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Results for “Targeted Gene Repair”

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At least 19 recordsLinked to original sources
PubMed2002

The 'biased' evolution of targeted gene repair.

Targeted gene repair is a powerful yet controversial technique invented to direct single-base changes in chromosomal genes. The reaction mechanism has only recently come to light, and parameters that confer a level of control on the frequency of repair are still being defined in model genetic systems. Regulatory factors, such as strand bias, vector length, and the phase of the cell cycle in which targeting events take place, are now appreciated. This knowledge will lead to development of this technique for everyday genomics applications and, hopefully, gene therapeutics.

Animals↗
PubMed2005

A tolerance of DNA heterology in the mammalian targeted gene repair reaction.

Targeted gene repair consists of at least two major steps, the pairing of an oligonucleotide to a site bearing DNA sequence complementarity followed by a nucleotide exchange reaction directed by the oligonucleotide. In this study, oligonucleotides with different structures were designed to target a stably integrated (mutant) enhanced green fluorescent protein (EGFP) gene and used to direct the repair of a single base mutation. We show that the efficiency of correction is influenced by the degree of DNA sequence homology existing between the oligonucleotide and target gene. Correction is reduced when a heterologous stretch of DNA sequence is placed in the center of the oligonucleotide and the mismatched base pair is then formed near the terminus. The negative impact of heterology is dependent on the type of DNA sequence inserted and on the size of the heterologous region. If the heterologous sequence is palindromic and adopts a secondary structure, the negative impact on the correction frequency is removed, and wild-type levels of repair are restored. Although differences in the efficiency of correction are observed in various cell types, the effect of structural changes on gene repair is consistent. These results reveal the existence of a directional-specific repair pathway that relies on the pairing stability of a bilateral complex and emphasize the importance of sequence homology between pairing partners for efficient catalysis of gene repair.

Animals↗
PubMed2006

The involvement of replication in single stranded oligonucleotide-mediated gene repair.

Targeted gene repair mediated by single-stranded oligonucleotides (SSOs) has great potential for use in functional genomic studies and gene therapy. Genetic changes have been created using this approach in a number of prokaryotic and eukaryotic systems, including mouse embryonic stem cells. However, the underlying mechanisms remain to be fully established. In one of the current models, the 'annealing-integration' model, the SSO anneals to its target locus at the replication fork, serving as a primer for subsequent DNA synthesis mediated by the host replication machinery. Using a lambda-Red recombination-based system in the bacterium Escherichia coli, we systematically examined several fundamental premises that form the mechanistic basis of this model. Our results provide direct evidence strongly suggesting that SSO-mediated gene repair is mechanistically linked to the process of DNA replication, and most likely involves a replication intermediate. These findings will help guide future experiments involving SSO-mediated gene repair in mammalian and prokaryotic cells, and suggest several mechanisms by which the efficiencies may be reliably and substantially increased.

DNA Mismatch Repair↗
PubMed2005

Reaction parameters of targeted gene repair in mammalian cells.

Targeted gene repair uses short DNA oligonucleotides to direct a nucleotide exchange reaction at a designated site in a mammalian chromosome. The widespread use of this technique has been hampered by the inability of workers to achieve robust levels of correction. Here, we present a mammalian cell system in which DLD-1 cells bearing integrated copies of a mutant eGFP gene are repaired by modified single-stranded DNA oligonucleotides. We demonstrate that two independent clonal isolates, which are transcribed at different levels, are corrected at different frequencies. We confirm the evidence of a strand bias observed previously in other systems, wherein an oligonucleotide designed to be complementary to the nontranscribed strand of the target directs a higher level of repair than one targeting the transcribed strand. Higher concentrations of cell oligonucleotides in the electroporation mixture lead to higher levels of correction. When the target cell population is synchronized into S phase then released before electroporation, the correction efficiency is increased within the entire population. This model system could be useful for pharmacogenomic applications of targeted gene repair including the creation of cell lines containing single-base alterations.

Animals↗
PubMed2003

Targeted gene repair -- in the arena.

The development of targeted gene repair is under way and, despite some setbacks, shows promise as an alternative form of gene therapy. This approach uses synthetic DNA molecules to activate and direct the cell's inherent DNA repair systems to correct inborn errors. The progress of this technique and its therapeutic potential are discussed in relation to the treatment of genetic diseases.

Animals↗
PubMed2002

Strand bias in targeted gene repair is influenced by transcriptional activity.

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 non-template strand is targeted.

Amino Acid Sequence↗
PubMed2006

Targeted gene repair: the ups and downs of a promising gene therapy approach.

As a novel form of molecular medicine based on direct actions over the genes, targeted gene repair has raised consideration recently above classical gene therapy strategies based on genetic augmentation or complementation. Targeted gene repair relies on the local induction of the cell's endogenous DNA repair mechanisms to attain a therapeutic gene conversion event within the genome of the diseased cell. Successful repair has been achieved both in vitro and in vivo with a variety of corrective molecules ranging from oligonucleotides (chimeraplasts, modified single-stranded oligonucleotides, triplex-forming oligonucleotides), to small DNA fragments (small fragment homologous replacement (SFHR)), and even viral vectors (AAV-based). However, controversy on the consistency and lack of reproducibility of early experiments regarding frequencies and persistence of targeted gene repair, particularly for chimeraplasty, has flecked the field. Nevertheless, several hurdles such as inefficient nuclear uptake of the corrective molecules, and misleading assessment of targeted repair frequencies have been identified and are being addressed. One of the key bottlenecks for exploiting the overall potential of the different targeted gene repair modalities is the lack of a detailed knowledge of their mechanisms of action at the molecular level. Several studies are now focusing on the assessment of the specific repair pathway(s) involved (homologous recombination, mismatch repair, etc.), devising additional strategies to increase their activity (using chemotherapeutic drugs, chimeric nucleases, etc.), and assessing the influence of the cell cycle in the regulation of the repair process. Until therapeutic correction frequencies for single gene disorders are reached both in cellular and animal models, precision and undesired side effects of this promising gene therapy approach will not be thoroughly evaluated.

Animals↗
PubMed2003

Low-dose bleomycin induces targeted gene repair frequency in cultured melan-c cells using chimeric RNA/DNA oligonucleotide transfection.

We attempted to raise the frequency of targeted gene repair attained by RNA/DNA chimeraplasty through activation of endogenous DNA recombination/repair system in cultured melan-c cells. For elevating the repair activity, either gamma-ray irradiation at 2 Gy or bleomycin addition at 5 nM to the culture medium was done. Subsequently, the tyrosinase-targeted chimeric RNA/DNA oligonucleotides were transfected. The frequency of targeted gene repair was assessed by three means, i.e., changes in cell color from transparent to dark black, restriction fragment band patterns, and DNA sequencing analysis. The results indicated that the gamma-ray irradiation did not improve the efficiency at all. In contrast, the low-dose bleomycin addition induced the frequency of targeted base conversion, compared with transfection with chimeric RDOs alone, although the frequency was still insufficient and varied from 0.1 to 10% of the total cultured cells.

Amino Acid Sequence↗
PubMed2005

Bleomycin stimulates targeted gene repair directed by single-stranded oligodeoxynucleotides in BHK-21 cells.

Synthetic single-stranded oligodeoxynucleotides (ssODNs) designed for single nucleotide mismatch can induce targeted gene repair (TGR) in chromosomal DNA. TGR directed by ssODNs is an ideal method for gene manipulation because it precisely exchanges single nucleotides in targeted DNA without additional modification to the genome. However, TGR efficiency is still insufficient for practical use. Here, we report that bleomycin (BLM), which breaks double-stranded DNAs, stimulates TGR directed by ssODNs in BHK-21 cells. To evaluate TGR efficiency, transformants of BHK-21 cells permanently expressing mutant EGFP genes were established. Microscopy and restriction fragment length polymorphism (RFLP) analysis clearly showed that the mutant EGFP gene was restored to a normal EGFP gene with an ssODN comple-mentary to a transcribed strand. TGR efficiency was measured by counting the number of GFP-fluorescent cells with a flow cytometer. TGR efficiency of the ssODN complementary to a transcribed strand was significantly higher than that of ssODN complementary to a non-transcribed strand, indicating that a strand bias existed. Exposure of cells to BLM stimulated TGR directed by ssODN in a dose-dependent manner, and high concentrations of BLM (750 ng/ml) abolished strand bias on TGR efficiency. These results suggest that BLM stimulates TGR directed by ssODNs in BHK-21 cells, probably due to activation of the DNA repair system.

Animals↗
PubMed2001

Targeted gene repair in mammalian cells using chimeric RNA/DNA oligonucleotides and modified single-stranded vectors.

Determining the function of newly discovered genes is at the center of the evolving field of genomics. With the elucidation of the human DNA sequence, the importance of single base changes to gene function has become apparent. In some cases, nucleotide alteration accounts for inherited disorders, but in other cases, subtle, even conservative, base changes can influence the function of a gene and its product. To identify how critical genetic changes alter function, molecular tools such as synthetic vectors have been created to direct nucleotide exchange. Some of these vectors, including chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides, have shown promise in the specific alteration of a single base at an exact position within the gene. Here, we describe the activity of the synthetic vectors in a mammalian cell system. The episomal target contains a mutation in the neomycin resistance gene fused to a reporter ligand-binding domain. Correction of the mutated base enables translation of the normal fusion product. This protein can now bind a ligand, resulting in the expression of the fusion protein visualized by green fluorescence. Hence, the activity of any similar vector can be measured easily (and in real time) using confocal microscopy. The system provides the basis for examining the effectiveness of new targeting molecules for creating or repairing single base alterations. In addition, genes suspected of affecting the frequency of repair can be tested through their expression in cells harboring the mutated target plasmid. Once the frequency of exchange in cells is established, the use of these vectors will become commonplace in a process designed to generate specific single base changes in genes involved in signal transduction. Such changes should help define functional domains within these proteins.

Animals↗
PubMed2006

Microsatellite instability and mismatch repair target gene mutations in cell lines and xenografts of prostate cancer.

BACKGROUND: Mismatch repair (MMR) and microsatellite instability (MSI) occur in prostate cancer, but their role has not been well documented. METHODS: In 6 cell lines and 22 xenografts from prostate cancer, PCR and gel electrophoresis were performed to detect MSI by analyzing microsatellite markers BAT-25 and BAT-26 and to detect frameshift mutations in mononucleotide repeats in BAX, IGF2R, MSH3, MSH6, and TGFBR2. RESULTS: Four of 6 (67%) cell lines and 3 of 22 (14%) xenografts showed MSI. At least 1 frameshift mutation was detected in the same 4 (67%) cell lines and in 5 of 22 (23%) xenografts. While MSH3 was frequently mutated in cell lines, BAX and TGFBR2 showed frequent alterations in both cell lines and xenografts. CONCLUSIONS: MSI related to MMR deficiency is relatively frequent in high-grade tumors, and mutations in MSI target genes involved in cell death and proliferation appeared to be selected for during prostatic carcinogenesis.

Animals↗
PubMed2006

Targeted gene repair activates Chk1 and Chk2 and stalls replication in corrected cells.

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.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2005

Gene therapy progress and prospects: targeted gene repair.

The capacity to correct a mutant gene within the context of the chromosome holds great promise as a therapy for inherited disorders but fulfilling this promise has proven to be challenging. However, steady progress is being made and the development of gene repair as a viable and robust approach is underway. Here, we present some of the recent advances that are helping to shape our thinking about the feasibility and the limitations of this technique. For the most part, these advances center on understanding the regulation of the reaction and validating its application in animal models.

DNA Repair↗
PubMed2002

Targeted gene repair and its application to neurodegenerative disorders.

Synthetic DNA oligonucleotides can direct the exchange of single nucleotides within coding regions of mammalian genes by hybridizing to their complementary sequence in the chromosome and creating a recombination joint structure with a single mismatched base pair. Inherent DNA repair processes recognize the mismatch and resolve it using the DNA sequence of the oligonucleotide vector as the template. This gene surgery approach can be used to repair mutations or to disrupt tri-nucleotide repeats in dysfunctional genes responsible for neurological disorders.

Animals↗
PubMed1999

Targeted gene repair directed by the chimeric RNA/DNA oligonucleotide in a mammalian cell-free extract.

Chimeric oligonucleotides consisting of RNA and DNA residues have been shown to catalyze site-directed genetic alteration in mammalian cells both in vitro and in vivo. Since the frequency of these events appears to be logs higher than the rates of gene targeting, a process involving homologous recombination, we developed a system to study the mechanisms of chimera-directed gene conversion. Using a mammalian cell-free extract and a genetic readout in Escherichia coli, we find that point mutations and single base deletions can be corrected at frequencies of approximately 0.1% and 0.005%, respectively. The reaction depends on an accurately designed chimera and the presence of functional hMSH2 protein. The results of genetic and biochemical studies reported herein suggest that the process of mismatch repair functions in site-directed gene correction.

Animals↗