Site-specific and directional gene replacement mediated by Cre recombinase

Trinh, Kham R.; Morrison, Sherie L.

doi:10.1016/s0022-1759(00)00250-7

Cited by 14 publications

(13 citation statements)

References 17 publications

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“…We have used AAV vector-mediated homologous targeting to introduce an expression cassette 24 followed by Cre recombinase-mediated, directional cassette exchange 31 to evaluate the potential of specific regulatory elements to attenuate or eliminate activation of the LMO2 protooncogene. We targeted LMO2 because it has been implicated in the pathogenesis of leukemia in 2 patients with X-SCID as a consequence of insertional mutagenesis by the therapeutic transgene vector.…”

Section: Discussionmentioning

confidence: 99%

“…Different loxP sites, the cis-acting sequences required for Cre-mediated recombination, were placed upstream and downstream of the introduced cassette to facilitate site-specific and directional cassette exchange. [29][30][31] Our experimental efforts have focused on the LMO2 gene, a well-known proto-oncogene that is involved in chromosomal translocations in human T-cell leukemias and results in perturbations of T lymphopoiesis and a predilection to lymphomas in transgenic mice. 32 As noted earlier, integration within or near the LMO2 gene has been implicated as a pathogenic event in 2 patients who developed leukemia after successful gene therapy for X-SCID.…”

Section: Introductionmentioning

confidence: 99%

“…31 As a test of our system, we assembled an LTR-DsRed2 expression cassette ( Figure 4A) that had an identical design to the original LTR-GFP cassette except the GFP coding sequences were replaced with those of DsRed2. Because Jurkat cells are not easily transfected with a conventional transfection protocol (efficiency ranging from 10% to 15% using LipofectAmine2000), a scAAV-Cre vector was used to obtain high-level Cre expression.…”

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confidence: 99%

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An experimental system for the evaluation of retroviral vector design to diminish the risk for proto-oncogene activation

Ryu

Evans-Galea

Gray

et al. 2008

Blood

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Pathogenic activation of the LMO2 protooncogene by an oncoretroviral vector insertion in a clinical trial for X-linked severe combined immunodeficiency (X-SCID) has prompted safety concerns. We used an adeno-associated virus vector to achieve targeted insertion of a ␥-retroviral long terminal repeat (LTR) driving a GFP expression cassette with flanking loxP sites in a human T-cell line at the precise location of vector integration in one of the patients with X-SCID. The LTR-GFP cassette was inserted into the first intron of the LMO2 gene, resulting in strong activation of LMO2. Cre-mediated cassette exchange was used to replace the original LTR-GFP cassette with one flanked by insulator elements leading to a several fold reduction in LMO2 expression. The LTR-GFP cassette was also replaced with a globin gene regulatory cassette that failed to activate the LMO2 gene in lymphoid cells. A ␥-retroviral vector with 2 intact LTRs resulted in activation of the LMO2 gene when inserted into the first intron, but a self-inactivating lentiviral vector with an internal cellular promoter and flanking insulator elements did not activate the LMO2 gene. Thus, this system is useful for comparing the safety profiles of vector cassettes with various regulatory elements for their potential for proto-oncogene activation. IntroductionRetroviral vectors are potentially useful for gene therapy of blood disorders because they are capable of permanently integrating a therapeutic gene into stem cells and achieving long-term expression in differentiating hematopoietic cells. The initial clinical trials for severe combined immunodeficiency (SCID) secondary to adenosine deaminase deficiency (ADA) were either targeted to T lymphocytes 1 or resulted in too low a frequency of gene transfer into stem cells to produce clinical benefit. 2 Improved transduction conditions have now resulted in clinical benefit after stem cell-targeted gene transfer for patients with SCID secondary to ADA deficiency 3 or secondary to a deficiency in the common ␥ chain of interleukin receptors (X-SCID). [4][5][6] In addition, 2 severely affected patients with chronic granulomatous disease (CGD) achieved clearance of life-threatening infections by gene-corrected granulocytes derived from transduced stem cells. 7 In the context of these successful clinical trials, unequivocal evidence of the genotoxicity of retroviral vector integration has emerged in that 3 of the patients with X-SCID in one clinical trial 8,9 have developed leukemia. In 2, activation of the proto-oncogene, LMO2, was directly implicated as a genotoxic event. 8 In addition, the 2 patients with CGD exhibited striking clonal dominance with most of their gene-corrected cells having insertions into or near one or more of the MDS1-EVI1, PRDM16, or SETBP1 loci. Evidence indicated that these retroviral insertion events had activated the nearby proto-oncogene. 7 This experience emphasizes that the genotoxicity of retroviral integration is a relevant factor to be considered in designing vectors for future clinic...

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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An experimental system for the evaluation of retroviral vector design to diminish the risk for proto-oncogene activation

Ryu

Evans-Galea

Gray

et al. 2008

Blood

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“…DNA excision can subsequently occur between any pair of compatible sites to restore the two original DNA molecules or to exchange the intervening DNA segments between the two DNA molecules. This process, termed recombinase-mediated cassette exchange (RMCE), can be employed to integrate transgenes directionally into predefined genome sites (Trinh and Morrison, 2000;Baer and Bode, 2001). RMCE using two oppositely oriented identical RS sites, a donor containing the R recombinase gene and a third RS site to limit random integration, resulted in cassette exchange between the donor and a previously placed target in tobacco (Nanto et al, 2005).…”

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confidence: 99%

Site-Specific Integration of Transgenes in Soybean via Recombinase-Mediated DNA Cassette Exchange

et al. 2009

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A targeting method to insert genes at a previously characterized genetic locus to make plant transformation and transgene expression predictable is highly desirable for plant biotechnology. We report the successful targeting of transgenes to predefined soybean (Glycine max) genome sites using the yeast FLP-FRT recombination system. First, a target DNA containing a pair of incompatible FRT sites flanking a selection gene was introduced in soybean by standard biolistic transformation. Transgenic events containing a single copy of the target were retransformed with a donor DNA, which contained the same pair of FRT sites flanking a different selection gene, and a FLP expression DNA. Precise DNA cassette exchange was achieved between the target and donor DNA via recombinase-mediated cassette exchange, so that the donor DNA was introduced at the locus previously occupied by the target DNA. The introduced donor genes expressed normally and segregated according to Mendelian laws.

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“…The Cre/loxP system does not require accessory factors to carry out recombination in vivo or in vitro, and studies have identified several hetero-specific loxP sequences that exclusively recombine with themselves, but not with wild-type lox (Hoess et al 1986;Sauer 1992;Lee and Saito 1998;Siegel et al 2001). The combination of WT loxP and 511 loxP has been successfully used for a double reciprocal cross-over reaction to transfer a DNA segment or gene to a predetermined target site (Bethke and Sauer 1997;Feng et al 1999;Trinh and Morrison 2000), and this has proved to be an important tool for the in vivo manipulation of eukaryotic genomes, including gene activation and deactivation studies in both mammalian cells and transgenic mice. Recent reports, however, have suggested recombination can occur between wild-type and 511 loxP sequences (Lee and Saito 1998;Feng et al 1999), the levels depending upon the orientation of the sites with respect to one another (Siegel et al 2001).…”

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confidence: 99%

Recombinatorial Cloning Using Heterologous Lox Sites

Siegel¹,

Velappan²,

Pavlik³

et al. 2004

Genome Res.

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Recombination systems based on and Cre/loxP have been described to facilitate gene transfer from one vector to another in a high-throughput fashion, avoiding the bottlenecks associated with traditional cloning. However, no system described to date is suitable for the cloning of affinity reagents selected from display libraries, which requires that the recombination signals flanking the affinity reagent are translated with a minimum impact on functionality. As affinity reagents will be essential tools in the functional characterization of proteomes, and display technologies represent the most effective means to generate such affinity reagents on a genomic scale, we developed a Cre/loxP-based system, using mutually exclusive heterologous loxP sites placed 5Ј (Lox 2372) and 3Ј (Lox WT) of an affinity reagent (scFv). The translated lox sites have minimal impact on scFv expression or functionality, and, in association with a conditionally lethal gene (SacB) permit efficient, high-fidelity transfer to destination vectors. This approach will considerably facilitate the high-throughput downstream use of affinity reagents selected by display technologies, as well as being widely applicable to general recombinatorial cloning for genomic purposes.

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Site-specific and directional gene replacement mediated by Cre recombinase

Cited by 14 publications

References 17 publications

An experimental system for the evaluation of retroviral vector design to diminish the risk for proto-oncogene activation

An experimental system for the evaluation of retroviral vector design to diminish the risk for proto-oncogene activation

Site-Specific Integration of Transgenes in Soybean via Recombinase-Mediated DNA Cassette Exchange

Recombinatorial Cloning Using Heterologous Lox Sites

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