Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery

Lombardo, Angelo; Genovese, Pietro; Beauséjour, Christian; Colleoni, Silvia; Lee, Y.-L.; Kim, K.A.; Ando, Dale; Urnov, Fyodor D.; Galli, Cesare; Gregory, Philip D.; Holmes, Michael C.; Naldini, Luigi

doi:10.1016/j.bcmd.2007.10.064

Cited by 187 publications

(281 citation statements)

References 51 publications

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“…In these cases, the reporter vector dose can be scaled down by transducing the cells at low MOI followed by selection for the low-affinity NGFR-expressing cells, possibly resulting in a decreased sensitivity for iPSC detection. To ensure robust reporting from a single integrant and eliminate concerns for random insertional mutagenesis one could target integration into a preselected genomic site using zinc finger nucleases, as we recently reported [29,30].…”

Section: Discussionmentioning

confidence: 99%

“…The establishment of these reporters has direct implications for iPSC technology, providing new tools to facilitate and accelerate patient-specific iPSCs generation and expansion. Although in this study we used an integrating LV experimental system, miRNA-regulated vectors can be further developed to include loxP sites for Cre-mediated excision [40], targeted integration into preselected genomic sites [29,30], or delivery in the context of nonintegrating lentiviruses [41]. In addition, we foresee further developments by coupling a similar miRNA-regulated system with suicide genes [41], which might become valuable instruments for harnessing the therapeutic potential of iPSCs.…”

Section: Discussionmentioning

confidence: 99%

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A microRNA-Based System for Selecting and Maintaining the Pluripotent State in Human Induced Pluripotent Stem Cells

et al. 2011

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Induced pluripotent stem cell (iPSC) technology has provided researchers with a unique tool to derive disease-specific stem cells for the study and possible treatment of degenerative disorders with autologous cells. The low efficiency and heterogeneous nature of reprogramming is a major impediment to the generation of personalized iPSC lines. Here, we report the generation of a lentiviral system based on a microRNA-regulated transgene that enables for the efficient selection of mouse and human pluripotent cells. This system relies on the differential expression pattern of the mature form of microRNA let7a in pluripotent versus committed or differentiated cells. We generated microRNA responsive green fluorescent protein and Neo reporters for specific labeling and active selection of the pluripotent cells in any culture condition. We used this system to establish Rett syndrome and Parkinson's disease human iPSCs. The presented selection procedure represents a straightforward and powerful tool for facilitating the derivation of patient-specific iPSCs.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A microRNA-Based System for Selecting and Maintaining the Pluripotent State in Human Induced Pluripotent Stem Cells

et al. 2011

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“…On the basis of these observations, the LTR enhancer element in the vectors was removed and the internal promoter added for a safer gene transfer. [17] Recently, Aiuti et al [8] reported the clinical outcome of gene therapy for adenosine deaminase deficiency (ADA) SCID. In this trial, ADA-expressing retroviral vectors were transfused into CD34+ bone marrow cells ex vivo and eight of ten patients have essentially been treated.…”

Section: Retroviral Vectorsmentioning

confidence: 99%

Advances in Gene Delivery Systems

et al. 2011

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The transfer of genes into cells, both in vitro and in vivo, is critical for studying gene function and conducting gene therapy. Methods that utilize viral and nonviral vectors, as well as physical approaches, have been explored. Viral vector-mediated gene transfer employs replication-deficient viruses such as retro-virus, adenovirus, adeno-associated virus and herpes simplex virus. A major advantage of viral vectors is their high gene delivery efficiency. The nonviral vectors developed so far include cationic liposomes, cationic polymers, synthetic peptides and naturally occurring compounds. These nonviral vectors appear to be highly effective in gene delivery to cultured cells in vitro but are significantly less effective in vivo. Physical methods utilize mechanical pressure, electric shock or hydrodynamic force to transiently permeate the cell membrane to transfer DNA into target cells. They are simpler than viral-and nonviral-based systems and highly effective for localized gene delivery. The past decade has seen significant efforts to establish the most desirable method for safe, effective and target-specific gene delivery, and good progress has been made. The objectives of this review are to (i) explain the rationale for the design of viral, nonviral and physical methods for gene delivery; (ii) provide a summary on recent advances in gene transfer technology; (iii) discuss advantages and disadvantages of each of the most commonly used gene delivery methods; and (iv) provide future perspectives.

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“…This will be possibly achieved through a combination of proper cytokine cocktails and more sophisticated gene transfer technologies, such as zinc-finger nucleases, enabling transgene integration in preselected genomic sites. 79,80 As the level of technological complexity increases, possibly enhancing the potency of T cell-based gene transfer, we will need to face the difficult issue of economic sustainability of gene therapy. Paradoxically, this will be probably achieved through the development of phase III clinical trials, able to demonstrate the long-term benefits of T cell-based gene therapy on morbidity, overall survival and quality of life of treated patients, thus enabling proper cost/benefit assessments.…”

Section: Prospectsmentioning

confidence: 99%