Regulation of targeted gene repair by intrinsic cellular processes

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.     

Genome engineering with zinc-finger nucleases

Zinc-finger nucleases (ZFNs) are targetable DNA cleavage reagents that have been adopted as gene-targeting tools. ZFN-induced double-strand breaks are subject to cellular DNA repair processes that lead to both targeted mutagenesis and targeted gene replacement at remarkably high frequencies. This article briefly reviews the history of ZFN development and summarizes applications that have been made to genome editing in many different organisms and situations. Considerable progress has been made in methods for deriving zinc-finger sets for new genomic targets, but approaches to design and selection are still being perfected. An issue that needs more attention is the extent to which available mechanisms of double-strand break repair limit the scope and utility of ZFN-initiated events. The bright prospects for future applications of ZFNs, including human gene therapy, are discussed.     

动物基因组编辑中提升CRISPR/Cas9介导的同源重组效率研究进展*

基因组编辑技术可以对DNA或RNA进行精准改造,极大地促进了生命科学的发展。CRISPR/Cas9系统在靶位点诱导DNA发生双链或单链损伤,细胞对损伤部位采用无供体模板的非同源末端连接(non-homologous end joining,NHEJ)或有供体模板的同源重组(homologous recombination,HR)修复。基于HR的基因组编辑策略通常被用于获得DNA的精准改造,而NHEJ在动物DNA损伤修复中起主导作用。为了提升HR效率,研究人员设计了多种方案,包括CRISPR/Cas9系统优化和DNA修复通路调控等。从DNA损伤修复途径、Cas9变体选择、sgRNA设计、供体模板设计、DNA修复途径相关蛋白功能调控、供体模板募集效率提升、细胞周期调控及编辑细胞生存效率提升等方面详细综述了相关研究成果,发现尚未开发出放之四海而皆准的HR提升策略,基于HR的基因组编辑需要针对具体案例制定个体化策略。旨在为动物基因组编辑中提升CRISPR/Cas9介导的HR效率研究提供理论参考,为动物基因功能分析、基因治疗和经济动物基因编辑育种提供帮助。     

Retrovirus-mediated DNA repair gene transfer into xeroderma pigmentosum cells: Perspectives for a gene therapy

The rare hereditary disease xeroderma pigmentosum (XP) is clinically characterized by extreme sun sensitivity and an increased predisposition for developing skin cancer. Cultured cells from XP patients exhibit hypersensitivity to ultraviolet (UV) radiation due to the defect in nucleotide excision repair (NER), and other cellular abnormalities. Seven genes identified in the classical XP forms, XPA to XPG, are involved in the NER pathway. In view of developing a strategy of gene therapy for XP, we devised recombinant retrovirus-carrying DNA repair genes for transfer and stable expression of these genes in cells from XP patients. Results showed that these retroviruses are efficient tools for transducing XP fibroblasts and correcting repair-defective cellular phenotypes by recovering normal UV survival, unscheduled DNA synthesis, and RNA synthesis after UV irradiation, and also other cellular abnormalities resulting from NER defects. These results imply that the first step of cellular gene therapy might be accomplished successfully.     

Targeting cancer-specific synthetic lethality in double-strand DNA break repair

There is interest in the use of DNA repair inhibitors as sensitizers of classic cytotoxic therapy against cancer. However, there is also risk – theoretical, at least – that such a strategy may increase the side effects of traditional therapies, including but not limited to treatment-related secondary malignancies. Before being brought to clinical application, therefore, important questions remain to be answered regarding how these therapies will be tailored to achieve benefit without concomitantly increasing harm. A potential solution may involve targeting so-called “synthetic lethalities” in tumor DNA repair pathways; taking advantage of defects acquired in DNA repair pathways during tumorigenesis by targeting alternative repair pathways on which the tumor critically depends. Conceivably, as repair pathways are functional in normal tissue, such targeted therapy should be relatively tumor-specific and non-toxic. We review here the rationale for this strategy, describe examples of its application, and outline potential strengths and weaknesses of this approach. For simplicity, a focus will be placed on the repair of double-strand breaks as a model system, but the conceptual framework is generally applicable to many other pathways of DNA repair.     

Cetuximab augments cytotoxicity with poly (adp-ribose) polymerase inhibition in head and neck cancer

Overexpression of the epidermal growth factor receptor (EGFR) is a hallmark of head and neck cancers and confers increased resistance and inferior survival rates. Despite targeted agents against EGFR, such as cetuximab (C225), almost half of treated patients fail this therapy, necessitating novel therapeutic strategies. Poly (ADP-Ribose) polymerase (PARP) inhibitors (PARPi) have gained recent attention due to their unique selectivity in killing tumors with defective DNA repair. In this study, we demonstrate that C225 enhances cytotoxicity with the PARPi ABT-888 in UM-SCC1, UM-SCC6, and FaDu head and neck cancer cells. The mechanism of increased susceptibility to C225 and PARPi involves C225-mediated reduction of non-homologous end-joining (NHEJ)- and homologous recombination (HR)-mediated DNA double strand break (DSB) repair, the subsequent persistence of DNA damage, and activation of the intrinsic apoptotic pathway. By generating a DSB repair deficiency, C225 can render head and neck tumor cells susceptible to PARP inhibition. The combination of C225 and the PARPi ABT-888 can thus be an innovative treatment strategy to potentially improve outcomes in head and neck cancer patients. Furthermore, this strategy may also be feasible for other EGFR overexpressing tumors, including lung and brain cancers.     

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.     

A comparison of synthetic oligodeoxynucleotides, DNA fragments and AAV-1 for targeted episomal and chromosomal gene repair

Background  

Current strategies for gene therapy of inherited diseases consist in adding functional copies of the gene that is defective. An attractive alternative to these approaches would be to correct the endogenous mutated gene in the affected individual. This study presents a quantitative comparison of the repair efficiency using different forms of donor nucleic acids, including synthetic DNA oligonucleotides, double stranded DNA fragments with sizes ranging from 200 to 2200 bp and sequences carried by a recombinant adeno-associated virus (rAAV-1). Evaluation of each gene repair strategy was carried out using two different reporter systems, a mutated eGFP gene or a dual construct with a functional eGFP and an inactive luciferase gene, in several different cell systems. Gene targeting events were scored either following transient co-transfection of reporter plasmids and donor DNAs, or in a system where a reporter construct was stably integrated into the chromosome.     

Functional lentiviral vectors for xeroderma pigmentosum gene therapy

Xeroderma pigmentosum (XP) is a genetic disease characterized by an autosomal-transmitted genodermatosis involving impaired DNA repair activity, where XP patients present severe sensitivity to sunlight (UVB radiation) and are highly victimized by skin cancer. Complementing XP genes by gene therapy is one potential strategy for helping XP patients. However, current viral-based protocols still lack long-term and stable expression, due to limited post-mitotic infection and gene silencing (in the case of retroviruses) or transient expression and activation of immune response (in the case of adenoviruses). Here we demonstrate that the use of third-generation lentiviral vectors can overcome some of these limitations, rescuing the aberrant phenotype in different categories of the disease (XPA, XPC and XPD). Our results show that lentiviruses are efficient tools to transduce XP fibroblasts and correct repair-defective cellular phenotypes by recovering proper gene expression, normal UV survival and unscheduled DNA synthesis after UV radiation. We propose lentiviral vectors as an attractive alternative for gene therapy protocols focusing on DNA repair genetic diseases.     

Role of impaired DNA repair in genotoxic susceptibility of patients with head and neck cancer

DNA repair is critical for genotoxic susceptibility and cancer development. Forty-seven patients with head and neck squamous cell carcinoma (HNSCC) and 38 healthy controls were enrolled in this study. Among the patients, 16 subjects had metastasis of HNSCC. The extent of DNA damage, including oxidative lesions, and efficiency of repair after genotoxic treatment with hydrogen peroxide were examined using the alkaline comet assay. HNSCC cells were sensitive to genotoxic treatment and displayed impaired DNA repair. In particular, lesions caused by hydrogen peroxide were repaired less effectively in cancer cells from patients with metastasis than in cells from healthy controls. We suggest that impaired DNA repair might play a role in genotoxic susceptibility of patients with head and neck cancer. Finally, as a consequence of this finding we have shown that treatment with DNA-reactive drugs could be considered as an effective therapy strategy for head and neck cancer.     

Prospects of chimeric RNA-DNA oligonucleotides in gene therapy

A strategy called targeted gene repair was developed to facilitate the process of gene therapy using a chimeric RNA-DNA oligonucleotide. Experiments demonstrated the feasibility of using the chimeric oligonucleotide to introduce point conversion in genes in vitro and in vivo. However, barriers exist in the low and/or inconstant frequency of gene repair. To overcome this difficulty, three main aspects should be considered. One is designing a more effective structure of the oligonucleotide. Trials have included lengthening the homologous region, displacing the mismatch on the chimeric strand and inventing a novel thioate-modified single-stranded DNA, which was demonstrated to be more active than the primary chimera in cell-free extracts. The second aspect is optimizing the delivery system. Producing synthetic carriers for efficient and specific transfection is demanding, especially for treatment in vivo where targeting is difficult. The third and most important aspect lies in the elucidation of the mechanism of the strategy. Investigation of the mechanism of strand exchange between the oligonucleotide molecule and double-stranded DNA in prokaryotes may greatly help to understand the mechanism of gene repair in eukaryotes. The development of this strategy holds great potential for the treatment of genetic defects and other purposes.     

Oligonucleotide-mediated gene targeting in human hepatocytes: implications of mismatch repair

Gene therapy using viral vectors for liver diseases, particularly congenital disorders, is besought with difficulties, particularly immunologic reactions to viral antigens. As a result, nonviral methods for gene transfer in hepatocytes have also been explored. Gene repair by small synthetic single-stranded oligodeoxynucleotides (ODNs) produces targeted alterations in the genome of mammalian cells and represents a great potential for nonviral gene therapy. To test the feasibility of ODN-mediated gene repair within chromosomal DNA in human hepatocytes, two new cell lines with stably integrated mutant reporter genes, namely neomycin and enhanced green fluorescent protein were established. Targeting theses cells with ODNs specifically designed for repair resulted in site-directed and permanent gene conversion of the single-point mutation of the reporter genes. Moreover, the frequency of gene alteration was highly dependent on the mitotic activity of the cells, indicating that the proliferative status is an important factor for successful targeting in human hepatocytes. cDNA array expression profiling of DNA repair genes under different cell culture conditions combined with RNA interference assay showed that mismatch repair (MMR) in actively growing hepatocytes imposes a strong barrier to efficient gene repair mediated by ODNs. Suppression of MSH2 activity in hepatocytes transduced with short hairpin RNAs (shRNAs) targeted to MSH2 mRNA resulted in 25- to 30-fold increase in gene repair rate, suggesting a negative effect of MMR on ODN-mediated gene repair. Taken together, these data suggest that under appropriate conditions nonviral chromosomal targeting may represent a feasible approach to gene therapy in liver disease.     

Plant homologue of human excision repair gene ERCC1 points to conservation of DNA repair mechanisms

Nucleotide excision repair (NER), a highly versatile DNA repair mechanism, is capable of removing various types of DNA damage including those induced by UV radiation and chemical mutagens. NER has been well characterized in yeast and mammalian systems but its presence in plants has not been reported. Here it is reported that a plant gene isolated from male germline cells of lily (Lilium longiflorum) shows a striking amino acid sequence similarity to the DNA excision repair proteins human ERCC1 and yeast RAD10. Homologous genes are also shown to be present in a number of taxonomically diverse plant genera tested, suggesting that this gene may have a conserved function in plants. The protein encoded by this gene is able to correct significantly the sensitivity to the cross-linking agent mitomycin C in ERCC1-deficient Chinese hamster ovary (CHO) cells. These findings suggest that the NER mechanism is conserved in yeast, animals and higher plants.     

Single-stranded oligonucleotide-mediated gene repair in mammalian cells has a mechanism distinct from homologous recombination repair

Single-stranded DNA oligonucleotide (SSO)-mediated gene repair has great potentials for gene therapy and functional genomic studies. However, its underlying mechanism remains unclear. Previous studies from other groups have suggested that DNA damage response via the ATM/ATR pathway may be involved in this process. In this study, we measured the effect of two ATM/ATR inhibitors caffeine and pentoxifylline on the correction efficiency in SSO-mediated gene repair. We also checked their effect on double-stranded break (DSB)-induced homologous recombination repair (HRR) as a control, which is well known to be dependent on the ATM/ATR pathway. We found these inhibitors could completely inhibit DSB-induced HRR, but could only partially inhibit SSO-mediated process, indicating SSO-mediated gene repair is not dependent on the ATM/ATR pathway. Furthermore, we found that thymidine treatment promotes SSO-mediated gene repair, but inhibits DSB-induced HRR. Collectively, our results demonstrate that SSO-mediated and DSB-induced gene repairs have distinct mechanisms.     

Increased Anticancer Efficacy of Intravesical Mitomycin C Therapy when Combined with a PCNA Targeting Peptide

Non–muscle-invasive bladder cancers (NMIBCs) are tumors confined to the mucosa or the mucosa/submucosa. An important challenge in treatment of NMIBC is both high recurrence and high progression rates. Consequently, more efficacious intravesical treatment regimes are in demand. Inhibition of the cell’s DNA repair systems is a new promising strategy to improve cancer therapy, and proliferating cell nuclear antigen (PCNA) is a new promising target. PCNA is an essential scaffold protein in multiple cellular processes including DNA replication and repair. More than 200 proteins, many involved in stress responses, interact with PCNA through the AlkB homologue 2 PCNA-interacting motif (APIM), including several proteins directly or indirectly involved in repair of DNA interstrand crosslinks (ICLs). In this study, we targeted PCNA with a novel peptide drug containing the APIM sequence, ATX-101, to inhibit repair of the DNA damage introduced by the chemotherapeutics. A bladder cancer cell panel and two different orthotopic models of bladder cancer in rats, the AY-27 implantation model and the dietary BBN induction model, were applied. ATX-101 increased the anticancer efficacy of the ICL-inducing drug mitomycin C (MMC), as well as bleomycin and gemcitabine in all bladder cancer cell lines tested. Furthermore, we found that ATX-101 given intravesically in combination with MMC penetrated the bladder wall and further reduced the tumor growth in both the slow growing endogenously induced and the rapidly growing transplanted tumors. These results suggest that ATX-101 has the potential to improve the efficacy of current MMC treatment in NMIBC.     

Cis and trans mechanisms of DNA repair.

DNA repair is essential for genetic stability and variability. Remarkable advances in the understanding of DNA repair by the molecular analysis of the substrate (gene repair) or the enzyme (repair genes), emphasize evolutionary conservation. Recent progress also stresses the interaction(s) between DNA repair and numerous other cellular metabolic processes, including non-nuclear and/or non-genetic responses.     

Sources of DNA Double-Strand Breaks and Models of Recombinational DNA Repair

DNA is subject to many endogenous and exogenous insults that impair DNA replication and proper chromosome segregation. DNA double-strand breaks (DSBs) are one of the most toxic of these lesions and must be repaired to preserve chromosomal integrity. Eukaryotes are equipped with several different, but related, repair mechanisms involving homologous recombination, including single-strand annealing, gene conversion, and break-induced replication. In this review, we highlight the chief sources of DSBs and crucial requirements for each of these repair processes, as well as the methods to identify and study intermediate steps in DSB repair by homologous recombination.