Prospects for homologous recombination in human gene therapy

Vega, Manuel A.

doi:10.1007/bf00200899

Cited by 23 publications

(16 citation statements)

References 89 publications

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“…The integration frequency into an attB site located on an EBV plasmid is several orders of magnitude higher than the frequency of random integration (1). The site-specificity of the enzyme also distinguishes it from the random integration mediated by retroviral integrases and most transposases.…”

Section: Discussionmentioning

confidence: 99%

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A phage integrase directs efficient site-specific integration in human cells

Groth

Olivares

Thyagarajan

et al. 2000

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

464

438

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The integrase from the Streptomyces phage C31 carries out efficient recombination between the attP site in the phage genome and the attB site in the host bacterial chromosome. In this paper, we show that the enzyme also functions in human cells. A plasmid assay system was constructed that measured intramolecular integration of attP into attB. This assay was used to demonstrate that in the presence of the C31 integrase, precise unidirectional integration occurs with an efficiency of 100% in Escherichia coli and >50% in human cells. This assay system was also used to define the minimal sizes of attB and attP at 34 bp and 39 bp, respectively. Furthermore, precise and efficient intermolecular integration of an incoming plasmid bearing attP into an established Epstein-Barr virus plasmid bearing attB was documented in human cells. This work is a demonstration of efficient, site-specific, unidirectional integration in mammalian cells. These observations form the basis for site-specific integration strategies potentially useful in a broad range of genetic engineering applications.

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Section: Discussionmentioning

confidence: 99%

“…Homologous recombination can provide great specificity in integration sites, but it occurs at too low a frequency to be optimal for genetic engineering in multicellular organisms (1). Enzymes of the site-specific recombinase family also share high specificity, and, in addition, they act with greater efficiency.…”

mentioning

confidence: 99%

A phage integrase directs efficient site-specific integration in human cells

Groth

Olivares

Thyagarajan

et al. 2000

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

464

438

View full text Add to dashboard Cite

show abstract

“…The practical application of homologous recombination between exogenous DNA and a chromosomal target (gene targeting) is making an enormous impact on mouse molecular genetics (4,5). Highly efficient gene targeting would provide the ideal form of gene therapy, allowing deleterious mutations to be corrected rather than merely compensating for them (42), but at present the efficiency of gene targeting is several orders of magnitude too low for this to be feasible. Improvements in the frequency of homologous recombination and reductions in the proportion of integration which occurs by illegitimate recombination would be required before homologous recombination could be used for gene therapy.…”

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confidence: 99%

Processing of DNA Prior to Illegitimate Recombination in Mouse Cells

Henderson

Simons

1997

Molecular and Cellular Biology

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In mammalian cells, the predominant pathway of chromosomal integration of exogenous DNA is random or illegitimate recombination; integration by homologous recombination is infrequent. Homologous recombination is initiated at double-strand DNA breaks which have been acted on by single-strand exonuclease. To further characterize the relationship between illegitimate and homologous recombination, we have investigated whether illegitimate recombination is also preceded by exonuclease digestion. Heteroduplex DNAs which included strand-specific restriction markers at each of four positions were generated. These DNAs were introduced into mouse embryonic stem cells, and stably transformed clones were isolated and analyzed to determine whether there was any strand bias in the retention of restriction markers with respect to their positions. Some of the mismatches appear to have been resolved by mismatch repair. Very significant strand bias was observed in the retention of restriction markers, and there was polarity of marker retention between adjacent positions. We conclude that DNA is frequently subjected to 533 exonuclease digestion prior to integration by illegitimate recombination and that the length of DNA removed by exonuclease digestion can be extensive. We also provide evidence which suggests that frequent but less extensive 335 exonuclease processing also occurs.Most DNA introduced into mammalian cells is lost; that which is stably maintained is usually retained by virtue of its integration into nonhomologous chromosomal DNA sites by so-called illegitimate recombination. If the exogenous DNA includes sequences which are homologous with chromosomal sequences, a small minority of cells which integrate DNA will do so by homologous recombination. The practical application of homologous recombination between exogenous DNA and a chromosomal target (gene targeting) is making an enormous impact on mouse molecular genetics (4, 5). Highly efficient gene targeting would provide the ideal form of gene therapy, allowing deleterious mutations to be corrected rather than merely compensating for them (42), but at present the efficiency of gene targeting is several orders of magnitude too low for this to be feasible. Improvements in the frequency of homologous recombination and reductions in the proportion of integration which occurs by illegitimate recombination would be required before homologous recombination could be used for gene therapy. For such improvements to be made, it will be important to understand the mechanisms of gene targeting and illegitimate recombination and the relationships between these processes.There have been numerous studies of the mechanism of homologous recombination in mammalian cells. In many cases, the recombination substrates were introduced into cells together; in these studies, homologous recombination occurs efficiently and prior to chromosomal integration (1,6,20). In contrast with gene targeting, such extrachromosomal homologous recombination is efficient. The mechanism of extrachromosomal h...

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“…Such applications include chromosome engineering [13], gene therapy [14] and the analysis of gene expression [I51 and gene function [16, 171. In the latter case, the use of HR to create somatic cell lines carrying mutations in specific genes would be useful for the genetic analysis of a wide variety of phenomena that can be studied in cell culture. Such an approach is particularly relevant for the analysis of human gene function, where the use of ES cells is not an option, or for the analysis of those mouse genes whose disruption might prevent the development of viable mice from ES cells.…”

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confidence: 99%

Gene targeting in human somatic cells

Porter

Itzhaki

1993

European Journal of Biochemistry

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The role, if any, of the human interferon‐inducible 6–16 gene in the establishment of a cellular antiviral state is unknown. To address this problem, and as part of a wider investigation of homologous recombination (HR) and its applications in somatic cells, we have been using HR to disrupt the 6–16 gene in human cell lines [Itzhaki, J. E. & Porter, A. C. G. (1991) Nucleic Acids Res. 19, 3835–3842.] We describe here the design and use of insertion and replacement‐type targeting constructs based on a promoterless bacterial gpt gene that is activated by HR with the 6–16 gene. In HeLa cells, both targeting constructs underwent extrachromosomal HR with a cotransfected plasmid carrying the 6–16 gene. In a previously targeted clone derived from the fibrosarcoma cell line HT1080, the replacement construct underwent HR with either the modified or the unmodified 6–16 allele. The latter events generated doubly disrupted (6–16–/–) clones that failed to express any detectable 6–16 messenger RNA in response to interferon. Plaque assays of infected 6–16–/– cells showed that expression of the 6–16 gene was not required for the induction by interferon of an antiviral state against encephalomyocarditis virus, semliki forest virus or cocal virus.

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Prospects for homologous recombination in human gene therapy

Cited by 23 publications

References 89 publications

A phage integrase directs efficient site-specific integration in human cells

A phage integrase directs efficient site-specific integration in human cells

Processing of DNA Prior to Illegitimate Recombination in Mouse Cells

Gene targeting in human somatic cells

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