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Eric B. Kmiec - One of the best experts on this subject based on the ideXlab platform.
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Regulation of targeted Gene Repair by intrinsic cellular processes.
BioEssays : news and reviews in molecular cellular and developmental biology, 2009Co-Authors: Julia Engstrom, Takayuki Suzuki, Eric B. KmiecAbstract:Targeted Gene alteration (TGA) is a strategy for correcting single base mutations in the DNA of human cells that cause inherited disorders. TGA aims to reverse a phenotype by Repairing the mutant base within the chromosome itself, avoiding the introduction of exogenous Genes. The process of how to accurately Repair a Genetic mutation is elucidated through the use of single-stranded DNA oligonucleotides (ODNs) that can enter the cell and migrate to the nucleus. These specifically designed ODNs hybridize to the target sequence and act as a beacon for nucleotide exchange. The key to this reaction is the frequency with which the base is corrected; this will determine whether the approach becomes clinically relevant or not. Over the course of the last five years, workers have been uncovering the role played by the cells in regulating the Gene Repair process. In this essay, we discuss how the impact of the cell on TGA has evolved through the years and illustrate ways that inherent cellular pathways could be used to enhance TGA activity. We also describe the cost to cell metabolism and survival when certain processes are altered to achieve a higher frequency of Repair.
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DNA replication, cell cycle progression and the targeted Gene Repair reaction.
Cell cycle (Georgetown Tex.), 2008Co-Authors: Julia Engstrom, Eric B. KmiecAbstract:Single-stranded oligonucleotides (ssODNs) can direct base changes in mammalian cells and influence changes in phenotype. The mechanism by which ssODNs alters the sequence is being revealed by studies carried out in model systems. In the long run, this information will provide the basis for clinical protocols designed to target Genetic diseases. It is now clear that DNA replication plays an important part in the Gene Repair reaction. Here, we examine Gene Repair as a function of the amount of cells passing through S phase. We find that cells in mid to late S are most amenable to Gene Repair, and reaction manipulations that enrich the population of cells in S phase naturally lead to elevated correction frequencies. Our data suggest that these intra-S sub phases support higher levels of Repair independent of transfection efficiencies or the rates of replication. A preliminary Gene expression profile of cells in the most amenable correction phase indicates that the levels of cyclin G2, cyclin H, CDK12A and CD...
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Recovery of cell cycle delay following targeted Gene Repair by oligonucleotides.
DNA repair, 2007Co-Authors: Luciana Ferrara, Timothy Schwartz, Hetal Parekh-olmedo, Julia U. Engstrom, Eric B. KmiecAbstract:We have previously shown that activation of the homologous recombinational Repair pathway leads to a block of cell division in corrected cells, possibly through the activity of checkpoint proteins Chk1 and Chk2. In this study, we examine the long-term impact of this stalling on the growth of cells that have enabled Gene Repair events. Using a mutated eGFP Gene as an episomal reporter, we show that corrected (eGFP-positive) cells contain only a few active replication templates 2 weeks after electroporation, yet do not display an apoptotic or senescent phenotype. By 6 weeks after electroporation, cells resume active replication with a cell cycle profile that is comparable to that of the non-corrected (eGFP-negative) population. These results indicate that the initial stalling is transient and eGFP-positive cells eventually resume a normal phenotypic growth pattern, allowing for passaging and expansion in vitro.
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Reduction of Gene Repair by selenomethionine with the use of single-stranded oligonucleotides.
BMC molecular biology, 2007Co-Authors: Timothy Schwartz, Eric B. KmiecAbstract:Background The Repair of single base mutations in mammalian Genes can be directed by single-stranded oligonucleotides in a process known as targeted Gene Repair. The mechanism of this reaction is currently being elucidated but likely involves a pairing step in which the oligonucleotide align in homologous register with its target sequence and a correction step in which the mutant base is replaced by endogenous Repair pathways. This process is regulated by the activity of various factors and proteins that either elevate or depress the frequency at which Gene Repair takes place.
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eLS - Chimera‐directed Gene Repair
Encyclopedia of Life Sciences, 2006Co-Authors: Eric B. KmiecAbstract:The information gained from the Human Genome Project must be formulated such that the function of newly discovered Genes can be elucidated. A tool to aid in defining Gene function is targeted Gene Repair, a technique that may also be valuable in human Gene therapy. Keywords: chimera; Gene Repair; chimeric RNA–DNA oligonucleotide; transfection; RAD51; yeast
Markus Grompe - One of the best experts on this subject based on the ideXlab platform.
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Induced Liver ReGeneration Enhances CRISPR/Cas9-Mediated Gene Repair in Tyrosinemia Type 1.
Human gene therapy, 2020Co-Authors: Qing Shuo Zhang, Amita Tiyaboonchai, Sean Nygaard, Kevin Baradar, Angela Major, Niveditha Balaji, Markus GrompeAbstract:The efficiency of Gene Repair by homologous recombination in the liver is enhanced by CRISP/Cas9 incision near the mutation. In this study, we explored interventions designed to further enhance in ...
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adeno associated virus Gene Repair corrects a mouse model of hereditary tyrosinemia in vivo
Hepatology, 2010Co-Authors: Nicole K. Paulk, Karsten Wursthorn, Zhongya Wang, Milton J. Finegold, Mark A. Kay, Markus GrompeAbstract:Adeno-associated virus (AAV) vectors are ideal for performing Gene Repair due to their ability to target multiple different genomic loci, low immunogenicity, capability to achieve targeted and stable expression through integration, and low mutagenic and oncogenic potential. However, many handicaps to Gene Repair therapy remain. Most notable is the low frequency of correction in vivo. To date, this frequency is too low to be of therapeutic value for any disease. To address this, a point-mutation–based mouse model of the metabolic disease hereditary tyrosinemia type I was used to test whether targeted AAV integration by homologous recombination could achieve high-level stable Gene Repair in vivo. Both neonatal and adult mice were treated with AAV serotypes 2 and 8 carrying a wild-type genomic sequence for Repairing the mutated Fah (fumarylacetoacetate hydrolase) Gene. Hepatic Gene Repair was quantified by immunohistochemistry and supported with reverse transcription polymerase chain reaction and serology for functional correction parameters. Successful Gene Repair was observed with both serotypes but was more efficient with AAV8. Correction frequencies of up to 10−3 were achieved and highly reproducible within typical dose ranges. In this model, Repaired hepatocytes have a selective growth advantage and are thus able to proliferate to efficiently repopulate mutant livers and cure the underlying metabolic disease. Conclusion: AAV-mediated Gene Repair is feasible in vivo and can functionally correct an appropriate selection-based metabolic liver disease in both adults and neonates. (HEPATOLOGY 2010.)
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Adeno-associated virus Gene Repair corrects a mouse model of hereditary tyrosinemia in vivo. Hepatology 51
2010Co-Authors: Nicole K. Paulk, Karsten Wursthorn, Zhongya Wang, Milton J. Finegold, Mark A. Kay, Markus GrompeAbstract:Adeno-associated virus (AAV) vectors are ideal for performing Gene Repair due to their ability to target multiple different genomic loci, low immunogenicity, capability to achieve targeted and stable expression through integration, and low mutagenic and oncogenic potential. However, many handicaps to Gene Repair therapy remain. Most notable is the low frequency of correction in vivo. To date, this frequency is too low to be of therapeutic value for any disease. To address this, a point-mutation–based mouse model of the metabolic disease hereditary tyrosinemia type I was used to test whether targeted AAV integration by homologous recombination could achieve high-level stable Gene Repair in vivo. Both neonatal and adult mice were treated with AAV serotypes 2 and 8 carrying a wild-type genomic sequence for Repairing the mutated Fah (fumarylacetoacetate hydrolase) Gene. Hepatic Gene Repair was quantified by immunohistochemistry and supported with reverse transcription polymerase chain reaction and serology for functional correction parameters. Successful Gene Repair was observed with both serotypes but was more efficient with AAV8. Correction frequencies of up to 10 �3 were achieved and highly reproducible within typical dose ranges. In this model, Repaired hepatocytes have a selective growth advantage and are thus able to proliferate to efficiently repopulate mutant livers and cure the underlying metabolic disease. Conclusion: AAV-mediated Gene Repair is feasible in vivo and can functionally correct an appropriate selection-based metabolic liver disease in both adults and neonates. (HEPATOLOGY 2010;51: 1200-1208.) Gene therapy is a promising means to cure many monogenic diseases. However, traditional Gene therapies are best suited to treat diseases of deficient or absent Gene products rather than those diseases Abbreviations: AAV, adeno-associated virus; AST, aspartate aminotransferase; dGE, diploid genome equivalent; FAH, fumarylacetoacetate hydrolase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; hAAT, human alpha-1 antitrypsin; HTI, hereditary tyrosinemia type I; LD-PCR, long-distance polymerase chain reaction
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Adeno‐associated virus Gene Repair corrects a mouse model of hereditary tyrosinemia in vivo
Hepatology (Baltimore Md.), 2009Co-Authors: Nicole K. Paulk, Karsten Wursthorn, Zhongya Wang, Milton J. Finegold, Mark A. Kay, Markus GrompeAbstract:Adeno-associated virus (AAV) vectors are ideal for performing Gene Repair due to their ability to target multiple different genomic loci, low immunogenicity, capability to achieve targeted and stable expression through integration, and low mutagenic and oncogenic potential. However, many handicaps to Gene Repair therapy remain. Most notable is the low frequency of correction in vivo. To date, this frequency is too low to be of therapeutic value for any disease. To address this, a point-mutation–based mouse model of the metabolic disease hereditary tyrosinemia type I was used to test whether targeted AAV integration by homologous recombination could achieve high-level stable Gene Repair in vivo. Both neonatal and adult mice were treated with AAV serotypes 2 and 8 carrying a wild-type genomic sequence for Repairing the mutated Fah (fumarylacetoacetate hydrolase) Gene. Hepatic Gene Repair was quantified by immunohistochemistry and supported with reverse transcription polymerase chain reaction and serology for functional correction parameters. Successful Gene Repair was observed with both serotypes but was more efficient with AAV8. Correction frequencies of up to 10−3 were achieved and highly reproducible within typical dose ranges. In this model, Repaired hepatocytes have a selective growth advantage and are thus able to proliferate to efficiently repopulate mutant livers and cure the underlying metabolic disease. Conclusion: AAV-mediated Gene Repair is feasible in vivo and can functionally correct an appropriate selection-based metabolic liver disease in both adults and neonates. (HEPATOLOGY 2010.)
Li Liu - One of the best experts on this subject based on the ideXlab platform.
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Genetic re‐engineering of Saccharomyces cerevisiae RAD51 leads to a significant increase in the frequency of Gene Repair in vivo
Nucleic acids research, 2004Co-Authors: Li Liu, Katie Maguire, Eric B. KmiecAbstract:Oligonucleotides can be used to direct the alteration of single nucleotides in chromosomal Genes in yeast. Rad51 protein appears to play a central role in catalyzing the reaction, most likely through its DNA pairing function. Here, we re-engineer the RAD51 Gene in order to produce proteins bearing altered levels of known activities. Overexpression of wild-type ScRAD51 elevates the correction of an integrated, mutant hygromycin resistance Gene approximately 3-fold. Overexpression of an altered RAD51 Gene, which encodes a protein that has a higher affinity for ScRad54, enhances the targeting frequency nearly 100-fold. Another mutation which increases the affinity of Rad51 for DNA was also found to increase Gene Repair when overexpressed in the cell. Other mutations in the Rad51 protein, such as one that reduces interaction with Rad52, has little or no effect on the frequency of Gene Repair. These data provide the first evidence that the Rad51 protein can be modified so as to increase the frequency of Gene Repair in yeast.
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310. Feasibility and Limitations of Target Gene Repair in Mammalian Cells
Molecular Therapy, 2004Co-Authors: Eric B. Kmiec, Li Liu, Hetal Parekh-olmedoAbstract:Targeted Gene Repair embodies some of the most attractive theoretical aspects of Gene Repair therapy, the correction of a Genetic mutation within the context of the host chromosome. Should such a strategy prove robust, some inherited metabolic disorders could be well positioned for successful treatment and perhaps even cure. In its most popular iteration, the Gene Repair pathway employs synthetic DNA (and RNA) oligonucleotides to direct a nucleotide exchange reaction at the site of the mutated base, while serving as a template for the incorporation of the new base. The mechanism of action is being elucidated and many hurdles remain before the technique reaches a level of maturity that will enable clinical applications. We have participated in the development of this field and now report on its status. More directly, we discuss what appears to be feasible and what appears to be limited. In this line of investigation, we have used several mammalian cell types and various delivery systems to explore how positional effects, context dependencies and the copy numbers of the targeted Genes impact the frequency of Gene Repair. And, we have examined the influence of cell cycle and the activation of DNA damage response pathways on the Gene Repair process. Robust data can be obtained from actively replicating mammalian cells as cells passing through S phase being more amenable to Gene Repair. The frequency of Gene Repair can be reproducibly enhanced by activation of the homologous recombination pathway in response to DNA damage. The position of the target Gene with regard to its replication status and/or its transcriptional status can also determine the level of Gene Repair enabled at that site. These data have been attained using FACS analyses of well-controlled reporter Genes which do not produce a functional protein unless the specific mutation is corrected. Then, cells judged as being phenotypically Repaired, are isolated by sorting and the target Gene sequenced to confirm the precision of the reaction. These factors have now been assessed in systems which model inherited diseases such as Fabry[rsquo]s Disease and Sickle Cell Anemia. Our results will be discussed and we will also provide the details of the cell systems, which provide a more scientifically sound basis for developing this potentially useful technique.
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The development and regulation of Gene Repair.
Nature reviews. Genetics, 2003Co-Authors: Li Liu, Hetal Parekh-olmedo, Eric B. KmiecAbstract:A technique that can direct the Repair of a Genetic mutation in a human chromosome using the DNA Repair machinery of the cell is under development. Although this approach is not as mature as other forms of Gene therapy and fundamental problems continue to arise, it promises to be the ultimate therapy for many inherited disorders. There is a continuing effort to understand the potential and the limitations of this controversial approach.
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Rad51p and Rad54p, but not Rad52p, elevate Gene Repair in Saccharomyces cerevisiae directed by modified single-stranded oligonucleotide vectors
Nucleic acids research, 2002Co-Authors: Li Liu, Shuqiu Cheng, Anja J. Van Brabant, Eric B. KmiecAbstract:Synthetic single-stranded DNA vectors have been used to correct point and frameshift mutations in episomal or chromosomal targets in the yeast Saccharomyces cerevisiae. Certain parameters, such as the length of the vector and the Genetic background of the organism, have a significant impact on the process of targeted Gene Repair, and point mutations are corrected at a higher frequency than frameshift mutations. Genetic analyses reveal that expression levels of the recombination/Repair Genes RAD51, RAD52 and RAD54 can affect the frequency of Gene Repair. Overexpression of RAD51 enhances the frequency 4-fold for correction of an episomal target and 5-fold for correction of a chromosomal target; overexpression of RAD54 is also effective in stimulating Gene Repair, to the same extent as RAD51 in the chromosomal target. In sharp contrast, RAD52 Gene expression serves to reduce Gene Repair activity in rescue experiments and in experiments where RAD52 is overexpressed in a wild-type strain. This may suggest an antagonist role for Rad52p. Consistent with this notion, the highest level of targeted Repair occurs when the RAD51 Gene is overexpressed in a strain of yeast deficient in RAD52 Gene function.
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Strand bias in targeted Gene Repair is influenced by transcriptional activity.
Molecular and cellular biology, 2002Co-Authors: Li Liu, Michael C. Rice, Miya Drury, Shuqiu Cheng, Howard B. Gamper, Eric B. KmiecAbstract:Modified single-stranded DNA oligonucleotides can direct nucleotide exchange in Saccharomyces cerevisiae. Point and frameshift mutations are corrected in a reaction catalyzed by cellular enzymes involved in various DNA Repair processes. The present model centers on the annealing of the vector to one strand of the helix, followed by the correction of the designated base. The choice of which strand to target is a reaction parameter that can be controlled, so here we investigate the properties of strand bias in targeted Gene Repair. An in vivo system has been established in which a plasmid containing an actively transcribed, but mutated, hygromycin-enhanced green fluorescent protein fusion Gene is targeted for Repair and upon conversion will confer hygromycin resistance on the cell. Overall transcriptional activity has a positive influence on the reaction, elevating the frequency. If the targeting vector is synthesized so that it directs nucleotide Repair on the nontranscribed strand, the level of Gene Repair is higher than if the template strand is targeted. We provide data showing that the targeting vector can be displaced from the template strand by an active T7 phage RNA polymerase. The strand bias is not influenced by which strand serves as the leading or lagging strand during DNA synthesis. These results may provide an explanation for the enhancement of Gene Repair observed when the nontemplate strand is targeted.
Luciana Ferrara - One of the best experts on this subject based on the ideXlab platform.
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Recovery of cell cycle delay following targeted Gene Repair by oligonucleotides.
DNA repair, 2007Co-Authors: Luciana Ferrara, Timothy Schwartz, Hetal Parekh-olmedo, Julia U. Engstrom, Eric B. KmiecAbstract:We have previously shown that activation of the homologous recombinational Repair pathway leads to a block of cell division in corrected cells, possibly through the activity of checkpoint proteins Chk1 and Chk2. In this study, we examine the long-term impact of this stalling on the growth of cells that have enabled Gene Repair events. Using a mutated eGFP Gene as an episomal reporter, we show that corrected (eGFP-positive) cells contain only a few active replication templates 2 weeks after electroporation, yet do not display an apoptotic or senescent phenotype. By 6 weeks after electroporation, cells resume active replication with a cell cycle profile that is comparable to that of the non-corrected (eGFP-negative) population. These results indicate that the initial stalling is transient and eGFP-positive cells eventually resume a normal phenotypic growth pattern, allowing for passaging and expansion in vitro.
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Targeted Gene Repair activates Chk1 and Chk2 and stalls replication in corrected cells.
DNA repair, 2006Co-Authors: Luciana Ferrara, Eric B. KmiecAbstract:Oligonucleotides (ODNs) can direct the exchange of single nucleotides at specific sites in the mammalian genome. It is Generally believed that the ODN aligns in homologous register with its complementary site in the target Gene and provides a template for the endogenous Repair machinery to alter the sequence of the Gene. We have been studying the initial phase of the reaction with particular emphasis on the cellular events that occur when the oligonucleotide enters the cell. Our results show that, following introduction of the oligonucleotide, the DNA-damage response pathway is activated, evidenced by the presence of phosphorylated p53, Chk1 and Chk2, respectively. As a result, progression of some of these cells through the cell cycle is slowed and those bearing corrected Genes all contain phosphorylated Chk1 and Chk2. In contrast, uncorrected cells contain much lower levels of these proteins in the activated state and pass through the cell cycle in a normal fashion. We suggest that Gene Repair directed by oligonucleotides activates a pathway that prevents corrected cells from proliferating in cell culture through the activation of Chk1 and Chk2. Our results impact the future use of Gene Repair for ex vivo Gene therapy applications.
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1026. Manipulation of S Phase Progression Can Counteract Cell Cycle Arrest of Corrected Cells during the Oligonucleotide-Directed Gene Repair Reaction
Molecular Therapy, 2006Co-Authors: Luciana Ferrara, Julia Engstrom, Eric B. KmiecAbstract:Top of pageAbstract We have previously shown that activation of the homologous recombinational Repair pathway leads to a block of cell division in corrected cells, possibly through the activity of checkpoint proteins Chk1 and Chk2. Our results show that corrected cells contain only a few active replication templates two weeks after electroporation, yet do not display an apoptotic or senescent phenotype. This observation is unique for the corrected cells, as non-corrected cells, which still contain substantial levels of transfected oligonucleotides, do not show a significant induction of Chk1/Chk2 and are able to divide at normal rates, resulting the apparent decrease of Gene Repair frequency as a function of time. We show that the addition of thymidine, which impedes cellular replication through the inhibition of ribonucleotide reductase, is able to maintain high levels of Repair over several days and counteracts the reduction of Gene Repair levels. In addition, we reveal that by six weeks after electroporation, corrected cells resume active replication with a cell cycle profile that is comparable to that of the non-corrected population. These results indicate that the initial stalling of corrected cells is transient and cells eventually resume a normal phenotypic growth pattern, allowing for passaging and expansion in vitro.
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1056. Suppression and Enhancement of Gene Repair by the Modulation of ATM Function in Mammalian Cells
Molecular Therapy, 2005Co-Authors: Julia Engstrom, Hetal Parekh-olmedo, Luciana Ferrara, Eric B. KmiecAbstract:Targeted Gene Repair utilizes oligonucleotides to direct single base alterations in the genomes of mammalian cells. These vectors are designed to hybridize to the site of interest and to create a single mismatch with the mutant base. Although the mechanism of the nucleotide exchange phase of the reaction remains to be fully elucidated, evidence suggests that the induction of homologous recombination (HR) in response to DNA damage elevates DNA pairing activity and results in increased levels of Gene Repair. The HR pathway is controlled by the activation of the cell cycle checkpoint regulator, Ataxia-Telangiectasia mutated (ATM), which is induced by either DNA damage or by the presence of single stranded DNA. The radio-sensitizer, caffeine, has previously been shown to inhibit ATM kinase activity, by interfering with the phosphorylation of downstream substrates. When caffeine was added to a Gene Repair reaction prior to the introduction of the oligonucleotide, the frequency of correction was reduced, reinforcing the importance of HR in Gene Repair. If, however, caffeine was added after the oligonucleotide, the correction frequency was enhanced suggesting a dual functionality for caffeine in the Gene Repair reaction. Thus, we utilize caffeine as a reagent and a tool to understand the regulation of this reaction in greater detail. ATM activation in cells exposed to 4mM caffeine was examined following oligonucleotide delivery. At 18 and 24 hours, the level of ATM activation was increased significantly and even at 48 hours post-delivery, when ATM activation form the introduction of the ssDNA vector disappears, activated ATM was still quite discernable in caffeine treated cells. This sustained ATM activation could be explained by a concurrent effect of caffeine on the Gene Repair reaction; the oligonucleotide is retained by the cell for a longer period than in untreated cells. At 16 hours post electroporation, both cell populations exhibited about a 70% uptake of the oligonucleotide (as measured by the fluorescence of a FAM conjugated oligonucleotide). However, between 16 and 48 hours, the non-treated cells exhibit a steep drop in oligonucleotide levels, as compared to the caffeine treated cells. This suggests that the oligonucleotide activates ATM in all cells but in cells treated with caffeine, it reactivates ATM through an extended cellular half-life. The use of caffeine as a tool for studying the regulatory pathways of Gene Repair lead us to conclude that the activation of ATM is a critical step in promoting successful Gene correction. On one hand ATM is required to induce HR which catalyses the first phase of Gene Repair (DNA pairing), but on the other hand, activated ATM can block the replication of corrected cells affecting the overall frequency of the reaction. We are now modulating the levels of ATM so that the balance can be tipped in favor of more robust levels of Gene Repair.
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384. Double-Stranded DNA Breaks Stimulate the Frequency of Targeted Gene Repair in Mammalian Cells
Molecular Therapy, 2005Co-Authors: Timothy Schwartz, Hetal Parekh-olmedo, Luciana Ferrara, Eric B. KmiecAbstract:We have reported that double-stranded breaks in DNA stimulate the Gene Repair activity of the homologous recombination pathway. This induction also leads to a stalling of DNA replication thereby slowing the cell cycle phase in which the Gene Repair reaction occurs most often. DNA cleavage was induced initially by using anticancer drugs like etoposide (VP16), camptothecin (CPT) or chemicals like hydroxyurea (HU) and methymethanesulfonate (MMS). The last of these agents, MMS, is an alkyaltion agent capable of inducing DNA lesions and subsequent single and double strand breaks (DSB). This damage activates the non-homologous end joining (NHEJ) and homologous recombination (HR) DNA Repair pathways, a response that has been shown to increase the frequency of oligonucleotide-directed Gene Repair. We show in DLD-1 cells that the conversion frequency of a mutant base pair to wild-type in an integrated enhanced green fluorescent protein Gene is increased by pretreatment with MMS. This stimulation is dose-dependent and correlates to the level of DSB induced by MMS treatment. These DSBs result in Rad51p nuclear-relocalization and foci formation, a specific marker for HR activation. Additionally the cells exhibit cell cycle delay in S phase due to the stalling of replication forks. Our data suggest that MMS-induced DNA damage elicits a cellular response that stimulates Gene Repair in mammalian cells.
Clifford J. Steer - One of the best experts on this subject based on the ideXlab platform.
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Reversing Age-Related DNA Damage Through Engineered DNA Repair
The Future of Aging, 2010Co-Authors: Clifford J. Steer, Betsy T. KrenAbstract:The last two decades have shown significant advances in our understanding of age- and disease-related alterations in the regulation of Gene expression and the underlying endogenous Gene Repair pathways. As a result, there have been some important strides in the development of engineered technologies for Repairing mutated or damaged DNA via the development, delivery, and integration of specific and selective modified DNA into precise locations. This chapter describes available strategies for engineered Gene Repair including homologous recombination, the use of ribozymes, antisense nucleotides and DNA ribonucleases, single strand replacement/ chimeraplasty, and triplex DNA. The evolution of these approaches together with RNA interference is discussed, and relevant mechanisms and pathways are described. Present demonstrations of the utility of each of these Gene modification approaches for therapeutic use imply that these methods can be employed to correct inherited and aging-related mutations and their consequences in future applications.
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Gene Repair and transposon-mediated Gene therapy.
Stem cells (Dayton Ohio), 2002Co-Authors: Paul D. Richardson, Betsy T. Kren, Lance B. Augustin, Clifford J. SteerAbstract:The main strategy of Gene therapy has traditionally been focused on Gene augmentation. This approach typically involves the introduction of an expression system designed to express a specific protein in the transfected cell. Both the basic and clinical sciences have Generated enough information to suggest that Gene therapy would eventually alter the fundamental practice of modern medicine. However, despite progress in the field, widespread clinical applications and success have not been achieved. The myriad deficiencies associated with Gene augmentation have resulted in the development of alternative approaches to treat inherited and acquired Genetic disorders. One, derived primarily from the pioneering work of homologous recombination, is Gene Repair. Simply stated, the process involves targeting the mutation in situ for Gene correction and a return to normal Gene function. Site-specific Genetic Repair has many advantages over augmentation although it too is associated with significant limitations. This review outlines the advantages and disadvantages of Gene correction. In particular, we discuss technologies based on chimeric RNA/DNA oligonucleotides, single-stranded and triplex-forming oligonucleotides, and small fragment homologous replacement. While each of these approaches is different, they all share a number of common characteristics, including the need for efficient delivery of nucleic acids to the nucleus. In addition, we review the potential application of a novel and exciting nonviral Gene augmentation strategy--the Sleeping Beauty transposon system.
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Gene therapy as an alternative to liver transplantation.
Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation S, 2002Co-Authors: Betsy T. Kren, Namita Roy Chowdhury, Jayanta Roy Chowdhury, Clifford J. SteerAbstract:Liver transplantation has become a well-recognized therapy for hepatic failure resulting from acute or chronic liver disease. It also plays a role in the treatment of certain inborn errors of metabolism that do not directly injure the liver. In fact, the liver maintains a central role in many inherited and acquired Genetic disorders. There has been a considerable effort to develop new and more effective Gene therapy approaches, in part, to overcome the need for transplantation as well as the shortage of donor livers. Traditional Gene therapy involves the delivery of a piece of DNA to replace the faulty Gene. More recently, there has been a growing interest in the use of Gene Repair to correct certain Genetic defects. In fact, targeted Gene Repair has many advantages over conventional replacement strategies. In this review, we will describe a variety of viral and nonviral strategies that are now available to the liver. The ever-growing list includes viral vectors, antisense and ribozyme technology, and the Sleeping Beauty transposon system. In addition, targeted Gene Repair with RNA/DNA oligonucleotides, small-fragment homologous replacement, and triplex-forming and single-stranded oligonucleotides is a long-awaited and potentially exciting approach. Although each method uses different mechanisms for Gene Repair and therapy, they all share a basic requirement for the efficient delivery of DNA.
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The application of dna Repair vectors to Gene therapy
Current opinion in biotechnology, 2002Co-Authors: Betsy T. Kren, Clifford J. SteerAbstract:The nature of DNA, the sequence of the human genome and our increased understanding of the Genetic basis of many inherited and acquired disorders have made the possibility of curing diseases a reality. The modulation of a host's genome is now the ultimate goal in the treatment of Genetic diseases. Historically, Gene therapy recognized two very different approaches: Gene replacement or augmentation and Gene Repair. Gene Repair precisely targets and corrects the chromosomal mutation responsible for a Genetic and/or acquired disorder. Many recent advances have been made in this area of research.
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Strategies for hepatic Gene correction.
Journal of drug targeting, 2002Co-Authors: Paul D. Richardson, Betsy T. Kren, Christian Thoma, Clifford J. SteerAbstract:Gene augmentation has been the paradigm in the majority of Gene therapy protocols but in recent years the potential of Repairing the mutated Gene in situ by targeted Gene correction has become a reality. In fact, targeted Gene Repair has many advantages over conventional replacement strategies, notably the possibility to treat dominant as well as recessive disorders, and the small molecular size of the pharmacologically active agent. Chimeric RNA/DNA oligonucleotides, small fragment homologous replacement, as well as triplex-forming and single-stranded oligonucleotides are all examples of the growing armamentarium for Gene Repair, and are the subject of this review. In addition, we have also included a discussion of the reawakened Sleeping Beauty (SB) transposon system as a novel non-viral Gene replacement strategy.