Unaltered repopulation properties of mouse hematopoietic stem cells transduced with lentiviral vectors

González‐Murillo, África; Lozano, M. Luz; Montini, Eugenio; Bueren, Juan A.; Güenechea, Guillermo

doi:10.1182/blood-2008-03-142661

Cited by 24 publications

(21 citation statements)

References 49 publications

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“…1 Conceivably, polyclonal fluctuation is only possible under conditions of a rich stem cell repertoire, and can hardly be observed when using suboptimal cytokines for ex vivo culture, as in previous studies by us and others. 23,24,35,36,46 Many previous studies used less advanced cytokine cocktails for ex vivo culture, and oligoclonal dominance of gammaretroviral or lentiviral vector-transduced HSCs occurred even in the absence of insertional lesions. 23,24 Interestingly, although Southern blot analysis of DNA harvested from bone marrow cells indicated oligoclonal dominance, the more sensitive integrome analyses still revealed polyclonal fluctuation, with detection of previously hidden clones even at late time points.…”

Section: Discussionmentioning

confidence: 99%

“…23,24,35,36,46 Many previous studies used less advanced cytokine cocktails for ex vivo culture, and oligoclonal dominance of gammaretroviral or lentiviral vector-transduced HSCs occurred even in the absence of insertional lesions. 23,24 Interestingly, although Southern blot analysis of DNA harvested from bone marrow cells indicated oligoclonal dominance, the more sensitive integrome analyses still revealed polyclonal fluctuation, with detection of previously hidden clones even at late time points. The use of optimized cytokine cocktails for in vitro amplification of HSCs may thus help to maintain homeostatic mechanisms by supporting polyclonal engraftment and delaying clonal restriction.…”

Section: Discussionmentioning

confidence: 99%

“…on May 9, 2018. by guest www.bloodjournal.org From Preclinical gammaretroviral and lentiviral gene-marking experiments, mostly performed under suboptimal cytokine conditions, resulted in clonal selection in vivo with or without insertional transformation. 23,24 Conversely, prolonged ex vivo cultivation of gammaretroviral vector-transduced hematopoietic cells has been associated with an increased risk of clonal outgrowth and malignant transformation in mice and nonhuman primates. 25,26 In the present study, we aimed to test the impact of ex vivo HSC expansion in STIF conditions on the induction of clonal dominance in the context of efficient lentiviral gene transfer.…”

Section: Introductionmentioning

confidence: 99%

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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: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Maetzig

Brugman

Bartels

et al. 2011

Blood

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“…They are also safer than other integrative vector systems because they do not have integration preference for transcriptional start sites, although they do integrate semi-randomly in transcriptionally active areas (Montini et al, 2006). In fact several studies have recently demonstrated the lower genotoxicity of LVs compared to oncoretroviral-based vectors (Montini et al, 2006;Gonzalez-Murillo et al, 2008). These characteristics have prompted scientists to propose LVs as one of the most interesting vectors for gene therapy strategies when stable gene expression is required.…”

Section: Lentiviral-based Vectors (Lv)mentioning

confidence: 99%

New Vectors for Stable and Safe Gene Modification

Martin¹,

Benabdellah²,

Cobo³

et al. 2011

Gene Therapy - Developments and Future Perspectives

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“…Lentiviral vectors have proven to be promising for therapeutic applications because of their ability to transduce non-dividing cells with integration of the encoded transgene into the host genome, and the decreased risk of insertional mutagenesis relative to the retroviral vectors. 7,8 Protocols for lentiviral transduction of HSCs have evolved over the years with a recent trend toward abbreviated culture times and minimal cytokine exposure, to avoid differentiation, and loss of long-term repopulating capacity after transplantation. [9][10][11][12] In addition to loss of repopulating capacity, transduction of HSCs has been shown to result in loss of homing capacity 13 and may have other detrimental effects.…”

Section: Introductionmentioning

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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Unaltered repopulation properties of mouse hematopoietic stem cells transduced with lentiviral vectors

Cited by 24 publications

References 49 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

New Vectors for Stable and Safe Gene Modification

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

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