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Antisense oligonucleotide- based drugs for splicing modulation were recently approved for various genetic diseases with unmet need. Here we aimed to generate skipping over exon 23 of the CFTR transcript, to eliminate the W1282X nonsense mutation and avoid RNA degradation induced by the nonsense mediated mRNA decay mechanism, allowing production of partially active CFTR proteins lacking exon 23.∼80 ASOs were screened in 16HBEge W1282X cells. ASO candidates showing significant exon skipping were assessed for their W1282X allele selectivity and the increase of CFTR protein maturation and function. The effect of a highly potent ASO candidates was further analyzed in well differentiated primary human nasal epithelial cells, derived from a W1282X homozygous patient.ASO screening led to identification of several ASOs that significantly decrease the level of CFTR transcripts including exon 23. These ASOs resulted in significant levels of mature CFTR protein and together with modulators restore the channel function following free uptake into these cells. Importantly, a highly potent lead ASOs, efficiently delivered by free uptake, was able to increase the level of transcripts lacking exon 23 and restore the CFTR function in cells from a W1282X homozygote patient.The highly efficient exon 23 skipping induced by free uptake of the lead ASO and the resulting levels of mature CFTR protein exhibiting channel function in the presence of modulators, demonstrate the ASO therapeutic potential benefit for CF patients carrying the W1282X mutation with the objective to advance the lead candidate SPL23-2 to proof-of-concept clinical study.

Expression of modules during carcinogenesis.

Functional enrichment in modules.

The last years have shown enormous advancement in the therapeutic potential of RNA-related treatments, specifically for antisense oligonucleotide (ASO)-based drugs, leading to increased numbers of ASO regulatory approvals. In this study we focus on SPL84, an inhaled ASO-based drug, developed for the treatment of the pulmonary disease, Cystic Fibrosis (CF). Pulmonary drug delivery is challenging, due to a variety of biological, physical, chemical, and structural barriers, especially when aiming to target the cell nucleus. The efficient distribution of SPL84 in the lungs, penetration into the cells and nucleus, and stability are critical parameters that will impact drug efficacy in a clinical setting. In this study, we demonstrate the proper distribution and cell and nucleus penetration of SPL84 in mouse and monkey lungs. In vivo and in vitro studies confirmed the stability and mobility of our inhaled ASO drug through CF patient-derived mucus and in lung lysosomal extracts. Our results, supported by a promising pre-clinical pharmacological effect, emphasize the high potential of SPL84 as an effective drug for the treatment of CF patients.In addition, successfully tackling the lung distribution of SPL84 and specific cell targeting offers huge opportunities for further development of SpliSense inhaled ASO-based drugs for unmet pulmonary diseases.

Background Antisense oligonucleotide (ASO)-based drugs for splicing modulation were recently approved for various genetic diseases with unmet need. Here we aimed to develop an ASO-based splicing modulation therapy for Cystic Fibrosis (CF) patients carrying the 3849+10 kb C-to-T splicing mutation in the CFTR gene. Methods We have screened, in FRT cells expressing the 3849+10 kb C-to-T splicing mutation, ~30 2′-O-Methyl-modified phosphorothioate ASOs, targeted to prevent the recognition and inclusion of a cryptic exon generated due to the mutation. The effect of highly potent ASO candidates on the splicing pattern, protein maturation and CFTR function was further analyzed in well differentiated primary human nasal and bronchial epithelial cells, derived from patients carrying at least one 3849+10 kb C-to-T allele. Results A highly potent lead ASO, efficiently delivered by free uptake, was able to significantly increase the level of correctly spliced mRNA and completely restore the CFTR function to wild type levels in cells from a homozygote patient. This ASO led to CFTR function with an average of 43% of wild type levels in cells from various heterozygote patients. Optimized efficiency of the lead ASO was further obtained with 2′-Methoxy Ethyl modification (2′MOE). Conclusion The highly efficient splicing modulation and functional correction, achieved by free uptake of the selected lead ASO in various patients, demonstrate the ASO therapeutic potential benefit for CF patients carrying splicing mutations and is aimed to serve as the basis for our current clinical development.

Gene therapy offers great promise for cystic fibrosis which has never been quite fulfilled due to the challenges of delivering sufficient amounts of the CFTR gene and expression persistence for a sufficient period of time in the lungs to have any effect. Initial trials explored both viral and non-viral vectors but failed to achieve a significant breakthrough. However, in recent years, new opportunities have emerged that exploit our increased knowledge and understanding of the biology of CF and the airway epithelium. New technologies include new viral and non-viral vector approaches to delivery, but also alternative nucleic acid technologies including oligonucleotides and siRNA approaches for gene silencing and gene splicing, described in this review, as presented at the 2019 annual European CF Society Basic Science meeting (Dubrovnik, Croatia). We also briefly discuss other emerging technologies including mRNA and CRISPR gene editing that are advancing rapidly. The future prospects for genetic therapies for CF are now diverse and more promising probably than any time since the discovery of the CF gene.

DNA replication is a complex process tightly regulated to ensure faithful genome duplication, and its perturbation leads to DNA damage and genomic instability. Replication stress is commonly associated with slow and stalled replication forks. Recently, accelerated replication has emerged as a non-canonical form of replication stress. However, the molecular basis underlying fork acceleration is largely unknown. Here, we show that mutated HRAS activation leads to increased topoisomerase 1 (TOP1) expression, causing aberrant replication fork acceleration and DNA damage by decreasing RNA-DNA hybrids or R-loops. In these cells, restoration of TOP1 expression or mild replication inhibition rescues the perturbed replication and reduces DNA damage. Furthermore, TOP1 or RNaseH1 overexpression induces accelerated replication and DNA damage, highlighting the importance of TOP1 equilibrium in regulating R-loop homeostasis to ensure faithful DNA replication and genome integrity. Altogether, our results dissect a mechanism of oncogene-induced DNA damage by aberrant replication fork acceleration., This research was supported by grants from the Israel Science Foundation (grant nos. 176/11 and 1284/18), the Israeli Centers of Research Excellence (I-CORE), and Gene Regulation in Complex Human Disease, Center No. 41/11, and by the ISF-NSFC joint program (grant no. 2535/16) to B.K. and by grants from the Agencia Estatal de Investigación from the Spanish Ministry of Science and Innovation (PID2019-104270GB-I00/BMC), the European Research Council (ERC2014 AdG669898 TARLOOP), the European Union (FEDER), and the Foundation “Vencer el Cancer” to A.A. The authors thank Dr. Naomi Melamed-Book for her assistance in confocal microscopy and the Mantoux Bioinformatics Institute of the Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, for assistance in deep sequencing and bioinformatics analysis. The authors thank the members of the Kerem lab for thoughtful discussions and advice.

Antisense oligonucleotide (ASO)-based drugs for splicing modulation were recently been approved for various genetic diseases with unmet need. Here we aimed to develop an ASO-based splicing modulation therapy for Cystic Fibrosis (CF) patients carrying the 3849+10kb C-to-T splicing mutation in the CFTR gene. We have screened, in FRT cells expressing this mutation, ~30 ASOs chemically modified with 2′-O-Methyl on a phosphrothioate backbone, targeted to prevent the recognition and inclusion of a cryptic exon generated due to the mutation. The screening identified five ASO candidates able to promote CFTR correct splicing and rescue channel activity. Further analyses in well differentiated primary human nasal and bronchial epithelial cells (HNEs, HBEs), derived from patients carrying at least one 3849+10kb C-to-T allele, led to the identification of a highly potent lead ASO. The ASO was efficiently delivered by free uptake into patients’ HNEs and HBEs and completely restored CFTR function to wild type levels in cells from a homozygous patient and led to 43±8% of wild type levels in cells from various heterozygous patients. Optimized efficiency was further obtained with 2’-Methoxy Ethyl chemical modification. The results demonstrate the therapeutic potential and clinical benefit of ASO-based splicing modulation for genetic diseases caused by splicing mutations.

DNA replication is a complex process that is tightly regulated to ensure faithful genome duplication, and its perturbation leads to DNA damage and genomic instability. Replication stress is commonly associated with slow and stalled replication forks. Recently, accelerated replication has emerged as a non-canonical form of replication stress. However, the molecular basis underlying fork acceleration is largely unknown. Here we show that increased topoisomerase 1 (TOP1) expression induces aberrant replication fork acceleration and DNA damage by decreasing RNA-DNA hybrids (R-loops). Degradation of R-loops by overexpression of RNaseH1 also accelerates replication and generates DNA damage. Furthermore, upregulation of TOP1 by activation of the mutated HRAS oncogene leads to fork acceleration and DNA damage in pre-senescent cells. In these cells, restoration of TOP1 expression level or mild replication inhibition rescues the perturbed replication and reduces DNA damage. These findings highlight the importance of TOP1 equilibrium in the regulation of R-loop homeostasis to ensure faithful DNA replication and genome integrity.

Common fragile sites (CFSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Several features were suggested to underlie CFS instability, however, these features are prevalent across the genome. Therefore, the molecular mechanisms underlying CFS instability remain unclear. Here, we explore the transcriptional profile and DNA replication timing (RT) under mild replication stress in the context of the 3D genome organization. The results reveal a fragility signature, comprised of a TAD boundary overlapping a highly transcribed large gene with APH-induced RT-delay. This signature enables precise mapping of core fragility regions in known CFSs and identification of novel fragile sites. CFS stability may be compromised by incomplete DNA replication and repair in TAD boundaries core fragility regions leading to genomic instability. The identified fragility signature will allow for a more comprehensive mapping of CFSs and pave the way for investigating mechanisms promoting genomic instability in cancer., Common fragile sites are regions susceptible to replication stress and are prone to chromosomal instability. Here, the authors, by analyzing the contribution of 3D chromatin organization, identify and characterize a fragility signature and precisely map these fragility regions.

Common fragile sties (CFSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Several features characterizing CFSs have been associated with their instability, however, these features are prevalent across the genome and do not account for all known CFSs. Therefore, the molecular mechanism underlying CFS instability remains unclear. Here, we explored the transcriptional profile and temporal order of DNA replication (replication timing, RT) of cells under replication stress conditions. We show that the RT of only a small portion of the genome is affected by replication stress, and that CFSs are enriched for delayed RT. We identified a signature for chromosomal fragility, comprised of replication stress-induced delay in RT of early/mid S-phase replicating regions within actively transcribed large genes. This fragility signature enabled precise mapping of the core fragility region. Furthermore, the signature enabled the identification of novel fragile sites that were not detected cytogenetically, highlighting the improved sensitivity of our approach for identifying fragile sites. Altogether, this study reveals a link between altered DNA replication and transcription of large genes underlying the mechanism of CFS expression. Thus, investigating the RT and transcriptional changes in cancer may contribute to the understanding of mechanisms promoting genomic instability in cancer.

Common fragile sites (CFSs) are chromosomal regions characterized as hotspots for breakage and chromosomal rearrangements following DNA replication stress. They are preferentially unstable in pre-cancerous lesions and during cancer development. Recently CFSs were found to be tissue- and even oncogene-induced specific, thus indicating an unforeseen complexity. Here we review recent developments in CFS research that shed new light on the molecular basis of their instability and their importance in cancer development.

The presence of dormant, microscopic cancerous lesions poses a major obstacle for the treatment of metastatic and recurrent cancers. While it is well-established that microRNAs play a major role in tumorigenesis, their involvement in tumor dormancy has yet to be fully elucidated. We established and comprehensively characterized pairs of dormant and fast-growing human osteosarcoma models. Using these pairs of mouse tumor models, we identified three novel regulators of osteosarcoma dormancy: miR-34a, miR-93, and miR-200c. This report shows that loss of these microRNAs occurs during the switch from dormant avascular into fast-growing angiogenic phenotype. We validated their downregulation in patients' tumor samples compared to normal bone, making them attractive candidates for osteosarcoma therapy. Successful delivery of miRNAs is a challenge; hence, we synthesized an aminated polyglycerol dendritic nanocarrier, dPG-NH2, and designed dPG-NH2-microRNA polyplexes to target cancer. Reconstitution of these microRNAs using dPG-NH2 polyplexes into Saos-2 and MG-63 cells, which generate fast-growing osteosarcomas, reduced the levels of their target genes, MET proto-oncogene, hypoxia-inducible factor 1α, and moesin, critical to cancer angiogenesis and cancer cells' migration. We further demonstrate that these microRNAs attenuate the angiogenic capabilities of fast-growing osteosarcomas in vitro and in vivo. Treatment with each of these microRNAs using dPG-NH2 significantly prolonged the dormancy period of fast-growing osteosarcomas in vivo. Taken together, these findings suggest that nanocarrier-mediated delivery of microRNAs involved in osteosarcoma tumor-host interactions can induce a dormant-like state.

Human pluripotent stem cells (hPSCs) frequently acquire chromosomal aberrations such as aneuploidy in culture. These aberrations progressively increase over time and may compromise the properties and clinical utility of the cells. The underlying mechanisms that drive initial genomic instability and its continued progression are largely unknown. Here, we show that aneuploid hPSCs undergo DNA replication stress, resulting in defective chromosome condensation and segregation. Aneuploid hPSCs show altered levels of actin cytoskeletal genes controlled by the transcription factor SRF, and overexpression of SRF rescues impaired chromosome condensation and segregation defects in aneuploid hPSCs. Furthermore, SRF downregulation in diploid hPSCs induces replication stress and perturbed condensation similar to that seen in aneuploid cells. Together, these results suggest that decreased SRF expression induces replicative stress and chromosomal condensation defects that underlie the ongoing chromosomal instability seen in aneuploid hPSCs. A similar mechanism may also operate during initiation of instability in diploid cells.

Common fragile sites (CFSs) are specific genomic regions in normal chromosomes that exhibit genomic instability under DNA replication stress. As replication stress is an early feature of cancer development, CFSs are involved in the signature of genomic instability found in malignant tumors. The landscape of CFSs is tissue-specific and differs under different replication stress inducers. Nevertheless, the features underlying CFS sensitivity to replication stress are shared. Here, we review the events generating replication stress and discuss the unique characteristics of CFS regions and the cellular responses aimed to stabilizing these regions.

Premature termination codons (PTC) originate from nucleotide substitution introducing an in-frame PTC. They induce truncated, usually non-functional, proteins, degradation of the PTC containing transcripts by the nonsense-mediated decay (NMD) pathway and abnormal exon skipping. Readthrough compounds facilitate near cognate amino-acyl-tRNA incorporation, leading potentially to restoration of a functional full-length protein. Splicing mutations can lead to aberrantly spliced transcripts by creating a cryptic splice site or destroying a normal site. Most mutations result in disruption of the open reading frame and activation of NMD. Antisense oligonucleotides are single stranded short synthetic RNA-like molecules chemically modified to improve their stability and ability to recognize their target RNAs and modify the splice site. This review focuses on recent developments in therapies aiming to improve the health of CF patients carrying nonsense or splicing mutations.

DNA replication stress is one of the early drivers enabling the ongoing acquisition of genetic changes arising during tumorigenesis. As such, it is a feature of most pre-malignant and malignant cells. In this review article, we focus on the early events initiating DNA replication stress and the preferential sensitivity of common fragile sites (CFSs) to this stress. CFSs are specific genomic regions within the normal chromosomal structure, which appear as gaps and breaks in the metaphase chromosomes of cells grown under mild replication stress conditions. The main characteristics predisposing CFSs to instability include late replication timing, delayed replication completion, failure to activate additional origins, origin paucity along large genomic regions, collision between replication and transcription complexes along large genes, and the presence of AT-dinucleotide rich sequences. The contribution of these features to instability at CFSs during early cancer development is discussed.

Genomic instability plays a key role in driving cancer development. It is already found in precancerous lesions and allows the acquisition of additional cancerous features. A major source of genomic instability in early stages of tumorigenesis is DNA replication stress. Normally, origin licensing and activation, as well as replication fork progression, are tightly regulated to allow faithful duplication of the genome. Aberrant origin usage and/or perturbed replication fork progression leads to DNA damage and genomic instability. Oncogene activation is an endogenous source of replication stress, disrupting replication regulation and inducing DNA damage. Oncogene-induced replication stress and its role in cancer development have been studied comprehensively, however its molecular basis is still unclear. Here, we review the current understanding of replication regulation, its potential disruption and how oncogenes perturb the replication and induce DNA damage leading to genomic instability in cancer.

Common fragile sites (CFSs) are regions within the normal chromosomal structure that were characterized as hotspots for genomic instability in cancer almost 30 years ago. In recent years, many efforts have been made to understand the basis of CFS fragility and their involvement in the genomic signature of instability found in malignant tumors. CFSs are among the first regions to undergo genomic instability during cancer development because of their intrinsic sensitivity to replication stress conditions, which result from oncogene expression. The preferred sensitivity of CFSs to replication stress stems from various mechanisms including: replication fork arrest at AT-rich repeats, origin paucity along large genomic regions, failure in activation of dormant origins, late replication timing, collision between replication and transcription along large genes, all leading to incomplete replication of the CFS region and resulting in chromosomal instability. Here we review shared and unique characteristics of CFSs, their underlying causes and implications, particularly for the development of cancer.

Colorectal cancer develops in a sequential, evolutionary process, leading to a heterogenic tumor. Comprehensive molecular studies of colorectal cancer have been previously performed; still, the process of carcinogenesis is not fully understood. We utilized gene expression patterns from 94 samples including normal, adenoma, and adenocarcinoma colon biopsies and performed a coexpression network analysis to determine gene expression trajectories of 8,000 genes across carcinogenesis. We found that the majority of gene expression changes occur in the transition from normal tissue to adenoma. The upregulated genes, known to be involved in cellular proliferation, included c-Myc along with its targets. In a cellular model system, we show that physiologic upregulation of c-Myc can lead to cellular proliferation without DNA replication stress. Our analysis also found that carcinogenesis involves a progressive downregulation of genes that are markers of colonic tissue and propose that this reflects a perturbed differentiation of colon cells during carcinogenesis. The analysis of miRNAs targets pointed toward the involvement of miR17 in the regulation of colon cell differentiation. Finally, we found that copy-number variations (CNV) enriched in colon adenocarcinoma tend to occur in genes whose expression changes already in adenoma, with deletions occurring in genes downregulated and duplications in genes upregulated in adenomas. We suggest that the CNVs are selected to reinforce changes in gene expression, rather than initiate them. Together, these findings shed new light into the molecular processes that underlie the transformation of colon tissue from normal to cancer and add a temporal context that has been hitherto lacking. Cancer Res; 74(19); 5532–40. ©2014 AACR.

One-third of monogenic inherited diseases result from premature termination codons (PTCs). Readthrough of in-frame PTCs enables synthesis of full-length functional proteins. However, extended variability in the response to readthrough treatment is found among patients, which correlates with the level of nonsense transcripts. Here, we aimed to reveal cellular pathways affecting this inter-patient variability. We show that activation of the unfolded protein response (UPR) governs the response to readthrough treatment by regulating the levels of transcripts carrying PTCs. Quantitative proteomic analyses showed substantial differences in UPR activation between patients carrying PTCs, correlating with their response. We further found a significant inverse correlation between the UPR and nonsense-mediated mRNA decay (NMD), suggesting a feedback loop between these homeostatic pathways. We uncovered and characterized the mechanism underlying this NMD-UPR feedback loop, which augments both UPR activation and NMD attenuation. Importantly, this feedback loop enhances the response to readthrough treatment, highlighting its clinical importance. Altogether, our study demonstrates the importance of the UPR and its regulatory network for genetic diseases caused by PTCs and for cell homeostasis under normal conditions.

Human pluripotent stem cells (hPSCs) frequently acquire chromosomal aberrations, including aneuploidy, during culture. Recently, we identified a replication stress-based mechanism leading to ongoing chromosomal instability in aneuploid hPSCs that may also operate during the initiation of instability in diploid cells.

In about 10% of patients worldwide and more than 50% of patients in Israel, cystic fibrosis results from nonsense mutations (premature stop codons) in the messenger RNA (mRNA) for the cystic fibrosis transmembrane conductance regulator (CFTR). PTC124 is an orally bioavailable small molecule that is designed to induce ribosomes to selectively read through premature stop codons during mRNA translation, to produce functional CFTR.This phase II prospective trial recruited adults with cystic fibrosis who had at least one nonsense mutation in the CFTR gene. Patients were assessed in two 28-day cycles. During the first cycle, patients received PTC124 at 16 mg/kg per day in three doses every day for 14 days, followed by 14 days without treatment; in the second cycle, patients received 40 mg/kg of PTC124 in three doses every day for 14 days, followed by 14 days without treatment. The primary outcome had three components: change in CFTR-mediated total chloride transport; proportion of patients who responded to treatment; and normalisation of chloride transport, as assessed by transepithelial nasal potential difference (PD) at baseline, at the end of each 14-day treatment course, and after 14 days without treatment. The trial was registered with who.int/ictrp, and with clinicaltrials.gov, number NCT00237380.Transepithelial nasal PD was evaluated in 23 patients in the first cycle and in 21 patients in the second cycle. Mean total chloride transport increased in the first treatment phase, with a change of -7.1 (SD 7.0) mV (p0.0001), and in the second, with a change of -3.7 (SD 7.3) mV (p=0.032). We recorded a response in total chloride transport (defined as a change in nasal PD of -5 mV or more) in 16 of the 23 patients in the first cycle's treatment phase (p0.0001) and in eight of the 21 patients in the second cycle (p0.0001). Total chloride transport entered the normal range for 13 of 23 patients in the first cycle's treatment phase (p=0.0003) and for nine of 21 in the second cycle (p=0.02). Two patients given PTC124 had constipation without intestinal obstruction, and four had mild dysuria. No drug-related serious adverse events were recorded.In patients with cystic fibrosis who have a premature stop codon in the CFTR gene, oral administration of PTC124 to suppress nonsense mutations reduces the epithelial electrophysiological abnormalities caused by CFTR dysfunction.

Common fragile sites are specific genomic loci that form constrictions and gaps on metaphase chromosomes under conditions that slow, but do not arrest, DNA replication. These sites have been shown to have a role in various chromosomal rearrangements in tumors. Different DNA damage response proteins were shown to regulate fragile site stability, including ataxia-telangiectasia and Rad3-related (ATR) and its effector Chk1. Here, we investigated the role of ataxia-telangiectasia mutated (ATM), the main transducer of DNA double-strand break (DSB) signal, in this regulation. We demonstrate that replication stress conditions, which induce fragile site expression, lead to DNA fragmentation and recruitment of phosphorylated ATM to nuclear foci at DSBs. We further show that ATM plays a role in maintaining fragile site stability, which is revealed only in the absence of ATR. However, the activation of ATM under these replication stress conditions is ATR independent. Following conditions that induce fragile site expression both ATR and ATM phosphorylate Chk1, suggesting that both proteins regulate fragile site expression probably via their effect on Chk1 activation. Our findings provide new insights into the interplay between ATR and ATM pathways in response to partial replication inhibition and in the regulation of fragile site stability.

Nonsense-mediated mRNA decay (NMD) is a mechanism, which selectively degrades transcripts carrying premature termination codons (PTCs) and a variety of physiologic transcripts containing NMD-inducing features. In a recent study, we have found variable NMD efficiency among nasal epithelial cells obtained from cystic fibrosis (CF) patients. This variability was found for CF transmembrane conductance regulator (CFTR) transcripts carrying the W1282X PTC, as well as for several NMD physiologic substrates. Here, we aimed to investigate the possibility that variability in NMD efficiency is a more generalized phenomenon and is not restricted to nasal epithelial cells. To investigate this possibility, we analyzed the NMD efficiency of both a CFTR constructs carrying the W1282X PTC and beta-globin constructs carrying the NS39 PTC, in HeLa and MCF7 cells. Variability in NMD efficiency was found for both constructs between the cells, such that in HeLa cells the NMD was highly efficient and in MCF7 the efficiency was significantly lower. Moreover, similar differences in the efficiency of NMD were found for five endogenous NMD physiologic transcripts. Altogether, our results demonstrate existence of cells in which NMD of all transcripts is efficient, whereas others in which the NMD is less efficient, suggesting that the efficiency of NMD is an inherent character of cells. Our results also suggest that variability in the efficiency of NMD is a general phenomenon and is not restricted to nasal epithelial cells. As NMD affects the level of many transcripts, variability in the NMD efficiency might play a role as a genetic modifier of different cellular functions.

Aminoglycosides can readthrough premature termination codons (PTCs), permitting translation of full-length proteins. Previously we have found variable efficiency of readthrough in response to the aminoglycoside gentamicin among cystic fibrosis (CF) patients, all carrying the W1282X nonsense mutation. Here we demonstrate that there are patients in whom the level of CF transmembrane conductance regulator (CFTR) nonsense transcripts is markedly reduced, while in others it is significantly higher. Response to gentamicin was found only in patients with the higher level. We further investigated the possibility that the nonsense-mediated mRNA decay (NMD) might vary among cells and hence governs the level of nonsense transcripts available for readthrough. Our results demonstrate differences in NMD efficiency of CFTR transcripts carrying the W1282X mutation among different epithelial cell lines derived from the same tissue. Variability was also found for 5 physiologic NMD substrates, RPL3, SC35 1.6 kb, SC35 1.7 kb, ASNS, and CARS. Importantly, our results demonstrate the existence of cells in which NMD of all transcripts was efficient and others in which the NMD was less efficient. Downregulation of NMD in cells carrying the W1282X mutation increased the level of CFTR nonsense transcripts and enhanced the CFTR chloride channel activity in response to gentamicin. Together our results suggest that the efficiency of NMD might vary and hence have an important role in governing the response to treatments aiming to promote readthrough of PTCs in many genetic diseases.

Cystic fibrosis (CF; OMIM #219700) is a common autosomal recessive disease. The spectrum and frequency of CFTR mutations vary significantly in different populations and ethnic groups. A genetic epidemiological study was conducted in the indigenous ethnic group of people known as the Karachais. They live in the Republic of Karachay-Cherkessia, which lies in the northwest of Russia's North Caucasus region. Karachai's are Turkic-speaking and consist of 194 thousand people (approximately 40% of the population of the Republic). Molecular genetic analysis was performed in 10 unrelated Karachai families with CF patients from three districts in the Republic. A high frequency of W1282X mutation was found (18 of 20 mutant alleles): eight patients were homozygous for the W1282X mutation, and two were compound heterozygous (the second alleles were R1066C and R709X). Analysis for 13 common CF mutations in the sample of 142 healthy Karachais identified two 1677delTA and two W1282X mutation carriers. Thus, the most common CFTR mutation, F508del, was not detected among the CF patients or in healthy Karachais. The most frequent mutation among Karachai patients is W1282X (90%). Its frequency in healthy Karachais is approximately 0.007. Haplotype analysis using the CFTR intragene DNA markers IVS1CA, IVS6aGATT, IVS8CA and IVS17bCA showed that the origins of the W1282X mutation in Karachay-Cherkessia and the Eastern European part of Russia are different.

Recurrent genomic instability in cancer is attributed to positive selection and/or the sensitivity of specific genomic regions to breakage. Among these regions are fragile sites (FSs), genomic regions sensitive to replication stress conditions induced by the DNA polymerase inhibitor aphidicolin. However, the basis for the majority of cancer genomic instability hotspots remains unclear. Aberrant oncogene expression induces replication stress, leading to DNA breaks and genomic instability. Here we map the cytogenetic locations of oncogene-induced FSs and show that in the same cells, each oncogene creates a unique fragility landscape that only partially overlaps with aphidicolin-induced FSs. Oncogene-induced FSs colocalize with cancer breakpoints and large genes, similar to aphidicolin-induced FSs. The observed plasticity in the fragility landscape of the same cell type following oncogene expression highlights an additional level of complexity in the molecular basis for recurrent fragility in cancer.

Common fragile sites are specific loci that form gaps and constrictions on metaphase chromosomes exposed to replication stress, which slows DNA replication. These sites have a role in chromosomal rearrangements in tumors; however, the molecular mechanism of their expression is unclear. Here we show that replication stress leads to focus formation of Rad51 and phosphorylated DNA-PKcs, key components of the homologous recombination (HR) and nonhomologous end-joining (NHEJ), double-strand break (DSB) repair pathways, respectively. Down-regulation of Rad51, DNA-PKcs, or Ligase IV, an additional component of the NHEJ repair pathway, leads to a significant increase in fragile site expression under replication stress. Replication stress also results in focus formation of the DSB markers, MDC1 and γH2AX. These foci colocalized with those of Rad51 and phospho-DNA-PKcs. Furthermore, γH2AX and phospho-DNA-PKcs foci were localized at expressed fragile sites on metaphase chromosomes. These findings suggest that DSBs are formed at common fragile sites as a result of replication perturbation. The repair of these breaks by both HR and NHEJ pathways is essential for chromosomal stability at these sites.

The clinical spectrum of cystic fibrosis (CF) is influenced by the cystic fibrosis transmembrane conductance regulator (CFTR) genotype. However, variable courses of the disease were demonstrated among patients with identical genotypes. Since siblings share identical CFTR mutations and environmental factors, they can serve as a model to assess the effect of modifier genes on disease expression, and also to evaluate cross-infection. The aim of this study was to compare disease expression among siblings with CF. All sibling pairs treated at 7 CF centers in Israel were included in the study. Data were collected from patients' medical charts. Fifty families with at least 2 siblings were identified. As expected, the second-born sibling was diagnosed at an earlier age compared to the first-born. The mode of CF presentation at diagnosis showed significant familial concordance. In the families where the first sibling presented with gastrointestinal manifestations, 79% of the second siblings also presented with gastrointestinal manifestations. When gastrointestinal manifestations were absent in the first sibling, only 12% of the second siblings presented with gastrointestinal manifestations (P < 0.0001). Likewise, when the first sibling presented with respiratory symptoms, 60% of the second siblings presented with the similar symptoms. However, when the first sibling presented without respiratory symptoms, only 12% of the second siblings presented with respiratory symptoms (P < 0.001). Meconium ileus (MI) was present in 20 patients (21%). In 10 families where the first-born sibling had MI, 8 (80%) of the subsequent siblings had MI. On the other hand, in the 39 families where the first-born sibling did not have MI, only 2 (5%) subsequent siblings had MI (P < 0.001). Pancreatic insufficiency (PI) also had high familial concordance (P < 0.0001). Percentile growth for weights and heights and lung function (FVC, FEV(1), and FEF(25-75)) at ages 7 and 10 years were similar between siblings. P. aeruginosa grew from sputum in 89% of our study patients. When P. aeruginosa was isolated from the first-born patient, 91% of the second siblings were also positive for P. aeruginosa, whereas when the initial sibling was not a carrier of P. aeruginosa, only 50% of subsequent siblings were positive (P < 0.0001). This familial concordance was not observed for S. aureus. By contrast, the age of first isolation of P. aeruginosa and S. aureus was significantly earlier in the second sibling than in the first for the two bacteria: 10.3 +/- 5.1 vs. 7.3 +/- 5.2 years (P < 0.05) for P. aeruginosa, and 11.5 +/- 5.4 years vs. 6.8 +/- 5.1 years (P < 0.05) for S. aureus. CF siblings tend to share similar phenotypes that are not mutation-dependent. The lack of variability between siblings in mode of initial CF presentation, rates of MI, pulmonary function, and nutritional status supports the role of modifier genes in the determination of these factors.

Patients with normal or borderline sweat tests present a diagnostic challenge. In spite of the availability of genetic analysis and measurement of nasal potential difference, there is still uncertainty in diagnosing cystic fibrosis in some patients. CA 19-9 is a tumor-associated antigen whose levels were previously found to be elevated in some cystic fibrosis patients. We investigated whether serum CA 19-9 levels can contribute to establishing the diagnosis of cystic fibrosis in patients with a borderline sweat test, and evaluated the influence of different clinical variables on CA 19-9 levels. Serum CA 19-9 levels were measured in 82 cystic fibrosis patients grouped according to their genotype and in 38 healthy individuals. Group A included 50 patients who carried two mutations previously found to be associated with a pathological sweat test and pancreatic insufficiency (DeltaF508, W1282X, G542X, N1303K, and S549R). Group B included 13 compound heterozygote cystic fibrosis patients who carried one mutation known to cause mild disease with a borderline or normal sweat test and pancreatic sufficiency (3849+10kb C-->T, 5T). Group C included 38 normal controls. Nineteen cystic fibrosis patients carried at least one unidentified mutation. An association between CA 19-9 levels and age, pulmonary function, pancreatic status, sweat chloride, previous pancreatitis, serum lipase, meconium ileus, distal intestinal obstruction, liver disease, and diabetes was investigated. The distribution of CA 19-9 levels was significantly different between the three groups ( p

10−15% of mutations in the CFTR gene affect its correct splicing. The disease severity in patients carrying these mutations is highly variable, correlated with the level of aberrantly spliced transcripts. We aim to develop a splicing mutationspecific therapy using antisense oligonucleotides (AOs), to modulate the splicing pattern in patients carrying common splicing mutations. We focused on a common splicing mutation, the 3849+10kb C-to-T, which leads to inclusion of an 84 bp cryptic exon between exons 22−23 in the mature mRNA. This cryptic exon contains an in-frame stop codon that leads to degradation of a significant fraction of the mRNA by the NMD pathway as well as to the production of prematurely truncated nonfunctional proteins. We designed 4 2′-O-methyl phosphorothioatemodified AOs, targeted to prevent the recognition of enhancer splice motifs in the cryptic exon or to mask the junctions between this exon and its flanking sequences. Epithelial cell line established from a nasal polyp of a CF patient carrying the 3849+10kb C-to-T mutation was transfected with 2 of the designed AOs. AOstreated cells showed a highly significant decrease in the level of aberrantly spliced CFTR mRNA, along with a significant increase in normal spliced CFTR mRNA levels, reflecting transcripts that under normal conditions are degraded by the NMD pathway. These results indicate that AOs targeted to mask splicing motifs around the cryptic exon generated due to the 3849+10kb C-to-T splicing mutation, can modulate increase the correct splicing. Further studies are required to investigate whether these AOS can restore the CFTR function and improve patients’ clinical state. WS1.6 OligoG normalizes the CF mucus phenotype

The diagnosis of cystic fibrosis (CF) is based on characteristic clinical and laboratory findings. However, a subgroup of patients present with an atypical phenotype that comprises partial CF phenotype, borderline sweat tests and one or even no common cystic fibrosis transmembrane conductance regulator (CFTR) mutations. The aim of this study was to evaluate the role of nasal potential difference (PD) measurements in the diagnosis of CF patients with an atypical presentation and in a population of patients suspected to have CF.Nasal PD was measured in 162 patients from four different groups: patients with classical CF (n=31), atypical phenotype (n=11), controls (n=50), and patients with questionable CF (n=70). The parameter, or combination of nasal PD parameters was calculated in order to best discriminate all CF patients (including atypical CF) from the non-CF group.The patients with atypical CF disease had intermediate values of PD measurements between the CF and non-CF groups. The best discriminate model that assigned all atypical CF patients as CF used: e(response to chloride-free and isoproterenol/response to amiloride)with a cut-off >0.70 to predict a CF diagnosis. When this model was applied to the group of 70 patients with questionable CF, 24 patients had abnormal PD similar to the atypical CF group. These patients had higher levels of sweat chloride concentration and increased rate of CFTR mutations.Nasal potential difference is useful in diagnosis of patients with atypical cystic fibrosis. Taking into account both the sodium and chloride transport elements of the potential difference allows for better differentiation between atypical cystic fibrosis and noncystic fibrosis patients. This calculation may assist in the diagnostic work-up of patients whose diagnosis is questionable.

Objectives: This study assessed the phenotypic variability of LQTS in carriers with the same and with different mutations in the LQT2 gene. Background: Mutations of ion-channel genes are known to cause the long QT syndrome (LQTS), a disorder associated with distinctive genotypic-specific electrocardiographic patterns and variable clinical expression. Methods: Clinical and electrocardiographic characteristics were assessed in five large LQTS families, each with a different mutation of the HERG gene (LQT2; n = 469, 69% genotyped, 102 carriers). One mutation was located on the N-terminus and the other four on the C-terminus of the HERG channel protein. Results: The QTc duration and the frequency of cardiac events (syncope and LQTS-related cardiac arrest/deatht were similar among carriers with the five HERG mutations. QTc was as variable in carriers of the same mutation as it was among carriers with different HERG mutations (P = 0.19). Qualitative assessment of the electrocardiograms revealed extensive intra-and interfamilial variability in T-vvave morphology. Among carriers with multiple electrocardiograms extending over 2 to 7 years, variation in QTc over time was minimal. A strong association was found between QTc and the occurrence of cardiac events in carriers of all five mutations. Conclusions: The clinical expression of LQTS was equally variable in carriers from families with the same or different HERG mutations. These findings highlight the complexity of the clinical phenotype in this Mendelian dominant disorder and suggest that one or more modifier genes contribute to the variable expression of this syndrome. A.N.E. 2002;7(1):40–46

Background —Multiple mutations of SCN5A , the gene that encodes the human Na + channel α-subunit, are linked to 1 form of the congenital long-QT syndrome (LQT-3). D1790G (DG), an LQT-3 mutation of the C-terminal region of the Na + channel α-subunit, alters steady-state inactivation of expressed channels but does not promote sustained Na + channel activity. Recently, flecainide, but not lidocaine, has been found to correct the disease phenotype, delayed ventricular repolarization, in DG carriers. Methods and Results —To understand the molecular basis of this difference, we studied both drugs using wild-type (WT) and mutant Na + channels expressed in HEK 293 cells. The DG mutation conferred a higher sensitivity to lidocaine (EC 50 , WT=894 and DG=205 μmol/L) but not flecainide tonic block in a concentration range that is not clinically relevant. In contrast, in a concentration range that is therapeutically relevant, DG channels are blocked selectively by flecainide (EC 50 , WT=11.0 and DG=1.7 μmol/L), but not lidocaine (EC 50 , WT=318.0 and DG=176 μmol/L) during repetitive stimulation. Conclusions —These results (1) demonstrate that the DG mutation confers a unique pharmacological response on expressed channels; (2) suggest that flecainide use–dependent block of DG channels underlies its therapeutic effects in carriers of this gene mutation; and (3) suggest a role of the Na + channel α-subunit C-terminus in the flecainide/channel interaction.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene containing a premature termination signal are expected to produce little or no CFTR chloride channels. It has been shown in vitro, that aminoglycoside antibiotics can increase the frequency of erroneous insertion of nonsense codons hence permitting the translation of CFTR alleles carrying missense mutations to continue reading to the end of the gene. This led to the appearance of functional CFTR channels at the apical plasma membrane. The aim of this research was to determine if topical application of gentamicin to the nasal epithelium of patients with cystic fibrosis (CF) carrying stop mutations can express, in vivo, functional CFTR channels. Nine CF patients carrying stop mutations (mean age 23 +/- 11 yr, range 12 to 46 yr) received gentamicin drops (0.3%, 3 mg/ml) three times daily intranasally for a total of 14 d. Nasal potential difference (PD) was measured before and after the treatment. Before gentamicin application all the patients had abnormal nasal PD typical of CF. After gentamicin treatment, significant repolarization of the nasal epithelium representing chloride transport was increased from -1 +/- 1 mV to -10 +/- 11 mV (p < 0. 001). In conclusion, gentamicin may influence the underlying chloride transport abnormality in patients with CF carrying stop mutations.

Disease severity varies among cystic fibrosis (CF) patients carrying the same cystic fibrosis transmembrane conductance regulator (CFTR) genotype and among organs of the same individual. It has been shown that the class V splicing mutation 3849 + 10 kb C--T produces both normal and aberrantly spliced CFTR transcripts. We analyzed the levels of normal CFTR messenger RNA (mRNA) in different organs of an aborted fetus carrying the 3849 + 10 kb C--T mutation, and found that they correlated with the histopathologic changes observed in these organs. We performed semiquantitative nondifferential reverse transcription-polymerase chain reaction on several organs from a 22-wk aborted CF fetus carrying the 3849 + 10 kb C--T mutation. A very low level (1%) of normal CFTR mRNA was detected in the severely affected ileum of this fetus. Higher levels were found in the histopathologically unaffected trachea (17%), colon (19%), and lung (26%). Thus, as early as in utero, the regulation of alternative splice-site selection is an important mechanism underlying variable CF severity. Understanding of the mechanisms regulating alternative splicing in different tissues will contribute to potential therapy for patients carrying splicing mutations in CF and other human disease genes.

Determination of pancreatic function is essential in cystic fibrosis. The most-reliable method is by measuring pancreatic enzymes in the duodenum following intravenous or oral stimulation. However, this is invasive, time consuming, and expensive. Indirect tests are non-invasive but lack accuracy. This study examines a simple test which combines pancreatic stimulation by Lundh meal and sequential serum lipase measurements. The test was performed on three groups: group A, 36 cystic fibrosis patients carrying two mutations associated with severe disease and pancreatic insufficiency (delta F508, W1282X, G542X, N1303K, S549R); group B, 8 compound heterozygote cystic fibrosis patients carrying one mutation causing mild disease with pancreatic sufficiency (3849 + 10 kb C--T); group C, 17 healthy individuals. Basal lipase levels were 2-16.5, 16.4-73, and 8.5-27.8 U/l in groups A, B, and C, respectively, with some overlapping between groups. There were three patterns of lipase activity (1) consistently low levels (group A) suggested a severely affected insufficient pancreas; (2) normal basal levels followed by a linear rise peaking 30 min after the meal (found in 16 of 17 healthy individuals and 3 patients of group B) reflecting an unaffected sufficient pancreas; (3) elevated lipase levels not influenced by the meal (5 patients of group B). This reflects an ongoing destructive process in the pancreas which will eventually result in conversion from pancreatic sufficiency to pancreatic insufficiency. Hence serum lipase activity prior to and 30 min after Lundh meal is a good indicator of pancreatic status allowing categorization of cystic fibrosis patients as pancreatic insufficient, pancreatic sufficient, or pancreatic sufficient with late conversion to insufficiency.

Disease severity varies among cystic fibrosis (CF) patients carrying the same CFTR genotype. Here we studied the mechanism underlying disease variability in individuals carrying a splicing CFTR mutation, 3849+10 kb C--T. This mutation was shown to produce both correctly and aberrantly spliced CFTR transcripts containing an additional cryptic exon. Semiquantitative nondifferential RT-PCR showed considerable variability in the level (0-28%) of aberrantly spliced RNA transcribed from the 3849+10 kb C--T mutation in nasal epithelium from 10 patients. A significant inverse correlation was found between the level of the aberrantly spliced CFTR transcripts and pulmonary function, expressed as FEV1 (r = 0.92, P0.0001). Patients with normal pulmonary function (FEV180% predicted) had lower levels of aberrantly spliced CFTR RNA (0 to 3%) than those with FEV180%, (9 to 28% aberrantly spliced RNA). Only aberrantly spliced CFTR RNA was detected in the lung of a patient with severe lung disease who underwent lung transplantation. Our results show that the severity of CF lung disease correlates with insufficiency of normal CFTR RNA. Thus, the regulation of alternative splice site selection may be an important mechanism underlying partial penetrance in CF. Further understanding of this regulation will contribute to potential therapy for patients carrying splicing mutations in human disease genes.