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At least 55 records · Page 3Linked to original sources
PubMed2004

Unmodified oligodeoxynucleotides require single-strandedness to induce targeted repair of a chromosomal EGFP gene.

BACKGROUND: A number of genetic defects in humans are due to point mutations in a single, often tightly regulated gene. Genetic treatment of such defects is preferably done by correcting only the altered base pair at the endogenous locus rather than by a gene replacement strategy involving viral vectors. Promisingly high repair rates have been achieved in some systems with the non-viral approach of transfecting chimeric RNA/DNA oligonucleotides (chimeraplasts). However, since this technique does not yet perform robustly, several parameters thought to be important in oligonucleotide-mediated gene repair were examined. METHODS: A series of transgenic HEK-293 cell clones has been established harboring high or low copy numbers of a point-mutated 'enhanced green fluorescent protein' (EGFP) gene as the target. At the level of single living cells, repair efficiencies were measured by fluorescence-activated cell sorting (FACS) regarding topology (single-stranded, double-stranded), exonuclease protection (four phosphorothioate linkages at both ends), polarity (sense, antisense), and length (13mer, 19mer, 35mer, 69mer) of the oligonucleotide. RESULTS: When targeting chromosomal loci, up to 0.2% corrected cells were obtained with single-stranded unmodified oligodeoxynucleotides, whereas a chimeraplast, its DNA analogue, and double-stranded DNA fragments were practically non-functional. Correction efficiencies correlated with target gene copy numbers. Modifying exonuclease resistance, polarity or length of single-stranded oligodeoxynucleotides did not enhance repair efficacy above the sub-percentage range. CONCLUSIONS: Successful chromosomal reporter gene repair in HEK-293 cells required an oligodeoxynucleotide to be single-stranded. In concert with the gene copy number correlation, functional interaction between the repair molecule and the target site seems to be one bottleneck in targeted gene repair.

Cell Line↗
PubMed2003

Failure to generate atheroprotective apolipoprotein AI phenotypes using synthetic RNA/DNA oligonucleotides (chimeraplasts).

BACKGROUND: Elevated plasma high-density lipoprotein (HDL), and its major constituent apolipoprotein AI (apoAI), are cardioprotective. Paradoxically, two natural variants of apoAI, termed apoAI(Milano) and apoAI(Paris), are associated with low HDL, but nevertheless provide remarkable protection against heart disease for heterozygous carriers and may even lead to longevity. Both variants arise from point mutations and have Arg(173) and Arg(151) to Cys substitutions, respectively, which allow disulphide-linked dimers to form. Potentially, synthetic RNA/DNA oligonucleotides (chimeraplasts) can permanently correct single point mutations in genomic DNA. Here, we use a variation of such targeted gene repair technology, 'gain-of-function chimeraplasty', and attempt to enhance the biological activity of apoAI by altering a single genomic base to generate the atheroprotective phenotypes, apoAI(Milano) and apoAI(Paris). METHODS: We targeted two cultured cell lines that secrete human apoAI, hepatoblastoma HepG2 cells and recombinant CHO-AI cells, using standard 68-mer chimeraplasts with polyethyleneimine (PEI) as carrier and then systematically varied several experimental conditions. As a positive control we targeted the dysfunctional APOE2 gene, which we have previously converted to wild-type APOE3. RESULTS: Conversion of wild-type apoAI to apoAI(Milano) proved refractory, with limited correction in CHO-AI cells only. However, a successful conversion to apoAI(Paris) was achieved, as demonstrated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and direct genomic sequencing. Unexpectedly, attempts with a new batch of 68-mer chimeraplast to enhance conversion, by using different delivery vehicles, including chemically modified PEI, failed to show a base change; nor could conversion be detected with an 80-mer or a 52-76-mer series. In contrast, when a co-culture of CHO-E2 and CHO-AI cells was co-targeted, a clear conversion of apoE2 to apoE3 was seen, whereas no apoAI(Paris) could be detected. When the individual chimeraplasts were analysed by denaturing electrophoresis only the active apoE2-to-E3 chimeraplast gave a sharp band. CONCLUSIONS: Our findings suggest that different batches of chimeraplasts have variable characteristics and that their quality may be a key factor for efficient targeting and/or base conversion. We conclude that, although an evolving technology with enormous potential, chimeraplast-directed gene repair remains problematical.

Animals↗
PubMed2002

High-frequency intermolecular homologous recombination during herpes simplex virus-mediated plasmid DNA replication.

Homologous recombination is a prominent feature of herpes simplex virus (HSV) type 1 DNA replication. This has been demonstrated and traditionally studied in experimental settings where repeated sequences are present or are being introduced into a single molecule for subsequent genome isomerization. In the present study, we have designed a pair of unique HSV amplicon plasmids to examine in detail intermolecular homologous recombination (IM-HR) between these amplicon plasmids during HSV-mediated DNA replication. Our data show that IM-HR occurred at a very high frequency: up to 60% of the amplicon concatemers retrieved from virion particles underwent intermolecular homologous recombination. Such a high frequency of IM-HR required that both plasmids be replicated by HSV-mediated replication, as IM-HR events were not detected when either one or both plasmids were replicated by simian virus 40-mediated DNA replication, even with the presence of HSV infection. In addition, the majority of the homologous recombination events resulted in sequence replacement or targeted gene repair, while the minority resulted in sequence insertion. These findings imply that frequent intermolecular homologous recombination may contribute directly to HSV genome isomerization. In addition, HSV-mediated amplicon replication may be an attractive model for studying intermolecular homologous recombination mechanisms in general in a mammalian system. In this regard, the knowledge obtained from such a study may facilitate the development of better strategies for targeted gene correction for gene therapy purposes.

Animals↗
PubMed2005

Gene repair in mammalian cells is stimulated by the elongation of S phase and transient stalling of replication forks.

The repair of point mutations directed by modified single-stranded DNA oligonucleotides is dependent on the activity of proteins involved in homologous recombination (HR). As a consequence, factors that stimulate homologous recombination, such as double strand breaks, can impact the frequency with which repair occurs. Here, we report that the stalling of replication forks can also activate the gene repair pathway and lead to an enhanced level of nucleotide exchange. The mammalian cell line, DLD-1, containing an integrated mutant eGFP gene, was used as an assay system to explore how replication fork activity affects the overall repair reaction. The addition of 2',3'-dideoxycytidine (ddC), a nucleoside analog that retards the rate of elongation and effectively stalls the replication fork, results in a lengthened S phase and an increased number of gene repair events. This stimulation was reversed when caffeine was added to the reaction at concentrations that block the homologous recombination pathway. In contrast, the nucleoside analog, 1-beta-D-arabinofuranosylcytosine which stops replication in these cells, failed to stimulate the gene repair reaction to any appreciable degree until the block is released and active replication resumes. Furthermore, overexpression of wild-type p53 which is known to bind transiently to stalled replication forks blocked the stimulatory effect of ddC. Overexpression of mutant p53 genes, deficient in the capacity to bind DNA, however, did not inhibit the reaction. Our results indicate that an expansion of S phase and a transient stalling of replication forks can increase the frequency of targeted gene repair.

Amino Acid Sequence↗
PubMed1999

Potential therapy paradigms for Marfan syndrome.

Marfan syndrome is the most common genetic disorder of the connective tissue with an estimated prevalence of 1:10,000. The disease is characterised by manifestations in the cardiovascular, skeletal and ocular systems. The most severe manifestations are those of the cardiovascular system: mitral valve prolapse and dilation of the aortic root, which may progress to aortic dissection, a common cause of mortality in patients. Marfan syndrome is a dominant genetic disorder caused by mutations in the gene coding for fibrillin-1, the FBN1 gene. Fibrillin, a 347 kDa glycoprotein, is found in most connective tissues and is a major component of the extracellular microfibrils. More than 100 different FBN1 mutations have been identified in individuals with Marfan syndrome, the majority of which are unique missense point mutations. Evidence suggests a dominant-negative mechanism of pathogenesis for the disorder, that is, the presence of the mutant fibrillin molecule interferes with the function of the normal protein. Therapies for dominant disorders such as Marfan syndrome (MFS) are likely to require both suppression of the disease allele expression and maintenance of expression of its wild-type counterpart. Thus, dominant genetic disorders present a unique therapeutic challenge. One approach to developing a therapy would be to use catalytic nucleic acid molecules. Antisense catalytic RNAs, or ribozymes, have been widely used to down-regulate or repair targeted gene expression respectively through the cleavage or trans-splicing of messenger RNA. Similarly, antisense DNA molecules or DNAzymes have been shown to be capable of cleaving target RNA molecules in a highly specific manner. This review will discuss the potential of catalytic nucleic acid molecules as therapeutic agents for MFS.

Journal Article↗
PubMed2001

In vivo and in vitro correction of the mdx dystrophin gene nonsense mutation by short-fragment homologous replacement.

Targeted genetic correction of mutations in cells is a potential strategy for treating human conditions that involve nonsense, missense, and transcriptional splice junction mutations. One method of targeted gene repair, single-stranded short-fragment homologous replacement (ssSFHR), has been successful in repairing the common deltaF508 3-bp microdeletion at the cystic fibrosis transmembrane conductance regulator (CFTR) locus in 1% of airway epithelial cells in culture. This study investigates in vitro and in vivo application of a double-stranded method variant of SFHR gene repair to the mdx mouse model of Duchenne muscular dystrophy (DMD). A 603-bp wild-type PCR product was used to repair the exon 23 C-to-T mdx nonsense transition at the Xp21.1 dys locus in cultured myoblasts and in tibialis anterior (TA) from male mdx mice. Multiple transfection and variation of lipofection reagent both improved in vitro SFHR efficiency, with successful conversion of mdx to wild-type nucleotide at the dys locus achieved in 15 to 20% of cultured loci and in 0.0005 to 0.1% of TA. The genetic correction of mdx myoblasts was shown to persist for up to 28 days in culture and for at least 3 weeks in TA. While a high frequency of in vitro gene repair was observed, the lipofection used here appeared to have adverse effects on subsequent cell viability and corrected cells did not express dystrophin transcript. With further improvements to in vitro and in vivo gene repair efficiencies, SFHR may find some application in DMD and other genetic neuromuscular disorders in humans.

Animals↗
PubMed2005

The distal sequence element of the selenocysteine tRNA gene is a tissue-dependent enhancer essential for mouse embryogenesis.

Appropriate expression of the selenocysteine tRNA (tRNA(Sec)) gene is necessary for the production of an entire family of selenoprotein enzymes. This study investigates the consequence of disrupting an upstream enhancer region of the mouse tRNA(Sec) gene (Trsp) known as the distal sequence element (DSE) by use of a conditional repair gene targeting strategy, in which a 3.2-kb insertion was introduced into the promoter of the gene. In the absence of DSE activity, homozygous mice failed to develop in utero beyond embryonic day 7.5 and had severely decreased levels of selenoprotein transcript. Cre-mediated removal of the selection cassette recovered DSE regulation of Trsp, restoring wild-type levels of tRNA(Sec) expression and allowing the generation of viable rescued mice. Further analysis of targeted heterozygous adult mice revealed that the enhancer activity of the DSE is tissue dependent since, in contrast to liver, heart does not require the DSE for normal expression of Trsp. Similarly, in mouse cell lines we showed that the DSE functions as a cell-line-specific inducible element of tRNA(Sec). Together, our data demonstrate that the DSE is a tissue-dependent regulatory element of tRNA(Sec) expression and that its activity is vital for sufficient tRNA(Sec) production during mouse embryogenesis.

Animals↗
PubMed2001

Nonviral gene therapy.

The last 10 years have seen substantial progress in the development and application of nonviral vectors in gene therapy. Several novel nonviral methods have been developed that approach viruses with respect to transfection efficiency. A variety of nonviral delivery systems that can be used for gene therapy in different clinical settings are also available. In this review article, we will detail all of the major nonviral vectors that are currently used in gene therapy while highlighting some recent developments, particularly the progress towards the understanding of the cellular and in vivo barriers in gene transfer. Recent advancement in achieving sustained and regulated gene expression will also be addressed. Finally, this review will briefly cover targeted gene repair using nonviral delivery systems. Their impact in gene therapy will also be discussed.

Adjuvants, Immunologic↗
PubMed2000

RecA stimulates sister chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in plants transformed by Agrobacterium.

Expression of the bacterial RecA protein in plants stimulates homologous recombination in tobacco. Here we show that RecA plays a direct role in DNA strand exchange in vivo. The number of sister chromatid exchanges (SCEs) was increased 2.4-fold over wild type in transgenic tobacco plants expressing a nuclear-targeted RecA (nt-RecA) protein and could not be increased further by DNA damage, which caused a doubling of the baseline SCE frequency in wild-type plants. Although gene targeting requires homologous recombination, the number of targeted gene replacements was not increased markedly by the presence of nt-RecA by using Agrobacterium-mediated transformation. However, the number of double-strand breaks that were repaired at both sides by homologous recombination was increased 3.3-fold. Stimulation of SCE and fidelity of double-strand break repair by nt-RecA, but not by gene targeting, suggests that the stimulatory activity of RecA is linked to active DNA synthesis. Therefore, nascent replication-associated single strands may be a prerequisite for RecA action in plant cells.

Base Sequence↗
PubMed2006

Constructing the eukaryotic expression vector to study preliminarily the functions of hammerhead ribozyme targeting base excision repair gene HOGG1.

OBJECTIVE: Adriamycin is widely used as an effective anti-tumor drug clinically treating a number of human cancers, but the effect of adriamycin is limited by drug resistance. The various kinds of investigations indicated that the anti-tumor activity of adriamycin resulted from drug-induced free radical formation. The free radicals could lead to oxidative DNA damage, and the lesion would be repaired by base excision repair (BER) pathway. Human 8-oxoguanine DNA glycosylase 1 (HOGG1) is a key enzyme on BER pathway. To study the influence and biological mechanism of the HOGG1 to adriamycin drug-sensitivity, the eukaryotic expression vector with gene of hammerhead ribozyme targeting HOGG1 mRNA would be constructed and identified, and then the change of drug-sensitivity in lung cancer A549 cells would be investigated. METHODS: According to computer design, two specific restriction site BamH I and EcoR I were added to both ends of the ribozyme gene, then the modified ribozyme gene was synthesized and cloned into the eukaryotic expression vector pcDNA3.1 (+). The positive recombinants were screened by ampicillin resistance, and plasmids were extracted from the positive recombinants and digested by BamH I and EcoR I , and then were analyzed by agarose gel electrophoresis and DNA sequencing. The recombinants were transiently transfected into A549 cells. The positive recombinants were identified by reverse transcription-polymerase chain reaction (RT-PCR) targeting to NEO gene, which was a neomycin resistance gene for selection of stable cell lines and only existed in vectors. The changes of HOGG1 mRNA in A549 cells were detected by RT-PCR. Then the cellular sensitivity to adriamycin was tested by comparison between untransfected cells and transfected cells by MTT assay. The adriamycin-induced DNA damage was investigated by comet assay or single cell gel electrophoresis (SCGE) between untransfected and transfected cells. RESULTS: The recombinants containing the ribozyme gene were successfully selected by restriction endonuclease digestion and agarose gel electrophoresis, and were further proved by DNA automatic sequencing. A549 cells containing the recombinants were identified by RT-PCR, because NEO genes were amplified only in cells transfected successfully. The expression of HOGG1 mRNA in A549 transfected with ribozyme gene was 36% significantly less than in control cells (P < 0.05). MTT assay showed that the sensitivity of transfected cells to adriamycin were significantly increased in comparison with untransfected cells (P < 0.05). The comet assay showed that the extent of DNA damage induced by adriamycin was worse in transfected cells than unrtransfected cells (P < 0.05), but there was no time-dependent reaction correlation observed in cells. CONCLUSION: The eukaryotic expression vector with gene of hammerhead ribozyme targeting HOGG1 mRNA was constructed successfully, and effectively inhibited the expression of HOGG1 gene in lung cancer A549 cells, and increased the cellular sensitivity to adriamycin, and will help to study deeply the functions of base excision repair genes-HOGG1.

Base Sequence↗
PubMed2002

Mechanisms underlying targeted gene correction using chimeric RNA/DNA and single-stranded DNA oligonucleotides.

Chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides have been developed for site-specific correction of episomal and chromosomal target genes. The gene repair approach relies on specific hybridization of the oligonucleotides to the target gene generating a mismatch with the targeted point mutation. Restored gene function is anticipated to occur through activation of endogenous repair systems that recognize the created mismatch. We present an overview of the gene correction results obtained in several target genes by employing various oligonucleotide designs and a discussion of the possible mechanisms underlying the gene correction techniques. Experimental data suggest that modified single-stranded oligonucleotides form intermediate three-stranded heteroduplexes involving the human RecA homologue, hRad51, whereas chimeric RNA/DNA oligonucleotides may participate in three or four-stranded intermediate structures. Protein factors such as hRad52, hRad54, hRPA, and p53 may modulate the heteroduplex formation and participate in the activation of the endogenous mismatch repair and/or nucleotide excision repair pathway(s). The efficiency of the gene correction process may furthermore be influenced by the differential recognition of mismatches by repair enzymes and possible sequence context effects.

Adenosine Triphosphatases↗
PubMed2004

Sequence-specific modification of mouse genomic DNA mediated by gene targeting techniques.

The major impact of the human genome sequence is the understanding of disease etiology with deduced therapy. The completion of this project has shifted the interest from the sequencing and identification of genes to the exploration of gene function, signalling the beginning of the post-genomic era. Contrasting with the spectacular progress in the identification of many morbid genes, today therapeutic progress is still lagging behind. The goal of all gene therapy protocols is to repair the precise genetic defect without additional modification of the genome. The main strategy has traditionally been focused on the introduction of an expression system designed to express a specific protein, defective in the transfected cell. But the numerous deficiencies associated with gene augmentation have resulted in the development of alternative approaches to treat inherited and acquired genetic disorders. Among these one is represented by gene repair based on homologous recombination (HR). Simply stated, the process involves targeting the mutation in situ for gene correction and for restoration of a normal gene function. Homologous recombination is an efficient means for genomic manipulation of prokaryotes, yeast and some lower eukaryotes. By contrast, in higher eukaryotes it is less efficient than in the prokaryotic system, with non-homologous recombination being 10-50 fold higher. However, recent advances in gene targeting and novel strategies have led to the suggestion that gene correction based on HR might be used as clinical therapy for genetic disease. This site-specific gene repair approach could represent an alternative gene therapy strategy in respect to those involving the use of retroviral or lentiviral vectors to introduce therapeutic genes and linked regulatory sequences into random sites within the target cell genome. In fact, gene therapy approaches involving addition of a gene by viral or nonviral vectors often give a short duration of gene expression and are difficult to target to specific populations of cells. The purpose of this paper is to review oligonucleotide-based gene targeting technologies and their applications on modifying the mouse genome.

Animals↗
PubMed2003

Pathogenesis of DNA repair-deficient cancers: a statistical meta-analysis of putative Real Common Target genes.

DNA mismatch repair deficiency is observed in about 15% of human colorectal, gastric, and endometrial tumors and in lower frequencies in a minority of other tumors thereby causing insertion/deletion mutations at short repetitive sequences, recognized as microsatellite instability (MSI). Evolution of tumors, including those with MSI, is a continuous process of mutation and selection favoring neoplastic growth. Mutations in microsatellite-bearing genes that promote tumor cell growth in general (Real Common Target genes) are assumed to be the driving force during MSI carcinogenesis. Thus, microsatellite mutations in these genes should occur more frequently than mutations in microsatellite genes without contribution to malignancy (ByStander genes). So far, only a few Real Common Target genes have been identified by functional studies. Thus, comprehensive analysis of microsatellite mutations will provide important clues to the understanding of MSI-driven carcinogenesis. Here, we evaluated published mutation frequencies on 194 repeat tracts in 137 genes in MSI-H colorectal, endometrial, and gastric carcinomas and propose a statistical model that aims to identify Real Common Target genes. According to our model nine genes including BAX and TGFbetaRII were identified as Real Common Targets in colorectal cancer, one gene in gastric cancer, and three genes in endometrial cancer. Microsatellite mutations in five additional genes seem to be counterselected in gastrointestinal tumors. Overall, the general applicability, the capacity to unlimited data analysis, the inclusion of mutation data generated by different groups on different sets of tumors make this model a useful tool for predicting Real Common Target genes with specificity for MSI-H tumors of different organs, guiding subsequent functional studies to the most likely targets among numerous microsatellite harboring genes.

Base Pair Mismatch↗
PubMed2003

Targeted gene modification in mismatch-repair-deficient embryonic stem cells by single-stranded DNA oligonucleotides.

Gene targeting through homologous recombination in murine embryonic stem (ES) cells is already strongly suppressed by DNA mismatch-repair (MMR)-dependent anti-recombination when targeting construct and target locus differ at <1% of the nucleotide positions. We demonstrate that MMR activity also raises a strong impediment to gene modification mediated by small synthetic DNA oligonucleotide sequences. In the absence of the DNA MMR gene MSH2, synthetic single-stranded deoxyribo-oligonucleotides can be used to site-specifically modify the ES cell genome. We show that PCR-based procedures can be used to identify and clone modified cells. By this method we have substituted a single codon in the retinoblastoma gene.

Amino Acid Substitution↗
PubMed1991

Double-strand gap repair in a mammalian gene targeting reaction.

To better understand the mechanism of homologous recombination in mammalian cells that facilitates gene targeting, we have analyzed the recombination reaction that inserts a plasmid into a homologous chromosomal locus in mouse embryonic stem cells. A partially deleted HPRT gene was targeted with various plasmids capable of correcting the mutation at this locus, and HPRT+ recombinants were directly selected in HAT medium. The structures of the recombinant loci were then determined by genomic Southern blot hybridizations. We demonstrate that plasmid gaps of 200, 600, and 2,500 bp are efficiently repaired during the integrative recombination reaction. Targeting plasmids that carry a double-strand break or gap in the region of DNA homologous to the target locus produce 33- to 140-fold more hypoxanthine-aminopterin-thymidine-resistant recombinants than did these same plasmids introduced in their uncut (supercoiled) forms. Our data suggest that double-strand gaps and breaks may be enlarged prior to the repair reaction since sequence heterologies carried by the incoming plasmids located close to them are often lost. These results extend the known similarities between mammalian and yeast recombination mechanisms and suggest several features of the insertional (O-type) gene targeting reaction that should be considered when one is designing mammalian gene targeting experiments.

Animals↗
PubMed1996

Mutations of E2F-4 trinucleotide repeats in colorectal cancer with microsatellite instability.

Genetic instability at microsatellites in some colorectal cancer (CRC) have been linked to the defects of human mismatch repair genes, but the targets of these defective genes have been largely unknown. We screened CRC specimens for alteration of E2F-4 gene by analyzing both cDNA and genomic sequences along with replication error (RER+) phenotype. Two out of 20 sporadic CRC patients showed RER+ phenotype. We found tumor-specific copy number alteration in 13 consecutive trinucleotide (CAG) repeats within the coding exon of E2F-4 exclusively in these 2 specimens. Thus, E2F-4 may be a clue of the target gene of defective repair genes in CRC with genetic instability in addition to the TGF-beta type II receptor gene.

Base Sequence↗