylguanine-DNA-methyltransferase and HOXB4

Cited by 31 publications

References 26 publications

Near-maximal expansions of hematopoietic stem cells in culture using NUP98-HOX fusions

Near-maximal expansions of hematopoietic stem cells in culture using NUP98-HOX fusions

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

Gene therapy with drug resistance genes

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