Rapid 1-hour transduction of whole bone marrow leads to long-term repopulation of murine recipients with lentivirus-modified hematopoietic stem cells

Kurre, Peter; Anandakumar, Ponni; Kiem, HP

doi:10.1038/sj.gt.3302659

Cited by 28 publications

(31 citation statements)

References 21 publications

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“…19 Interestingly, recently described cytokine cocktails such as STIF (stem cell factor [SCF], thrombopoietin [TPO], insulin-like growth factor-2 [IGF-2], and fibroblast growth factor-1 [FGF-1]), which mimic conditions found in fetal liver, support efficient in vitro amplification of murine and human HSCs. 20 Increasing the number of transplantable HSCs in vitro should promote therapeutic outcome by accelerated engraftment and multilineage reconstitution, and may create a basis for pretransplantation quality control.…”

Section: Introductionmentioning

confidence: 99%

Polyclonal fluctuation of lentiviral vector–transduced and expanded murine hematopoietic stem cells

Maetzig

Brugman

Bartels

et al. 2011

Blood

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Gene therapy has proven its potential to cure diseases of the hematopoietic system. However, severe adverse events observed in clinical trials have demanded improved gene-transfer conditions. Whereas progress has been made to reduce the genotoxicity of integrating gene vectors, the role of pretransplantation cultivation is less well investigated. We IntroductionHematopoietic stem cells (HSCs) are characterized by their ability to self-renew and differentiate into all cell types of the hematopoietic system. At physiologic steady-state conditions, only a limited number of HSCs are active at any given time, thus preserving a large fraction of quiescent cells as a reservoir for long-term regeneration. 1 As demonstrated by retroviral gene marking in transplanted mice or nonhuman primates, cycling HSCs are either periodically replaced (clonal succession), maintained as a constant minor pool (clonal maintenance), or follow a mixture of succession, maintenance, and cyclic reactivation (clonal fluctuation). [2][3][4][5] The latter depends on a rich HSC pool and thus requires optimized HSC culture and gene-transfer conditions to be maintained in gene-therapy protocols.Targeting HSCs with integrating gene vectors guarantees lifelong expression of the therapeutic transgene in all progeny cells, and has been effective for the cure of monogenetic diseases such as X-linked severe combined immunodeficiency, chronic granulomatous disease, adenosine deaminase deficiency, and adrenoleukodystrophy. [6][7][8][9] In the first 2 conditions, murine leukemia virus-derived gammaretroviral vector integrations in the vicinity of protooncogenes have led to premalignant clonal dominance and malignant transformation, 7,8 and similar observations have been made in murine models. 10,11 Some of the integration sites that mediate malignant transformation in humans (eg, EVI1, CCND2, and LMO2) can also be found in the Retroviral Tagged Cancer Gene Database, 12 a collection of integration sites of mouse tumors induced by replicating murine leukemia virus. Genes identified in multiple independent tumors are referred to as common insertion sites and are potentially linked to tumor initiation.To improve transduction of quiescent HSCs, HIV-1-derived lentiviral vectors have been developed and evaluated in preclinical and clinical studies. 9,13,14 In contrast to gammaretroviral vectors, which preferentially integrate close to transcription start sites, CpG islands, and DNaseI-hypersensitive sites, lentiviral vectors preferentially integrate within active transcription units. 15,16 In assays detecting transformed mutants, this pattern was found to be 3-to 10-fold safer than the gammaretroviral preferences, 17,18 most likely related to a reduced frequency of enhancer-mediated gene activation.The ability of lentiviral vectors to transduce nondividing cells allows for the use of short, "stemness"-preserving protocols for genetic modification of HSCs. 19 Interestingly, recently described cytokine cocktails such as STIF (stem cell factor [SCF], thrombopoietin ...

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Section: Introductionmentioning

confidence: 99%

Polyclonal fluctuation of lentiviral vector–transduced and expanded murine hematopoietic stem cells

Maetzig

Brugman

Bartels

et al. 2011

Blood

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“…First, we -BM or LSK with a GFP lentivirus. We performed a single round of exposure to a significant excess of viral particles (6,11,12). The transduction efficacy was >75% while the glioma-mediated attraction of the HPC in vitro or in vivo was unaltered (Figs.…”

Section: Discussionmentioning

confidence: 99%

“…Retroviral and lentiviral vectors have been used to achieve genetic correction of patient-derived autologous HPC (9,10). In this regard, lentiviral vectors provided advantages because of their ability to stably transduce nondividing cells and thus enabling short ex vivo culture durations (11,12).…”

Section: Intravenously (Iv) Injected Human Cd34mentioning

confidence: 99%

Glioma tropism of lentivirally transduced hematopoietic progenitor cells

et al. 2010

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Abstract. Experimental gliomas attract hematopoietic progenitor cells (HPC) in vivo. HPC are therefore promising candidates for a cell-based delivery of therapeutic molecules to experimental gliomas. A therapeutic application requires efficient genetic manipulation of the cellular vector and a lack of tumorigenicity. Here, we studied the impact of lentiviral transduction on the glioma tropism of human or murine HPC. Transduction of human or murine HPC with a GFP lentivirus (lenti-GFP) did not interfere with the gliomamediated attraction of HPC. Bone marrow reconstitution of C57Bl/6 mice with syngeneic GFP-transgenic lineagedepleted bone marrow cells (lin -BM) was as efficient as reconstitution with syngeneic lin -BM transduced ex vivo with lenti-GFP. SMA-560 gliomas growing orthotopically in lenti-GFP-reconstituted VM/Dk mice recruited GFP-positive bone marrow-derived cells. Thus, lentiviral transduction did not interfere with the attraction of exogenously injected HPC or endogenous bone marrow-derived cells by experimental gliomas. Lenti-GFP-HPC implanted directly into tumor-free brains were not tumorigenic. The intravenous injection of lenti-GFP-HPC in glioma-bearing mice did not alter the survival of otherwise untreated animals and had no impact on the survival benefit conferred by cerebral irradiation. Taken together, genetic manipulation of HPC with lenti-GFP neither made these cells tumorigenic nor interfered with their glioma tropism.

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“…The results of the secondary transplantation experiment for the SLAM-HSCs are similar to those obtained after transduction, transplantation and secondary transplantation of KSL-HSC. [22][23][24] It is important to note that the populations used in this study were not purified HSCs, which require a much more stringent isolation strategy, but rather populations highly enriched for HSCs that would provide practical advantages for HSC-directed gene transfer studies. Nevertheless, the relative HSC content of each population is important for interpretation of our results.…”

Section: Slam-enriched Hematopoietic Stem Cells P Laje Et Almentioning

confidence: 99%

“…To estimate the relative HSC content, we calculated repopulating units (RUs) 25,26 relative to the standard repopulating ability of 1 Â 10 5 competitor cells, according With recognition of the ability of lentiviral vectors to transduce non-dividing cells, it has become apparent that, although minimal stimulation of HSC is necessary to avoid apoptosis, a brief exposure to two hematopoietic growth factors provides adequate support for transduction without loss of repopulating capacity. 22,27 Other factors, such as the addition of recombinant fibronectin or polybrene, have been shown to improve the transduction efficiency without affecting the behavior of the transduced cells. 28,29 This study recognizes the contribution of multiple other investigators and incorporated those features and modifications to design a brief and abbreviated transduction protocol that was highly efficient in transducing HSCs.…”

Section: Slam-enriched Hematopoietic Stem Cells P Laje Et Almentioning

confidence: 99%

SLAM-enriched hematopoietic stem cells maintain long-term repopulating capacity after lentiviral transduction using an abbreviated protocol

2009

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Gene transfer to long-term repopulating hematopoietic stem cells (HSCs) using integrating viral vectors is an important goal in gene therapy. The SLAM (signaling lymphocyte activation molecule)-family receptors have recently been used for the isolation of highly enriched murine HSCs. This HSC enrichment protocol is relatively simple, and results in an HSC population with comparable repopulating capacity to c-kit + lin À Sca-1 + (KSL) HSCs. The capacity to withstand genetic manipulation and, most importantly, to maintain long-term repopulating capacity of SLAM-enriched HSC populations has not been reported. In this study, SLAMenriched HSCs were assessed for transduction efficiency and in vivo long-term repopulating capacity after lentiviral transduction using an abbreviated transduction protocol and KSL-enriched HSCs as a reference population. SLAM-and KSL-enriched HSCs were efficiently transduced by lentiviral vector using a simple protocol that involves minimal in vitro manipulation and no pre-stimulation. SLAM-HSCs are at least equal to KSL-HSCs with respect to efficiency of transduction and maintenance of long-term repopulating capacity. Although there was a reduction in repopulating capacity related to enrichment and culture manipulations relative to freshly isolated bone marrow (BM) cells, no detrimental effects were identified on long-term competitive capacity related to transduction, as transduced cells maintained stable levels of chimerism in competition with nontransduced cells and freshly isolated BM cells. These results support the SLAM-HSC enrichment protocol as a simple and efficient method for HSC enrichment for gene transfer studies.

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Rapid 1-hour transduction of whole bone marrow leads to long-term repopulation of murine recipients with lentivirus-modified hematopoietic stem cells

Cited by 28 publications

References 21 publications

Polyclonal fluctuation of lentiviral vector–transduced and expanded murine hematopoietic stem cells

Polyclonal fluctuation of lentiviral vector–transduced and expanded murine hematopoietic stem cells

Glioma tropism of lentivirally transduced hematopoietic progenitor cells

SLAM-enriched hematopoietic stem cells maintain long-term repopulating capacity after lentiviral transduction using an abbreviated protocol

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