Batsheva Kerem

Hebrew University of Jerusalem | HUJI · Department of Genetics

Background CFTR protein structures bound to each of Elexacaftor/Tezacaftor/Ivacaftor (ETI) were recently established. We aimed to use this data to predict and assess responses to ETI and each of its components in intestinal organoids derived from patients carrying rare CFTR mutations, not yet approved for ETI, based on their mutation location withi...

Common fragile sites (CFS) are specific genomic regions prone to chromosomal instability under conditions of DNA replication stress. CFSs manifest as breaks, gaps, and constrictions on metaphase chromosomes under mild replication stress. These replication-sensitive CFS regions are preferentially unstable during cancer development, as reflected by t...

Recent advances in the therapeutic potential of RNA-related treatments, specifically for antisense oligonucleotide (ASO)-based drugs, have led 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 delive...

Background: Elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) significantly improves health outcomes in people with cystic fibrosis (pwCF) carrying one or two F508del mutations. According to in vitro assays performed in FRT cells, 178 additional mutations respond to ELX/TEZ/IVA. The N1303K mutation is not included in this list of mutations. Recent in...

Most people with Cystic Fibrosis (PwCF) harbor Cystic Fibrosis Transmembrane Conductance (CFTR) mutations that respond to highly effective CFTR modulators (HEM); however, a small fraction of non-responsive variants will require alternative approaches for treatment. Furthermore, the long-term goal to develop a cure for CF will require novel therapeu...

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 Fibros...

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 m...

Background 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 pro...

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 F...

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...

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. Oncogene expression triggers replicative stress that can lead to genetic instability, driving cancer progression. Thus, revealing the molecular basis for oncogene-induced replication...

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 tr...

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 br...

Common fragile sites (CFSs) are genomic regions prone to breakage under replication stress conditions recurrently rearranged in cancer. Many CFSs are enriched with AT-dinucleotide rich sequences (AT-DRSs) which have the potential to form stable secondary structures upon unwinding the double helix during DNA replication. These stable structures can...

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 underlyi...

Common fragile sites (CFSs) are specific genomic regions in normal chromosomes that exhibit genomic instability under DNA replication stress. Since 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 un...

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 progress...

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

Aneuploidy is a hallmark of cancer and underlies genetic disorders characterized by severe developmental defects, yet the molecular mechanisms explaining its effects on cellular physiology remain elusive. Here we show, using a series of human cells with defined aneuploid karyotypes, that gain of a single chromosome increases genomic instability. Ne...

Supplementary Figures 1-10 and Supplementary Tables 1-2

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. Her...

Oncogene expression can lead to replication stress and genome instability. Recently, we identified oncogene-induced fragile sites (FSs) and revealed that the landscape of recurrent fragility in the same cell type is dynamic. This implies an additional level of complexity in the molecular basis of recurrent fragility in cancer.

Chromosomal instability in early cancer stages is caused by replication stress. One mechanism by which oncogene expression induces replication stress is to drive cell proliferation with insufficient nucleotide levels. Cancer development is driven by alterations in both genetic and environmental factors. Here, we investigated whether replication str...

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...

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...

Colorectal cancer (CRC) develops in a sequential, evolutionary process, leading to a heterogenic tumor. Comprehensive molecular studies of CRC have been previously performed; still the process of carcinogenesis isn't fully understood. We utilized gene expression patterns from 94 samples including normal, adenoma and adenocarcinoma colon biopsies, a...

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 revea...

Personalized medicine promises that medical decisions, practices and products are tailored to the individual patient. Cystic fibrosis, an inherited disorder of ion transport in exocrine glands, is the first successful example of customized drug development for mutation-specific therapy. This monograph reports on the contributions of the basic and c...

Genome instability is a hallmark of cancer. Common fragile sites (CFSs) are specific regions in the human genome that are sensitive to replication stress and are prone to genomic instability in different cancer types. Here we molecularly cloned a new CFS, FRA11H, in 11q13. The genomic region of FRA11H harbors a hotspot of chromosomal breakpoints fo...

Supplementary Figure and Tables

Despite recent progress in defining the ciliome, the genetic basis for many cases of primary ciliary dyskinesia (PCD) remains elusive. We evaluated five children from two unrelated, consanguineous Palestinian families who had PCD with typical clinical features, reduced nasal nitric oxide concentrations, and absent dynein arms. Linkage analyses reve...

RT PCR analysis of LRRC6 expression in RNAi silenced cells. (A) LRRC6 expression in LRRC6-specific shRNA transfected airway epithelial cells (B) Immunoblot analyses of airway epithelial cells transfected with three different LRRC6-specific shRNA or non-targeted shRNA (NT) sequences and nontransfected control cells (M). (C) En face images of LRRC6 i...

Perturbed DNA replication in early stages of cancer development induces chromosomal instability preferentially at fragile sites. However, the molecular basis for this instability is unknown. Using DNA combing, we studied the replication dynamics along two common fragile sites on chromosome 16, FRA16C and FRA16D. We found in FRA16C that under normal...

Common fragile sites (CFSs) were characterized almost 30 years ago as sites undergoing genomic instability in cancer. Recently, in vitro studies have found that oncogene-induced replication stress leads to CFS instability. In vivo, CFSs were found to be preferentially unstable during early stages of cancer development and to leave a unique signatur...

Perturbed DNA replication in early stages of cancer development induces chromosomal instability preferentially at fragile sites. However, the molecular basis for this instability is unknown. Here, we show that even under normal growth conditions, replication fork progression along the fragile site, FRA16C, is slow and forks frequently stall at AT-r...

Several diseases have been clinically or genetically related to cystic fibrosis (CF), but a consensus definition is lacking. Here, we present a proposal for consensus guidelines on cystic fibrosis transmembrane conductance regulator (CFTR)-related disorders (CFTR-RDs), reached after expert discussion and two dedicated workshops. A CFTR-RD may be de...

Chromosomal instability in early cancer stages is caused by stress on DNA replication. The molecular basis for replication perturbation in this context is currently unknown. We studied the replication dynamics in cells in which a regulator of S phase entry and cell proliferation, the Rb-E2F pathway, is aberrantly activated. Aberrant activation of t...

Approximately one-third of the alleles causing genetic diseases carry premature termination codons (PTCs). Therapeutic approaches for mutations generating in-frame PTCs are aimed at promoting translational readthrough of the PTC, to enable the synthesis and expression of full-length functional proteins. Interestingly, readthrough studies in tissue...

Introduction: Molecular combing is a process whereby many molecules of DNA (hundreds of kb) are identically stretched and aligned on a silanized glass surface, in a size and sequence independent manner. The addition of nucleotide analogs (for example, CldU and IdU) to cell cultures, allows detection of the replication pattern at different sites in...

Non-Homologous End Joining (NHEJ) is one of the two major pathways of DNA Double Strand Breaks (DSBs) repair. Mutations in human NHEJ genes can lead to immunodeficiency due to its role in V(D)J recombination in the immune system. In addition, most patients carrying mutations in NHEJ genes display developmental anomalies which are likely the result...

Approximately one-third of alleles causing genetic diseases carry premature termination codons (PTCs), which lead to the production of truncated proteins. The past decade has seen considerable interest in therapeutic approaches aimed at readthrough of in-frame PTCs to enable synthesis of full-length proteins. However, attempts to readthrough PTCs i...

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 t...

It is often challenging for the clinician interested in cystic fibrosis (CF) to interpret molecular genetic results, and to integrate them in the diagnostic process. The limitations of genotyping technology, the choice of mutations to be tested, and the clinical context in which the test is administered can all influence how genetic information is...

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, inc...

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 vari...

Fragile sites are specific genomic loci that form gaps, constrictions and breaks on chromosomes exposed to replication stress conditions. In the father of a patient with Beckwith-Wiedemann syndrome and a pure truncation of 18q22-qter, a new aphidicolin-sensitive fragile site on chromosome 18q22.2 (FRA18C) is described. The region in 18q22 appears h...

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 w...