Insertion of Retroviral Vectors in NOD/SCID Repopulating Human Peripheral Blood Progenitor Cells Occurs Preferentially in the Vicinity of Transcription Start Regions and in Introns

Laufs, Stephanie; Nagy, K. Zsuzsanna; Giordano, Frank A.; Hotz‐Wagenblatt, Agnes; Zeller, W. J.; Früehauf, Stefan

doi:10.1016/j.ymthe.2004.08.001

Cited by 86 publications

(64 citation statements)

References 38 publications

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“…Gamma-retroviral vectors have a propensity to integrate into active chromatin regions and around promoters (1)(2)(3)(4), where insertion of LTR transcriptional enhancers may interfere with normal gene regulation (5) and eventually contribute to malignant transformation (6)(7)(8). The occurrence of leukemia in patients treated by gene therapy for X-linked severe combined immunodeficiency (X-SCID) has been correlated with insertional activation of a T cell proto-oncogene (9).…”

mentioning

confidence: 99%

Retroviral vector integration deregulates gene expression but has no consequence on the biology and function of transplanted T cells

Recchia

Bonini

Magnani

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

175

150

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The use of retroviral vectors in gene therapy has raised safety concerns for the genotoxic risk associated with their uncontrolled insertion into the human genome. We have analyzed the consequences of retroviral transduction in T cells from leukemic patients treated with allogeneic stem cell transplantation and donor lymphocytes genetically modified with a suicide gene (HSV-TK). Retroviral vectors integrate preferentially within or near transcribed regions of the genome, with a preference for sequences around promoters and for genes active in T cells at the time of transduction. Quantitative transcript analysis shows that one fifth of these integrations affect the expression of nearby genes. However, transduced T cell populations maintain remarkably stable gene expression profiles, phenotype, biological functions, and immune repertoire in vivo, with no evidence of clonal selection up to 9 yr after administration. Analysis of integrated proviruses in transduced cells before and after transplantation indicates that integrations interfering with normal T cell function are more likely to lead to clonal ablation than expansion in vivo. Despite the potentially dangerous interactions with the T cell genome, retroviral integration has therefore little consequence on the safety and efficacy of T cell transplantation.donor lymphocyte infusion ͉ gene therapy ͉ graft-versus-host disease ͉ insertional mutagenesis ͉ retroviral integration

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mentioning

confidence: 99%

Retroviral vector integration deregulates gene expression but has no consequence on the biology and function of transplanted T cells

Recchia

Bonini

Magnani

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

175

150

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“…44 In insertion site analyses our group could show that lentiviral vectors in contrast to gammaretroviral vectors integrated at a significantly lower rate within a distance of 10 kb from the transcription start site. 45,46 Therefore, the probability of affecting oncogenes or tumor suppressor genes might be lower by the use of lentiviral vectors. In a model with tumor-susceptible mice transplantation of gammaretroviral-transduced hematopoietic cells resulted in an accelerated tumorigenic process, whereas with lentiviral vectors no additional adverse events were detected.…”

Section: Discussionmentioning

confidence: 99%

Chemoprotection of human hematopoietic stem cells by simultaneous lentiviral overexpression of multidrug resistance 1 and O6-methylguanine-DNA methyltransferaseP140K

et al. 2009

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Myelotoxicity is a dose-limiting effect of many chemotherapeutic regimens. Thus, there is great interest in protecting human hematopoietic stem cells by the transfer of drug resistance genes. The main focus of this study was the simultaneous overexpression of multidrug resistance 1 (MDR1) and the O 6 -benzylguanine (O 6 -BG)-resistant mutant MGMT P140K (O 6 -methylguanine-DNA methyltransferase) with a bicistronic lentiviral vector (HR 0 SIN-MDR1-IRES-MGMT P140K ), with regard to the capability to convey chemoprotection in the leukemia cell line, HL60, and human hematopoietic stem cells (CD34 + ). Combination therapy with O 6 -BG/1-(2-chloroethyl)-3-(4-amino-2-methyl pyrimidine-5-yl)methyl-1-nitrosourea) (ACNU) plus paclitaxel showed a significant survival advantage of HL60 cells transduced with this combination vector. In CD34 + cells, monotherapy with O 6 -BG/temozolomide (TMZ) resulted in an increased percentage of MGMT-positive cells (vs untreated cells) after transduction with HR 0 SIN-MDR1-IRES-MGMT P140K (28.3%). For combination therapy with O 6 -BG/temozolomide plus paclitaxel the increase was higher with the combination vector (52.8%) than with a vector expressing MGMT P140K solely (29.1%). With regard to MDR1-positive cells the protective effect of the combination vector (88.5%) was comparable to the single vector HR 0 SIN-MDR1 (90.0%) for monotherapy with paclitaxel and superior for combination therapy with O 6 -BG/temozolomide plus paclitaxel (84.6 vs 69.7%). In conclusion, the combination vector presents simultaneous protective effects of two drugresistance genes, offering an opportunity to increase the cancer therapeutic index.

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“…Physical and biological properties of the target DNA and the retrovirus life cycle appear to generate a preference for insertion in the vicinity of active genes. It appears that vectors based on lentiviruses and spumaviruses demonstrate slightly altered insertion profiles to γ-retroviruses [106][107][108][109][110][111]. For instance, HIV-1 appears to integrate within the coding region of genes more frequently than γ-retroviruses which demonstrate a preference for the 5′ end of genes in the vicinity of enhancer/promoter regions.…”

Section: Vector Induced Insertional Mutagenesis and Clonal Selectionmentioning

confidence: 99%

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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Insertion of Retroviral Vectors in NOD/SCID Repopulating Human Peripheral Blood Progenitor Cells Occurs Preferentially in the Vicinity of Transcription Start Regions and in Introns

Cited by 86 publications

References 38 publications

Retroviral vector integration deregulates gene expression but has no consequence on the biology and function of transplanted T cells

Retroviral vector integration deregulates gene expression but has no consequence on the biology and function of transplanted T cells

Chemoprotection of human hematopoietic stem cells by simultaneous lentiviral overexpression of multidrug resistance 1 and O6-methylguanine-DNA methyltransferaseP140K

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

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