Targeted Gene Repair Research Articles

375. Targeted Gene Repair Induces the DNA Damage Response Pathways through the Activation of ATM, and the Stalling of Replication Forks Leading to a Blockage of Inherited Correction

Top of pageAbstract We have been investigating the mechanism of targeted gene repair in mammalian cells using an eGFP reporter system. The protocol involves the electroporation of single-stranded oligonucleotides into an adenocarcinoma cell line containing integrated copies of a mutant eGFP gene. Correction of the point mutation is directed by the oligonucleotide in a reaction involving members of the homologous recombination (HR) pathway. The electroporation of single-stranded vector and the presence of free ends of DNA induces HR as the cell perceives that DNA damage has occurred. This response sets in motion a cascade of events including the activation of ATM and the cessation of DNA replication. Surprisingly, we find that cells bearing a corrected allele, verified at the phenotypic and genotypic levels are preferentially stalled in S phase and at the S/G2 border. Uncorrected cells resume active replication and mitosis, which essentially leads to a dilution of corrected cells as a function of time. Thus, perceptions that the gene repair has failed in certain systems can be accounted for by such a dilution phenomenon. These results also help explain the variable levels of correction seen during the early days of gene repair. Most importantly, these observations clearly have a long-term and profound effect on the potential for using synthetic oligonucleotides in ex vivo gene therapy applications.

1056. Suppression and Enhancement of Gene Repair by the Modulation of ATM Function in Mammalian Cells

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.

385. Gene Correction in the Absence of DNA Repair: A Universal Model Involving Homologous Pairing and DNA Replication

Our laboratory has been studying the mechanism and regulation of targeted gene repair in mammalian cells. Using a robust cell line where the genomic correction frequencies average 1%, we have been able to modulate the process of DNA replication and observe its effect on the gene repair reaction. In our hands, gene correction occurs predominantly in S phase cells especially when the movement of the replication fork is delayed. Reminiscent of its role in homeologous recombination, several labs, including ours, have observed that the mismatch repair pathway appears to interfere with gene repair activity. Taking all of these observations into consideration, we now propose a universal model for gene repair in which the oligonucleotide becomes incorporated into the newly synthesized daughter strand at or near a stalled replication fork. Assimilation of the oligonucleotide creates a single mismatched base pair which, in the case of a MMR-deficient cell line, is tolerated through a single round of replication. During a second round, the replication machinery uses the oligonucleotide as the parental template and synthesizes the second strand of a fully converted allele. This model explains a multitude of unrelated observations that surround the variability of this technique. The integrated model of gene repair will be presented with examples from yeast and mammalian cells that strongly support its validity.

384. Double-Stranded DNA Breaks Stimulate the Frequency of Targeted Gene Repair in Mammalian Cells

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.

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.

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-β- 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.

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.

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

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. 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. 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. 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.

DNA damage and homologous recombination regulate the frequency and process of targeted gene repair

DNA damage and homologous recombination regulate the frequency and process of targeted gene repair

Enhanced oligonucleotide-directed gene targeting in mammalian cells following treatment with DNA damaging agents

Targeted gene repair, a form of oligonucleotide-directed mutagenesis, employs end-modified single-stranded DNA oligonucleotides to mediate single-base changes in chromosomal DNA. In this work, we use a specific 72-mer to direct the repair of a mutated eGFP gene stably integrated in the genome of DLD-1 cells. Corrected cells express eGFP that can be identified and quantitated by FACS. The repair of this mutant gene is dependent on the presence of a specifically designed oligonucleotide and the frequency with which the mutation is reversed is affected by the induction of DNA damage. We used hydroxyurea, VP16 (etoposide), and thymidine to modulate the rate of DNA replication through the stalling of the replication forks or the introduction of lesions. Addition of hydroxyurea or VP16 before the electroporation of the oligonucleotide, results in an accumulation of double-strand breaks (DSB) whose repair is facilitated by either nonhomologous end joining (NHEJ) or homologous recombination (HR). The addition of thymidine results in DNA damage within replication forks, damage that is repaired through the process of homologous recombination. Our data suggest that gene repair activity is elevated when DNA damage induces or activates the homologous recombination pathway.

DNA replication and transcription direct a DNA strand bias in the process of targeted gene repair in mammalian cells.

The repair of point mutations can be directed by modified single-stranded DNA oligonucleotides and regulated by cellular activities including homologous recombination, mismatch repair and transcription. Now, we report that DNA replication modulates the gene repair process by influencing the frequency with which either DNA strand is corrected. An SV40-virus-based system was used to investigate the role of DNA synthesis on gene repair in COS-1 cells. We confirm that transcription exerts a strand bias on the gene repair process even when correction takes place on actively replicating templates. We were able to distinguish between the influences of transcription and replication on strand bias by changing the orientation of a gene encoding enhanced green fluorescent protein relative to the origin of replication, and confirmed the previously observed bias towards the untranscribed strand. We report that DNA replication can increase the level of untranscribed strand preference only if that strand also serves as the lagging strand in DNA synthesis. Furthermore, the effect of replication on gene repair frequency and strand bias appears to be independent of certain mismatched base pairs and oligonucleotide length.

310. Feasibility and Limitations of Target Gene Repair in Mammalian Cells

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.

317. DNA Damage and Homologous Recombination Regulate the Frequency and Process of Targeted Gene Repair

The pathway followed by targeted gene repair is not fully elucidated. In its simplest iteration, the process is viewed as being initiated by the introduction of short (25–75 bases) synthetic single-stranded oligonucleotides into a mammalian cell. The oligonucleotide aligns in homologous register with the target gene using the underlying sequence complementarity as a guide. Once positioned properly, a single mismatched base pair is created between the oligonucleotide and the target site. The structural changes and altered DNA conformation that clearly take place during the pairing phase attract proteins involved in DNA repair pathway. Here we demonstrate the involvement of the homologous recombination (HR) process in gene repair. By using a phenotypic readout assay system, where an eGFP gene containing a single point mutation is integrated into DLD-1 cells, we observe between 0.7 and 1.5% correction of the mutation. Gene repair is assessed by FACS analyses, cell sorting and confirmatory DNA sequencing of the target gene. When the cells are arrested in S phase as a result of DNA damage incurred by the presence of hydroxyurea or thymidine, the level of gene repair increases 3 to 4 fold. Addition of the anticancer drug VP16 (etopside) or Camptothecin (CPT) induces a similar response for both episomal or chromosomal targets. Synchronized cells are also more amenable to the gene repair reaction once released into S phase and a synergistic effect is seen when cell synchronization is coupled to the addition of HU or thymidine or CPT. These results suggest that DNA damage at the replication fork stimulates the homologous recombination pathway to act upon strand lesions (double strand breaks, etc.), enabling a more efficient gene repair reaction to take place. Agents or enzymes that induce the double strand breaks repair response are also quite effective in regulating and elevating the frequency of gene repair. Taken together, we propose that the factors and/or protein activated by recombination induced by replication inhibition (RIRI), particularly those involved in HR, are capable of enhancing the frequency of gene repair in mammalian cells.

316. Cystic Fibrosis Airway Epithelial Cells Transfection

The pathway of targeted gene repair may consist of two major steps, the pairing of a short modified single-stranded oligonucleotide with the target site followed by a repair event that reverses a point mutation. In this study, we show that the level of correction is influenced by the position of the mismatched base pair at the target site relative to the oligonucleotide. Vectors were designed to target an eGFP gene containing a single point mutation and integrated stability into DLD-1 cells. 72-mers are introduced into these cells by electroporation and expression of the corrected eGFP mutation is detected by FACS analyses. Oligonucleotides with a number of structural variants were designed to target the mutant eGFP gene. Appreciable levels of gene repair occur when the oligonucleotide contains the base that creates the mismatch with the target site in the center, whereas the efficiency of correction decreases less than two fold when the mismatch is located near the 3′ end of the oligonucleotide. However, correction directed by an oligonucleotide which creates a mismatch near its 5′ end is reduced up to 17 fold, depending on the vector design. The same directionality is observed even when these positioned mismatches are separated by 18 bases of sequence heterology. These results lead us to propose a directional specific repair pathway consisting of proteins involved in both homologous recombination and mismatch repair. Thus, the pairing stability of the repair complex likely plays a role in the successful application of this technique and these types of factors must be taken into consideration when designing protocols for targeted gene repair.

315. Targeted Gene Repair Exhibits a Polar Bias in the Directionality of Correction

The restoration of gene function through targeted correction of specific mutations guarantees that the correction is physiologically regulated and is expressed in “cell-appropriate” fashion. Such an approach has significant potential as a gene therapy strategy. Previous in vitro studies carried out in human airway epithelial cells showed both genomic and phenotypic correction of the most common cystic fibrosis transmembrane conductance regulator (CFTR) mutation, a 3-bp deletion in exon 10 (ΔF508), following introduction of small fragment homologous replacement (SFHR) comprised of WT CFTR sequences into transformed CF airway epithelial cells (Kunzelmann. et al 1996, Goncz, et al 1998, 2001, 2002)1,2,3,4. To enhance the efficacy of gene correction for therapeutic application, the mode of DNA delivery witch is an actual limit of gene correction was evaluated in terms of transfection parameters. We compared both physical and chemical DNA delivery approaches as measured by Enhance Green Fluorescent Protein (eGFP) gene expression. Chemically, we compared lipofectamine (Invitrogen), Superfect (Qiagen), Effectene (Qiagen) and a newly developed lipid, KLM47. For physical delivery, we used the AMAXA Nucleofector System. Our studies demonstrated that the nature (i.e., chemical composition) of lipids as well as the lipid/DNA (i.e., +/−) ratio modulate the transfection efficacy for each cell line. We also demonstrated that these cell lines derived from the airway epithelium have different lipid/DNA ratio requirements for optimal transfection. When compared to the AMAXA system, we observed a much higher efficiency of transfection (50–60%) than was observed with the chemical cocktails (5–10%). These studies are intriguing and suggest a physical delivery system may provide an effective means of delivering DNA to the nucleus and that ex vivo DNA delivery to pluripotent stem cells as a therapeutic modality.

305. Regulation of Targeted Gene Repair by DNA Replication and by DNA Replication Arrest

High frequencies of oligonucleotide-mediated gene repair events are linked to the cellular activities of homologous recombination, DNA repair, transcription and now, DNA replication. In the process of gene repair, oligonucleotides are designed to hybridize to either the transcribed or nontranscribed strand of DNA where the correction events reveal a strand bias favoring the nontranscribed strand. We now report that DNA replication plays a role in dictating the gene repair phenomenon of strand bias. We constructed mutant eGFP episomal targets lying adjacent to an active SV40 origin of replication and transformed them into COS1 cells. By changing the orientation of the eGFP gene relative to the origin of replication we were able to effectively separate the effects of the concurrently active processes of transcription and replication. In addition to the already reported bias towards the nontranscribed strand, a replication bias towards the lagging strand was uncovered. Replication bias remained intact and reproducible even when both the mismatch and the oligonucleotide were altered. To further examine the replication bias, identical episomal constructs were made substituting the eGFP gene with other target genes including a kanamycin resistance gene with a single base pair substitution or a tetracycline resistance gene with either a single base pair substitution or deletion. Bacterial readout assays allowed us to quantitate the correction values per plasmid rather than per cell. Two of the three bacterial genes exhibited a lagging strand bias indicating replication's influence upon the process. However, because this effect is not universal, it also indicates that the cellular effects on oligonucleotide-mediated repair are multifactoral; replication is clearly one of the number of processes involved. The balance of the process of replication, transcription, and DNA context most certainly influence the relative success of gene repair as well as the favored strand bias. Our results establish a gene repair hierarchy at the DNA level relative to the ongoing activities of transcription and replication.

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.

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.

Targeted genetic repair: an emerging approach to genetic therapy.

The last two decades have heralded a remarkable increase in our understanding of the genetic basis of disease and the endogenous mechanisms responsible for the repair of genomic DNA and the processing of RNA. Targeted gene repair is a powerful yet controversial technique developed to direct base changes in chromosomal genes, while RNA repair is an emerging strategy to alter the coding content of messenger RNAs. This Perspective series examines the inspired techniques for facilitating the simple correction of genetic defects in what would represent a major shift in the paradigm of clinical science, and the hurdles that need to be overcome in order to make clinical use of molecular therapeutics. Human genetic information is encoded in the sequences of nucleic acids found inside our cells. During the past two decades, it has become increasingly apparent that such instructions are not fixed, but rather that molecular processes exist that can revise them. Recently, a number of investigators have been exploring whether the ability to revise RNA or DNA sequences can be used to repair mutant genetic instructions, to treat inherited disorders such as cystic fibrosis and sickle cell disease, as well as to revise pathogenic genes associated with cancers and infectious diseases. This repair approach has received increasing attention because of the safety and efficacy issues encountered with more traditional gene therapy strategies where additional copies of therapeutic genes are delivered to and expressed in transduced cells. Most notably, the recent observation that retroviral gene transfer apparently induced leukemia in two children treated for X-linked SCIDs has raised significant safety concerns for traditional gene add-back strategies (1). In contrast to the traditional approach to gene therapy, genetic repair strategies attempt to directly correct endogenous genetic mistakes rather than deliver extra copies of genes to cells. Thus by analogy, genetic repair methods are similar to word processors that correct misspelled words within their intended written context, whereas gene add-back approaches are similar to editors who prepare corrected versions of defective sentences and then randomly insert them into the text without amending the original written mistake. Genetic repair strategies may have significant therapeutic and safety advantages over traditional gene therapy approaches for the treatment of many genetic disorders. Firstly, because the mutant genetic instructions are directly repaired, the corrected RNAs and/or DNAs will be maintained in their native sequence context and be regulated by their endogenous regulatory machinery. Secondly, in the instance where the mutant gene encodes a deleterious or dominant-negative mutant protein, repair of the mutant should simultaneously engender the regulated production of the wild-type protein while eliminating or reducing expression of the deleterious gene product. Finally, genetic repair strategies attempt to repair defective instructions in a site-specific manner. Therefore, once adequately developed, these strategies will result in less random mutagenesis of the genome and lead to fewer mutagenic side effects than do methods that randomly insert genes into the genome (1). In this Perspective series, six articles will update the reader on the progress toward and the hurdles that remain for developing such genetic repair strategies. The first half of the series will focus on approaches to RNA repair, while the latter will describe methods for DNA repair. As therapeutic modalities, RNA and DNA repair have different advantages and weaknesses. For example, RNA repair may represent a safer approach to genetic correction than DNA repair because the revision of unintended target RNA will not result in permanent genetic change within a cell since RNAs undergo continual turnover in vivo. However, the limited half-life of the amended instructions also necessitates that RNA repair strategies have to continually repair the mutant RNAs emerging from mutant DNA. By contrast, DNA repair will amend the cell’s genetic blueprint, and such repair need occur only once to permanently correct the products expressed from the repaired gene in the treated cell and its progeny. However, since any revised DNA will be stably maintained and propagated, the specificity of DNA repair is a major safety consideration because genes that are unintentionally revised will also be maintained and propagated.

Strand Bias in Targeted Gene Repair Is Influenced by Transcriptional Activity

Strand Bias in Targeted Gene Repair Is Influenced by Transcriptional Activity