Pub Date : 2007-03-01DOI: 10.1142/S1568558607000058
D. Pearce, D. Bonnet
A fundamental problem in cancer research is the identification of the cell type capable of initiating and sustaining the growth of the neoplastic clone in vivo. The key to solving this problem lies on the observation made over 40 years ago that tumors are heterogeneous and thus might be maintained only by a rare subset of cells called "cancer stem cells" (CSCs). However, the proof of this principle was only possible after the development of modern research tools for investigating the behavior of defined cell populations in vivo. The blood-related cancer leukemia was the first disease where human CSCs, or leukemic stem cells (LSCs), were isolated. The development of quantitative xenotransplantation assays using immune-deficient mouse recipients to detect primitive human hematopoietic stem cells (HSCs) with in vivo repopulating ability and the adaptation of this model to leukemia have been instrumental. Leukemia can now be viewed as aberrant hematopoietic processes initiated by rare LSCs that have maintained or reacquired the capacity for indefinite proliferation through accumulated mutations and/or epigenetic changes. Yet, despite their critical importance, much remains to be learned about the developmental origin of LSC and the mechanisms responsible for their emergence in the course of the disease. This report will review our current knowledge on normal and leukemic stem cell development and finally demonstrate how these discoveries provide a paradigm for identification of CSCs from solid tumors. By a careful comparative analysis of the properties of CSCs and of their normal counterparts, it should be possible to pinpoint critical features amenable to efficient anti-CSC therapies.
{"title":"LEUKEMIA STEM CELLS: STUDYING THE ROOT OF LEUKEMIA","authors":"D. Pearce, D. Bonnet","doi":"10.1142/S1568558607000058","DOIUrl":"https://doi.org/10.1142/S1568558607000058","url":null,"abstract":"A fundamental problem in cancer research is the identification of the cell type capable of initiating and sustaining the growth of the neoplastic clone in vivo. The key to solving this problem lies on the observation made over 40 years ago that tumors are heterogeneous and thus might be maintained only by a rare subset of cells called \"cancer stem cells\" (CSCs). However, the proof of this principle was only possible after the development of modern research tools for investigating the behavior of defined cell populations in vivo. The blood-related cancer leukemia was the first disease where human CSCs, or leukemic stem cells (LSCs), were isolated. The development of quantitative xenotransplantation assays using immune-deficient mouse recipients to detect primitive human hematopoietic stem cells (HSCs) with in vivo repopulating ability and the adaptation of this model to leukemia have been instrumental. Leukemia can now be viewed as aberrant hematopoietic processes initiated by rare LSCs that have maintained or reacquired the capacity for indefinite proliferation through accumulated mutations and/or epigenetic changes. Yet, despite their critical importance, much remains to be learned about the developmental origin of LSC and the mechanisms responsible for their emergence in the course of the disease. This report will review our current knowledge on normal and leukemic stem cell development and finally demonstrate how these discoveries provide a paradigm for identification of CSCs from solid tumors. By a careful comparative analysis of the properties of CSCs and of their normal counterparts, it should be possible to pinpoint critical features amenable to efficient anti-CSC therapies.","PeriodicalId":93646,"journal":{"name":"Gene therapy and regulation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2007-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1142/S1568558607000058","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64014749","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2007-03-01DOI: 10.1142/S156855860700006X
A. Sepp, F. Ghadessy, Y. Choo
Engineered DNA-binding proteins, in particular zinc finger proteins (ZFPs), have broad-ranging applications in gene therapy. An engineered ZFP transcription activator targeted to the VEGF locus is currently undergoing clinical trials for the induction of angiogenesis. A number of ZFP gene switches have been developed which allow highly controllable regulation of therapeutic transgene expression based on small molecule inducers/repressors. Finally, engineered ZFP nucleases have been used to correct a gene sequence in a living cell by stimulating homologous DNA recombination, suggesting a new, highly targeted approach to gene therapy. All these approaches rely on DNA-binding protein engineering, which in the past has mainly been achieved by selection using phage display. However, a convenient cell-free selection method known as in vitro compartmentalization (IVC) has previously been used to engineer DNA-binding proteins with enzymatic activities (e.g. polymerase and methylase), and the method has recently been extended to the engineering of sequence-specific ZFP DNA-binders. Below we describe the IVC procedure and review the progress made in applying this to the problem of facilitating the engineering of DNA-binding proteins.
{"title":"CELL-FREE SELECTION OF DNA-BINDING PROTEINS FOR FUTURE GENE THERAPY APPLICATIONS ∗","authors":"A. Sepp, F. Ghadessy, Y. Choo","doi":"10.1142/S156855860700006X","DOIUrl":"https://doi.org/10.1142/S156855860700006X","url":null,"abstract":"Engineered DNA-binding proteins, in particular zinc finger proteins (ZFPs), have broad-ranging applications in gene therapy. An engineered ZFP transcription activator targeted to the VEGF locus is currently undergoing clinical trials for the induction of angiogenesis. A number of ZFP gene switches have been developed which allow highly controllable regulation of therapeutic transgene expression based on small molecule inducers/repressors. Finally, engineered ZFP nucleases have been used to correct a gene sequence in a living cell by stimulating homologous DNA recombination, suggesting a new, highly targeted approach to gene therapy. All these approaches rely on DNA-binding protein engineering, which in the past has mainly been achieved by selection using phage display. However, a convenient cell-free selection method known as in vitro compartmentalization (IVC) has previously been used to engineer DNA-binding proteins with enzymatic activities (e.g. polymerase and methylase), and the method has recently been extended to the engineering of sequence-specific ZFP DNA-binders. Below we describe the IVC procedure and review the progress made in applying this to the problem of facilitating the engineering of DNA-binding proteins.","PeriodicalId":93646,"journal":{"name":"Gene therapy and regulation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2007-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1142/S156855860700006X","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64014763","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2004-12-01DOI: 10.1163/1568558043967481
U. Lakshmipathy, Luke Hammer, C. Verfaillie
Multipotent adult progenitor cells (MAPC) are stem cells isolated primarily from adult bone marrow that have the ability to differentiate in vitro into cells with phenotypic and functional characteristics of cells from the three germ layers, namely endoderm, mesoderm and neuroectoderm. In addition, MAPCs proliferate for extended periods of time without obvious senescence. Hence, MAPCs may be ideal cells for therapy of genetic disorders, provided that the main impediment to gene therapy, namely efficient gene transfer and persistent gene expression, can be overcome. Most commercially available lipid-based methods that very highly efficiently transfect cell lines and primary cells, fail to transfect MAPC. However, 10–15% transfection of MAPC can be achieved using Superfect. However, this approach requires high density culture of MAPCs which subsequently differentiate. Between 12 and 15% transfection is achieved in MAPCs using electroporation, but this is highly toxic to MAPCs. Here, using transient expression of an enhanced green fluorescent protein (EGFP) gene, we report that nucleofection results in a transfection efficiency of over 25% with mouse MAPC without perturbing the conditions suitable for MAPC growth and maintenance, and with significantly less toxicity than electroporation. Similar results were seen for rat and human MAPC. Efficient transfection of MAPC by nucleofection offers an appealing non-viral mode of gene delivery and can be used to overexpress genes of interest in these cells. In addition, it should easily accomodate emerging site-specific integration technology, thereby culminating eventually in MAPC-mediated long-term gene therapy for genetic disorders.
{"title":"Method — A nonviral gene transfer method for transfecting multipotent adult progenitor cells (MAPC)","authors":"U. Lakshmipathy, Luke Hammer, C. Verfaillie","doi":"10.1163/1568558043967481","DOIUrl":"https://doi.org/10.1163/1568558043967481","url":null,"abstract":"Multipotent adult progenitor cells (MAPC) are stem cells isolated primarily from adult bone marrow that have the ability to differentiate in vitro into cells with phenotypic and functional characteristics of cells from the three germ layers, namely endoderm, mesoderm and neuroectoderm. In addition, MAPCs proliferate for extended periods of time without obvious senescence. Hence, MAPCs may be ideal cells for therapy of genetic disorders, provided that the main impediment to gene therapy, namely efficient gene transfer and persistent gene expression, can be overcome. Most commercially available lipid-based methods that very highly efficiently transfect cell lines and primary cells, fail to transfect MAPC. However, 10–15% transfection of MAPC can be achieved using Superfect. However, this approach requires high density culture of MAPCs which subsequently differentiate. Between 12 and 15% transfection is achieved in MAPCs using electroporation, but this is highly toxic to MAPCs. Here, using transient expression of an enhanced green fluorescent protein (EGFP) gene, we report that nucleofection results in a transfection efficiency of over 25% with mouse MAPC without perturbing the conditions suitable for MAPC growth and maintenance, and with significantly less toxicity than electroporation. Similar results were seen for rat and human MAPC. Efficient transfection of MAPC by nucleofection offers an appealing non-viral mode of gene delivery and can be used to overexpress genes of interest in these cells. In addition, it should easily accomodate emerging site-specific integration technology, thereby culminating eventually in MAPC-mediated long-term gene therapy for genetic disorders.","PeriodicalId":93646,"journal":{"name":"Gene therapy and regulation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2004-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1163/1568558043967481","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64796194","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2004-12-01DOI: 10.1163/1568558043967463
H. Kawasaki, Y. Fukuda, K. Taira
Small RNAs, such as short-interfering RNAs (siRNAs) and microRNAs (miRNAs), regulate gene expression in a sequence-dependent manner in animals and plants. These small RNAs are generated from long double-stranded (ds) RNAs or pre-miRNAs by the ribonuclease Dicer and become incorporated into RNA interference (RNAi)-induced silencing complexes (RISCs) or miRNA ribonucleoprotein complexes (miRNPs). These complexes have the ability to cleave target mRNAs with perfect base-pairing and inhibit the translation of target mRNAs with partial base-pairing. In addition, small RNAs can also act on the chromosomes in the nucleus. In this process, it is known that siRNAs targeted to CpG islands within promoters can induce RNA-directed DNA methylation and play a role in heterochromatic gene silencing. Thus, small RNAs can regulate gene expression at both the transcriptional and post-transcriptional levels. In this review, we focus on small RNA-mediated transcriptional gene silencing and on its role and potential therapeutic applications in chromosome maintenance and gene regulation, including epigenetic regulation.
{"title":"Small RNA-mediated chromatin modification and transcriptional gene silencing","authors":"H. Kawasaki, Y. Fukuda, K. Taira","doi":"10.1163/1568558043967463","DOIUrl":"https://doi.org/10.1163/1568558043967463","url":null,"abstract":"Small RNAs, such as short-interfering RNAs (siRNAs) and microRNAs (miRNAs), regulate gene expression in a sequence-dependent manner in animals and plants. These small RNAs are generated from long double-stranded (ds) RNAs or pre-miRNAs by the ribonuclease Dicer and become incorporated into RNA interference (RNAi)-induced silencing complexes (RISCs) or miRNA ribonucleoprotein complexes (miRNPs). These complexes have the ability to cleave target mRNAs with perfect base-pairing and inhibit the translation of target mRNAs with partial base-pairing. In addition, small RNAs can also act on the chromosomes in the nucleus. In this process, it is known that siRNAs targeted to CpG islands within promoters can induce RNA-directed DNA methylation and play a role in heterochromatic gene silencing. Thus, small RNAs can regulate gene expression at both the transcriptional and post-transcriptional levels. In this review, we focus on small RNA-mediated transcriptional gene silencing and on its role and potential therapeutic applications in chromosome maintenance and gene regulation, including epigenetic regulation.","PeriodicalId":93646,"journal":{"name":"Gene therapy and regulation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2004-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1163/1568558043967463","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64796124","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2004-12-01DOI: 10.1163/1568558043967472
M. Asparuhova, R. Kole, D. Schümperli
The importance of alternative splicing for the diversity of the proteome and the large number of genetic diseases that are due to splicing defects call for methods to modulate alternative splicing decisions. Although splicing can be modulated by antisense oligonucleotides, this approach is confronted with problems of efficient delivery and the need for repeated administrations of large amounts of the oligonucleotides. Therefore we have developed methods allowing us to modulate splicing with the help of modified derivatives of the U7 small nuclear RNA involved in histone RNA 3′ end processing. Its nuclear accumulation as a stable ribonucleoprotein particle makes U7 snRNA especially useful for this purpose. In particular, U7 derivatives containing two tandem antisense sequences directed against targets upstream and downstream of an exon can induce the efficient and specific skipping of that exon. U7 expression cassettes have been successfully introduced into a great number of cell lines, primary cells or tissues with the help of lentiviral and adeno-associated viral vectors. Examples of these therapeutic strategies in the fields of β-thalassemia, Duchenne muscular dystrophy and HIV/AIDS are discussed.
{"title":"Antisense derivatives of U7 and other small nuclear RNAs as tools to modify pre-mRNA splicing patterns","authors":"M. Asparuhova, R. Kole, D. Schümperli","doi":"10.1163/1568558043967472","DOIUrl":"https://doi.org/10.1163/1568558043967472","url":null,"abstract":"The importance of alternative splicing for the diversity of the proteome and the large number of genetic diseases that are due to splicing defects call for methods to modulate alternative splicing decisions. Although splicing can be modulated by antisense oligonucleotides, this approach is confronted with problems of efficient delivery and the need for repeated administrations of large amounts of the oligonucleotides. Therefore we have developed methods allowing us to modulate splicing with the help of modified derivatives of the U7 small nuclear RNA involved in histone RNA 3′ end processing. Its nuclear accumulation as a stable ribonucleoprotein particle makes U7 snRNA especially useful for this purpose. In particular, U7 derivatives containing two tandem antisense sequences directed against targets upstream and downstream of an exon can induce the efficient and specific skipping of that exon. U7 expression cassettes have been successfully introduced into a great number of cell lines, primary cells or tissues with the help of lentiviral and adeno-associated viral vectors. Examples of these therapeutic strategies in the fields of β-thalassemia, Duchenne muscular dystrophy and HIV/AIDS are discussed.","PeriodicalId":93646,"journal":{"name":"Gene therapy and regulation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2004-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1163/1568558043967472","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64796181","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2004-12-01DOI: 10.1163/1568558043967454
M. Cannon, C. Pinkert, I. Trounce
The characterization of mitochondrial diseases has proceeded rapidly since the first descriptions of mitochondrial DNA (mtDNA)-linked disease mutations appeared in the late 1980s. To elucidate mechanisms of a variety of mitochondrial disorders and disease, both in vitro and in vivo modeling systems have been exploited. To produce these models, numerous approaches have been undertaken due to the difficulty associated with targeted mutagenesis and directed modification of the mitochondrial genome. Currently available models of mitochondrial disease are discussed in this paper, including our xenomitochondrial mice. In this model, mitochondria from one donor species are transferred to another. By doing so, cells and animals were generated with varying levels of heteroplasmy (or homoplasmy) for the introduced mitochondrial genomes. This caused graded variations in electron transport chain (ETC) dysfunction which were dependent upon the evolutionary divergence between donor and recipient. The protocol in generating these models involved the utilization of rhodamine-6G (R6G) to remove or eliminate endogenous mtDNA from the recipient cells. This paper will highlight the process and the implications of R6G treatment of mouse embryonic stem (ES) cells to create transmitochondrial cybrids. We summarize the history and mechanism of action of R6G as well as the future prospects for xenomitochondrial models toward increasing our understanding of mitochondrial biology and the dynamic interplay in signaling between mitochondria and the nucleus.
{"title":"Xenomitochondrial embryonic stem cells and mice: modeling human mitochondrial biology and disease","authors":"M. Cannon, C. Pinkert, I. Trounce","doi":"10.1163/1568558043967454","DOIUrl":"https://doi.org/10.1163/1568558043967454","url":null,"abstract":"The characterization of mitochondrial diseases has proceeded rapidly since the first descriptions of mitochondrial DNA (mtDNA)-linked disease mutations appeared in the late 1980s. To elucidate mechanisms of a variety of mitochondrial disorders and disease, both in vitro and in vivo modeling systems have been exploited. To produce these models, numerous approaches have been undertaken due to the difficulty associated with targeted mutagenesis and directed modification of the mitochondrial genome. Currently available models of mitochondrial disease are discussed in this paper, including our xenomitochondrial mice. In this model, mitochondria from one donor species are transferred to another. By doing so, cells and animals were generated with varying levels of heteroplasmy (or homoplasmy) for the introduced mitochondrial genomes. This caused graded variations in electron transport chain (ETC) dysfunction which were dependent upon the evolutionary divergence between donor and recipient. The protocol in generating these models involved the utilization of rhodamine-6G (R6G) to remove or eliminate endogenous mtDNA from the recipient cells. This paper will highlight the process and the implications of R6G treatment of mouse embryonic stem (ES) cells to create transmitochondrial cybrids. We summarize the history and mechanism of action of R6G as well as the future prospects for xenomitochondrial models toward increasing our understanding of mitochondrial biology and the dynamic interplay in signaling between mitochondria and the nucleus.","PeriodicalId":93646,"journal":{"name":"Gene therapy and regulation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2004-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1163/1568558043967454","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64795732","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2004-12-01DOI: 10.1163/1568558043967517
J. Mattick
Proteins and their products are the analog components of cells. However the number of protein-coding genes in humans is not markedly different from that of a simple nematode worm, despite the vast differences in their developmental complexity. On the other hand, most of the human genome is transcribed, mainly into non-protein-coding RNAs. Both logic and emerging evidence suggest that these RNAs are not junk, but form an extensive regulatory network that was a necessary adaptation to solve the vastly expanded regulatory requirements of complex organisms, and that these RNAs now comprise a hidden layer of feed-forward control signals that direct the epigenetic trajectories of differentiation and development.
{"title":"Digital RNA regulation of complex organisms","authors":"J. Mattick","doi":"10.1163/1568558043967517","DOIUrl":"https://doi.org/10.1163/1568558043967517","url":null,"abstract":"Proteins and their products are the analog components of cells. However the number of protein-coding genes in humans is not markedly different from that of a simple nematode worm, despite the vast differences in their developmental complexity. On the other hand, most of the human genome is transcribed, mainly into non-protein-coding RNAs. Both logic and emerging evidence suggest that these RNAs are not junk, but form an extensive regulatory network that was a necessary adaptation to solve the vastly expanded regulatory requirements of complex organisms, and that these RNAs now comprise a hidden layer of feed-forward control signals that direct the epigenetic trajectories of differentiation and development.","PeriodicalId":93646,"journal":{"name":"Gene therapy and regulation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2004-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1163/1568558043967517","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64796154","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2004-12-01DOI: 10.1163/1568558043967490
T. A. Wilkinson, W. Tan, S. A. Chow
Gene therapy approaches that involve the permanent insertion of therapeutic genes into host chromosomal DNA have many desirable features and show considerable promise for success in the clinic. One major drawback of these approaches is that any unintended insertion events from the therapy can potentially have detrimental effects in patients, as demonstrated by the development of malignancies in both animal and human studies. Therefore, directing the integration of foreign genes into "safe sites" within the genome is highly desirable for these approaches. In retroviral-based vector systems, the viral enzyme integrase (IN) catalyzes the insertion of a desired transgene nonspecifically into the host cell genome. Efforts to engineer IN to recognize specific target DNA sequences within the genome, and thereby improve the safety of future generations of retroviral-based vectors, are described. Recent results using fusion protein constructs of IN and E2C, a designed polydactyl zinc-finger protein that specifically recognizes an 18-base pair DNA sequence, are highlighted in this review. Encouraging results have been generated in vitro, and additional studies are ongoing in mammalian cell systems. The long-term goal of these efforts is the development of effective retroviral vectors that can safely deliver therapeutics in a gene therapy setting.
{"title":"Safe delivery of therapeutic genes into specific chromosomal sites using engineered retroviral integrase","authors":"T. A. Wilkinson, W. Tan, S. A. Chow","doi":"10.1163/1568558043967490","DOIUrl":"https://doi.org/10.1163/1568558043967490","url":null,"abstract":"Gene therapy approaches that involve the permanent insertion of therapeutic genes into host chromosomal DNA have many desirable features and show considerable promise for success in the clinic. One major drawback of these approaches is that any unintended insertion events from the therapy can potentially have detrimental effects in patients, as demonstrated by the development of malignancies in both animal and human studies. Therefore, directing the integration of foreign genes into \"safe sites\" within the genome is highly desirable for these approaches. In retroviral-based vector systems, the viral enzyme integrase (IN) catalyzes the insertion of a desired transgene nonspecifically into the host cell genome. Efforts to engineer IN to recognize specific target DNA sequences within the genome, and thereby improve the safety of future generations of retroviral-based vectors, are described. Recent results using fusion protein constructs of IN and E2C, a designed polydactyl zinc-finger protein that specifically recognizes an 18-base pair DNA sequence, are highlighted in this review. Encouraging results have been generated in vitro, and additional studies are ongoing in mammalian cell systems. The long-term goal of these efforts is the development of effective retroviral vectors that can safely deliver therapeutics in a gene therapy setting.","PeriodicalId":93646,"journal":{"name":"Gene therapy and regulation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2004-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1163/1568558043967490","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64795991","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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