Restoration Of Gene Function Research Articles

Rapid functional activation of horizontally transferred eukaryotic intron-containing genes in the bacterial recipient.

Horizontal gene transfer has occurred across all domains of life and contributed substantially to the evolution of both prokaryotes and eukaryotes. Previous studies suggest that many horizontally transferred eukaryotic genes conferred selective advantages to bacterial recipients, but how these eukaryotic genes evolved into functional bacterial genes remained unclear, particularly how bacteria overcome the expressional barrier posed by eukaryotic introns. Here, we first confirmed that the presence of intron would inactivate the horizontally transferred gene in Escherichiacoli even if this gene could be efficiently transcribed. Subsequent large-scale genetic screens for activation of gene function revealed that activation events could rapidly occur within several days of selective cultivation. Molecular analysis of activation events uncovered two distinct mechanisms how bacteria overcome the intron barrier: (i) intron was partially deleted and the resulting stop codon-removed mutation led to one intact foreign protein or (ii) intron was intactly retained but it mediated the translation initiation and the interaction of two split small proteins (derived from coding sequences up- and downstream of intron, respectively) to restore gene function. Our findings underscore the likelihood that horizontally transferred eukaryotic intron-containing genes could rapidly acquire functionality if they confer a selective advantage to the prokaryotic recipient.

Repair of CRISPR-guided RNA breaks enables site-specific RNA excision in human cells.

Genome editing with CRISPR RNA-guided endonucleases generates DNA breaks that are resolved by cellular DNA repair machinery. However, analogous methods to manipulate RNA remain unavailable. We show that site-specific RNA breaks generated with type-III CRISPR complexes are repaired in human cells and that this repair can be used for programmable deletions in human transcripts to restore gene function. Collectively, this work establishes a technology for precise RNA manipulation with potential therapeutic applications.

Molecular and clinical aspects relevant for counseling individuals with clonal hematopoiesis of indeterminate potential.

Clonal hematopoiesis of indeterminate potential (CHIP) has fascinated the medical community for some time. Discovered about a decade ago, this phenomenon links age-related alterations in hematopoiesis not only to the later development of hematological malignancies but also to an increased risk of early-onset cardiovascular disease and some other disorders. CHIP is detected in the blood and is characterized by clonally expanded somatic mutations in cancer-associated genes, predisposing to the development of hematologic neoplasms such as MDS and AML. CHIP-associated mutations often involve DNA damage repair genes and are frequently observed following prior cytotoxic cancer therapy. Genetic predisposition seems to be a contributing factor. It came as a surprise that CHIP significantly elevates the risk of myocardial infarction and stroke, and also contributes to heart failure and pulmonary hypertension. Meanwhile, evidence of mutant clonal macrophages in vessel walls and organ parenchyma helps to explain the pathophysiology. Besides aging, there are some risk factors promoting the appearance of CHIP, such as smoking, chronic inflammation, chronic sleep deprivation, and high birth weight. This article describes fundamental aspects of CHIP and explains its association with hematologic malignancies, cardiovascular disorders, and other medical conditions, while also exploring potential progress in the clinical management of affected individuals. While it is important to diagnose conditions that can lead to adverse, but potentially preventable, effects, it is equally important not to stress patients by confronting them with disconcerting findings that cannot be remedied. Individuals with diagnosed or suspected CHIP should receive counseling in a specialized outpatient clinic, where professionals from relevant medical specialties may help them to avoid the development of CHIP-related health problems. Unfortunately, useful treatments and clinical guidelines for managing CHIP are still largely lacking. However, there are some promising approaches regarding the management of cardiovascular disease risk. In the future, strategies aimed at restoration of gene function or inhibition of inflammatory mediators may become an option.

Genetic conversion of a split-drive into a full-drive element

The core components of CRISPR-based gene drives, Cas9 and guide RNA (gRNA), either can be linked within a self-contained single cassette (full gene-drive, fGD) or be provided in two separate elements (split gene-drive, sGD), the latter offering greater control options. We previously engineered split systems that could be converted genetically into autonomous full drives. Here, we examine such dual systems inserted at the spo11 locus that are recoded to restore gene function and thus organismic fertility. Despite minimal differences in transmission efficiency of the sGD or fGD drive elements in single generation crosses, the reconstituted spo11 fGD cassette surprisingly exhibits slower initial drive kinetics than the unlinked sGD element in multigenerational cage studies, but then eventually catches up to achieve a similar level of final introduction. These unexpected kinetic behaviors most likely reflect differing transient fitness costs associated with individuals co-inheriting Cas9 and gRNA transgenes during the drive process.

Circulating Plasma miRNA Homologs in Mice and Humans Reflect Familial Cerebral Cavernous Malformation Disease.

Patients with familial cerebral cavernous malformation (CCM) inherit germline loss of function mutations and are susceptible to progressive development of brain lesions and neurological sequelae during their lifetime. To date, no homologous circulating molecules have been identified that can reflect the presence of germ line pathogenetic CCM mutations, either in animal models or patients. We hypothesize that homologous differentially expressed (DE) plasma miRNAs can reflect the CCM germline mutation in preclinical murine models and patients. Herein, homologous DE plasma miRNAs with mechanistic putative gene targets within the transcriptome of preclinical and human CCM lesions were identified. Several of these gene targets were additionally found to be associated with CCM-enriched pathways identified using the Kyoto Encyclopedia of Genes and Genomes. DE miRNAs were also identified in familial-CCM patients who developed new brain lesions within the year following blood sample collection. The miRNome results were then validated in an independent cohort of human subjects with real-time-qPCR quantification, a technique facilitating plasma assays. Finally, a Bayesian-informed machine learning approach showed that a combination of plasma levels of miRNAs and circulating proteins improves the association with familial-CCM disease in human subjects to 95% accuracy. These findings act as an important proof of concept for the future development of translatable circulating biomarkers to be tested in preclinical studies and human trials aimed at monitoring and restoring gene function in CCM and other diseases.

Rapid assessment of the temporal function and phenotypic reversibility of neurodevelopmental disorder risk genes in Caenorhabditis elegans.

ABSTRACTRecent studies have indicated that some phenotypes caused by decreased function of select neurodevelopmental disorder (NDD) risk genes can be reversed by restoring gene function in adulthood. However, few of the hundreds of risk genes have been assessed for adult phenotypic reversibility. We developed a strategy to rapidly assess the temporal requirements and phenotypic reversibility of NDD risk gene orthologs using a conditional protein degradation system and machine-vision phenotypic profiling in Caenorhabditis elegans. We measured how degrading and re-expressing orthologs of EBF3, BRN3A and DYNC1H1 at multiple periods throughout development affect 30 morphological, locomotor, sensory and learning phenotypes. We found that phenotypic reversibility was possible for each gene studied. However, the temporal requirements of gene function and degree of rescue varied by gene and phenotype. This work highlights the critical need to assess multiple windows of degradation and re-expression and a large number of phenotypes to understand the many roles a gene can have across the lifespan. This work also demonstrates the benefits of using a high-throughput model system to prioritize NDD risk genes for re-expression studies in other organisms.

AAV-delivered suppressor tRNA overcomes a nonsense mutation in mice.

Gene therapy is a potentially curative medicine for many currently untreatable diseases, and recombinant adeno-associated virus (rAAV)is the most successful gene delivery vehicle for in vivo applications1-3. However, rAAV-based gene therapy suffers from several limitations, such as constrained DNA cargo size and toxicities caused by non-physiological expression of a transgene4-6. Here we show that rAAV delivery of a suppressor tRNA (rAAV.sup-tRNA) safely and efficiently rescued a genetic disease in a mouse model carrying a nonsense mutation, and effects lasted for more than 6 months after a single treatment. Mechanistically, this was achieved through a synergistic effect of premature stop codon readthrough and inhibition of nonsense-mediated mRNA decay. rAAV.sup-tRNA had a limited effect on global readthrough at normal stop codons and did not perturb endogenous tRNA homeostasis, as determined by ribosome profiling and tRNA sequencing, respectively. By optimizing the AAV capsid and the route of administration, therapeutic efficacy in various target tissues was achieved, including liver, heart, skeletal muscle and brain. This study demonstrates the feasibility of developing a toolbox of AAV-delivered nonsense suppressor tRNAs operating on premature termination codons (AAV-NoSTOP) to rescue pathogenic nonsense mutations and restore gene function under endogenous regulation. As nonsense mutations account for 11% of pathogenic mutations, AAV-NoSTOP can benefit a large number of patients. AAV-NoSTOP obviates the need to deliver a full-length protein-coding gene that may exceed the rAAV packaging limit, elicit adverse immune responses or cause transgene-related toxicities. It therefore represents a valuable addition to gene therapeutics.

Progress in Gene Editing Tools and Their Potential for Correcting Mutations Underlying Hearing and Vision Loss.

Blindness and deafness are the most frequent sensory disorders in humans. Whatever their cause — genetic, environmental, or due to toxic agents, or aging — the deterioration of these senses is often linked to irreversible damage to the light-sensing photoreceptor cells (blindness) and/or the mechanosensitive hair cells (deafness). Efforts are increasingly focused on preventing disease progression by correcting or replacing the blindness and deafness-causal pathogenic alleles. In recent years, gene replacement therapies for rare monogenic disorders of the retina have given positive results, leading to the marketing of the first gene therapy product for a form of childhood hereditary blindness. Promising results, with a partial restoration of auditory function, have also been reported in preclinical models of human deafness. Silencing approaches, including antisense oligonucleotides, adeno-associated virus (AAV)–mediated microRNA delivery, and genome-editing approaches have also been applied to various genetic forms of blindness and deafness The discovery of new DNA- and RNA-based CRISPR/Cas nucleases, and the new generations of base, prime, and RNA editors offers new possibilities for directly repairing point mutations and therapeutically restoring gene function. Thanks to easy access and immune-privilege status of self-contained compartments, the eye and the ear continue to be at the forefront of developing therapies for genetic diseases. Here, we review the ongoing applications and achievements of this new class of emerging therapeutics in the sensory organs of vision and hearing, highlighting the challenges ahead and the solutions to be overcome for their successful therapeutic application in vivo.

Machine learning based CRISPR gRNA design for therapeutic exon skipping.

Restoring gene function by the induced skipping of deleterious exons has been shown to be effective for treating genetic disorders. However, many of the clinically successful therapies for exon skipping are transient oligonucleotide-based treatments that require frequent dosing. CRISPR-Cas9 based genome editing that causes exon skipping is a promising therapeutic modality that may offer permanent alleviation of genetic disease. We show that machine learning can select Cas9 guide RNAs that disrupt splice acceptors and cause the skipping of targeted exons. We experimentally measured the exon skipping frequencies of a diverse genome-integrated library of 791 splice sequences targeted by 1,063 guide RNAs in mouse embryonic stem cells. We found that our method, SkipGuide, is able to identify effective guide RNAs with a precision of 0.68 (50% threshold predicted exon skipping frequency) and 0.93 (70% threshold predicted exon skipping frequency). We anticipate that SkipGuide will be useful for selecting guide RNA candidates for evaluation of CRISPR-Cas9-mediated exon skipping therapy.

Protein functionality as a potential bottleneck for somatic revertant variants

Somatic genome stability has been considered the rule, rather than the exception; yet, somatic mutations are driving factors in many diseases with a genetic background. Conversely, somatic reversion of germline mutations has been shown to mitigate pathogenicity by partially or completely restoring gene function. Such somatic revertant variants have been observed across a broad spectrum of immunodeficiencies and often result in atypical phenotypes that present a diagnostic and therapeutic challenge.

Novel Non-integrating DNA Nano-S/MAR Vectors Restore Gene Function in Isogenic Patient-Derived Pancreatic Tumor Models

We describe herein non-integrating minimally sized nano-S/MAR DNA vectors, which can be used to genetically modify dividing cells in place of integrating vectors. They represent a unique genetic tool, which avoids vector-mediated damage. Previous work has shown that DNA vectors comprising a mammalian S/MAR element can provide persistent mitotic stability over hundreds of cell divisions, resisting epigenetic silencing and thereby allowing sustained transgene expression. The composition of the original S/MAR vectors does present some inherent limitations that can provoke cellular toxicity. Herein, we present a new system, the nano-S/MAR, which drives higher transgene expression and has improved efficiency of establishment, due to the minimal impact on cellular processes and perturbation of the endogenous transcriptome. We show that these features enable the hitherto challenging genetic modification of patient-derived cells to stably restore the tumor suppressor gene SMAD4 to a patient-derived SMAD4 knockout pancreatic cancer line. Nano-S/MAR modification does not alter the molecular or phenotypic integrity of the patient-derived cells in cell culture and xenograft mouse models. In conclusion, we show that these DNA vectors can be used to persistently modify a range of cells, providing sustained transgene expression while avoiding the risks of insertional mutagenesis and other vector-mediated toxicity.

Editing aberrant splice sites efficiently restores β-globin expression in β-thalassemia

AbstractThe thalassemias are compelling targets for therapeutic genome editing in part because monoallelic correction of a subset of hematopoietic stem cells (HSCs) would be sufficient for enduring disease amelioration. A primary challenge is the development of efficient repair strategies that are effective in HSCs. Here, we demonstrate that allelic disruption of aberrant splice sites, one of the major classes of thalassemia mutations, is a robust approach to restore gene function. We target the IVS1-110G>A mutation using Cas9 ribonucleoprotein (RNP) and the IVS2-654C>T mutation by Cas12a/Cpf1 RNP in primary CD34+ hematopoietic stem and progenitor cells (HSPCs) from β-thalassemia patients. Each of these nuclease complexes achieves high efficiency and penetrance of therapeutic edits. Erythroid progeny of edited patient HSPCs show reversal of aberrant splicing and restoration of β-globin expression. This strategy could enable correction of a substantial fraction of transfusion-dependent β-thalassemia genotypes with currently available gene-editing technology.

Functional Screening for Pathways Regulating Chemoresistance and Sensitivity in Leukemia Stem Cells

INTRODUCTIONOn average 5 recurrent mutations are present in each patient with acute myeloid leukemia (AML). Many mutated genes are implicated as tumor suppressor genes, but their contribution to leukemia stem cell (LSC) survival and chemoresistance is often unknown. We hypothesized that ectopic expression of the wildtype sequence of these genes will restore the function of the tumor suppressor gene and will lead to reduced clonal expansion and increased chemosensitivity.AIMTo evaluate the contribution of recurrently mutated genes to leukemia stem cell survival and chemoresistance in human AML cells in vitro and in vivo.METHODSWe performed a loss-of-function screening in primary human AML cells and cell lines by lentiviral expression of a pool of 22 wildtype genes associated with AML pathogenesis, which can restore gene function of a repressed pathway or a dysfunctional tumor suppressor gene. The 22 full-length cDNAs were labelled with a genetic barcode, which can be amplified with a common primer for all 22 genes. The readout of the screening was reduced representation of the barcode DNA after in vitro culture or in vivo growth in patient-derived xenograft (PDX) models, which was amplified from DNA and quantified by next-generation sequencing (NGS).Five PDX models with favorable, normal or complex cytogenetics and 3-5 recurrent mutations per model were screened in order to identify cDNAs that could potentially limit the proliferative capacity of LSCs in vivo (5 mice per model). Nine to 16 weeks after transplantation, DNA from blood, bone marrow and spleen were analyzed by NGS. We also screened two CD34+-enriched cord blood samples in vitro. After transduction with the cDNA pool, cells were cultured for 11 days and the barcode representation was analyzed by NGS at days 2, 4, 7, 9 and 11.Finally, we screened the human leukemia cell lines U937and PB14, a newly established cell line from an AML patient with mutations in FLT3, NPM1, RAD21, GSE1, and ROBO2. Cells were treated with cytarabine, doxorubicin or venetoclax for a 3-day period, followed by a 4-day recovery period to allow outgrowth of resistant clones and accumulation of cDNAs that conferred drug resistance.RESULTSOur loss-of-function screening with overexpressed wildtype genes revealed that expression of ETV6 and PTPN11 depleted LSCs in 3 of 5 PDX models and that expression of CEBPA and KDM6A depleted the progeny of normal CD34+ cells in cord blood. ASXL1, EZH2, CUX1, SMC1A and SMC3 had a general negative effect on stem cell self-renewal in both leukemic and normal CD34+ cells.Relative frequencies of the leukemia-specific genes ETV6 and PTPN11 were reduced 3 to 16-fold and 2 to 3-fold, respectively. Both genes were not mutated in the 3 patients' diagnostic samples, but had reduced RNA expression by 10-70% compared to healthy control peripheral blood mononuclear cells. Relative frequencies of cord blood-specific CEBPA was reduced 4-fold and KDM6A 5-fold.We then evaluated whether activation of a repressed pathway can increase sensitivity to cytarabine, doxorubicin or venetoclax in U937 and PB14 cells after 1 or 2 weeks of treatment. All three drugs showed better cytotoxic effects upon p53 expression in both cell lines by a factor of 1.4 to 2.5 fold. Cytarabine and venetoclax improved elimination of leukemic cells that had been transduced with U2AF1 in PB14 cells, which are U2AF1 wildtype. Venetoclax improved elimination of U937 cells that had been transduced with ETV6 or KDM6A, which are wildtype for these genes, while RNA expression was reduced more than 50% in these cells compared to other leukemic cell lines (NB4 and MV4-11).CONCLUSIONFunctional cDNA screening in PDX models in vivo is feasible and can reveal selective vulnerabilities of leukemic compared to normal stem cells. Our approach was validated by the finding that p53 expression improved chemosensitivity for all drugs tested in two leukemia cell lines, which is expected from the known function of p53 as a critical tumor suppressor gene.Similarly, overexpression of the transcriptional corepressor ETV6 in leukemia cells with low ETV6 expression was found to inhibit leukemia stem cell proliferation in vivo and to sensitize U937 cells to venetoclax. Therefore, activation of ETV6 should be explored as a novel strategy to inhibit LSCs and improve treatment response. DisclosuresGanser:Novartis: Membership on an entity's Board of Directors or advisory committees. Heuser:Janssen: Consultancy; StemLine Therapeutics: Consultancy; Bayer Pharma AG: Consultancy, Research Funding; Tetralogic: Research Funding; Sunesis: Research Funding; Daiichi Sankyo: Research Funding; Karyopharm: Research Funding; BergenBio: Research Funding; Astellas: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding.

Octopamine and Tyramine Contribute Separately to the Counter-Regulatory Response to Sugar Deficit in Drosophila.

All animals constantly negotiate external with internal demands before and during action selection. Energy homeostasis is a major internal factor biasing action selection. For instance, in addition to physiologically regulating carbohydrate mobilization, starvation-induced sugar shortage also biases action selection toward food-seeking and food consumption behaviors (the counter-regulatory response). Biogenic amines are often involved when such widespread behavioral biases need to be orchestrated. In mammals, norepinephrine (noradrenalin) is involved in the counterregulatory response to starvation-induced drops in glucose levels. The invertebrate homolog of noradrenalin, octopamine (OA) and its precursor tyramine (TA) are neuromodulators operating in many different neuronal and physiological processes. Tyrosine-ß-hydroxylase (tßh) mutants are unable to convert TA into OA. We hypothesized that tßh mutant flies may be aberrant in some or all of the counter-regulatory responses to starvation and that techniques restoring gene function or amine signaling may elucidate potential mechanisms and sites of action. Corroborating our hypothesis, starved mutants show a reduced sugar response and their hemolymph sugar concentration is elevated compared to control flies. When starved, they survive longer. Temporally controlled rescue experiments revealed an action of the OA/TA-system during the sugar response, while spatially controlled rescue experiments suggest actions also outside of the nervous system. Additionally, the analysis of two OA- and four TA-receptor mutants suggests an involvement of both receptor types in the animals' physiological and neuronal response to starvation. These results complement the investigations in Apis mellifera described in our companion paper (Buckemüller et al., 2017).

Primary Ciliary Dyskinesia: An Update on Clinical Aspects, Genetics, Diagnosis, and Future Treatment Strategies.

Primary ciliary dyskinesia (PCD) is an orphan disease (MIM 244400), autosomal recessive inherited, characterized by motile ciliary dysfunction. The estimated prevalence of PCD is 1:10,000 to 1:20,000 live-born children, but true prevalence could be even higher. PCD is characterized by chronic upper and lower respiratory tract disease, infertility/ectopic pregnancy, and situs anomalies, that occur in ≈50% of PCD patients (Kartagener syndrome), and these may be associated with congenital heart abnormalities. Most patients report a daily year-round wet cough or nose congestion starting in the first year of life. Daily wet cough, associated with recurrent infections exacerbations, results in the development of chronic suppurative lung disease, with localized-to-diffuse bronchiectasis. No diagnostic test is perfect for confirming PCD. Diagnosis can be challenging and relies on a combination of clinical data, nasal nitric oxide levels plus cilia ultrastructure and function analysis. Adjunctive tests include genetic analysis and repeated tests in ciliary culture specimens. There are currently 33 known genes associated with PCD and correlations between genotype and ultrastructural defects have been increasingly demonstrated. Comprehensive genetic testing may hopefully screen young infants before symptoms occur, thus improving survival. Recent surprising advances in PCD genetic designed a novel approach called “gene editing” to restore gene function and normalize ciliary motility, opening up new avenues for treating PCD. Currently, there are no data from randomized clinical trials to support any specific treatment, thus, management strategies are usually extrapolated from cystic fibrosis. The goal of treatment is to prevent exacerbations, slowing the progression of lung disease. The therapeutic mainstay includes airway clearance maneuvers mainly with nebulized hypertonic saline and chest physiotherapy, and prompt and aggressive administration of antibiotics. Standardized care at specialized centers using a multidisciplinary approach that imposes surveillance of lung function and of airway biofilm composition likely improves patients’ outcome. Pediatricians, neonatologists, pulmonologists, and ENT surgeons should maintain high awareness of PCD and refer patients to the specialized center before sustained irreversible lung damage develops. The recent creation of a network of PCD clinical centers, focusing on improving diagnosis and treatment, will hopefully help to improve care and knowledge of PCD patients.

Gene based therapies for kidney regeneration

In this review we provide an overview of the expanding molecular toolbox that is available for gene based therapies and how these therapies can be used for a large variety of kidney diseases. Gene based therapies range from restoring gene function in genetic kidney diseases to steering complex molecular pathways in chronic kidney disorders, and can provide a treatment or cure for diseases that otherwise may not be targeted. This approach involves the delivery of recombinant DNA sequences harboring therapeutic genes to improve cell function and thereby promote kidney regeneration. Depending on the therapy, the recombinant DNA will express a gene that directly plays a role in the function of the cell (gene addition), that regulates the expression of an endogenous gene (gene regulation), or that even changes the DNA sequence of endogenous genes (gene editing). Some interventions involve permanent changes in the genome whereas others are only temporary and leave no trace. Efficient and safe delivery are important steps for all gene based therapies and also depend on the mode of action of the therapeutic gene. Here we provide examples on how the different methods can be used to treat various diseases, which technologies are now emerging (such as gene repair through CRISPR/Cas9) and what the opportunities, perspectives, potential and the limitations of these therapies are for the treatment of kidney diseases.

Abstract 3988: Detection of novel t(12;17)(p12;p13) in treatment-refractory/relapsed acute myeloid leukemia by anchored multiplex PCR(AMP)-based next-generation sequencing

Abstract Gene fusions are an important class of mutations and have been shown to drive many genetic diseases. While several technologies can be used to detect fusions, anchored multiplex PCR (AMP) next generation sequencing (NGS)-based detection offers the advantages of novel fusion detection and the ability to multiplex multitudinous genes. Here we report application of this technology in the evaluation of a 56-year-old man diagnosed with AML (2013). He was treated initially with Vidaza and later with CLAG-M induction (2014) with complete response and consolidation CLA (2015). He relapsed (4/2015) and was reinduced with 7+3 (Ida). He relapsed again and was reinduced with FCT but bone marrow biopsy showed persistent disease (75% blasts). Karyotype showed 46, XY, t(12;17)(p12;p13)[20]. It was postulated that ETV6 oncogene was involved based on 12p13 locus. The partner gene was unknown in this patient with multiply relapsed/refractory disease. AMP technology (Archer FusionPlex) confirmed involvement of the expected ETV6. However, more importantly, employing this technology lead to the discovery of a novel fusion partner, HIC1. The identity of the novel fusion partners was supported by the breakpoints originally observed by karyotype analysis. AMP promotes discovery of novel fusions by targeting one (known) of two genes involved in the fusion event. HIC1 regulates cellular growth and serves as a tumor suppressor gene. Inactivation of HIC1 has been associated with aggressive disease and poor survival in other cancer types. Interestingly, in vitro restoration of gene function by 5-aza-dC resulted in decreased cell proliferation and tumor aggressiveness in pancreatic cancer and head and neck squamous cell carcinoma suggesting the prospect of a therapeutic target. This discovery provides new insight into a possible leukemogenic pathway in AML and potential for targeted therapy. Citation Format: Laura Johnson, Katelyn Trifilo, Helen Wang, Brian Kudlow, Eric Padron, Peter R. Pappenhausen, Mohammad Hussaini. Detection of novel t(12;17)(p12;p13) in treatment-refractory/relapsed acute myeloid leukemia by anchored multiplex PCR(AMP)-based next-generation sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3988.

Novel small molecules potentiate premature termination codon readthrough by aminoglycosides.

Nonsense mutations introduce premature termination codons and underlie 11% of genetic disease cases. High concentrations of aminoglycosides can restore gene function by eliciting premature termination codon readthrough but with low efficiency. Using a high-throughput screen, we identified compounds that potentiate readthrough by aminoglycosides at multiple nonsense alleles in yeast. Chemical optimization generated phthalimide derivative CDX5-1 with activity in human cells. Alone, CDX5-1 did not induce readthrough or increase TP53 mRNA levels in HDQ-P1 cancer cells with a homozygous TP53 nonsense mutation. However, in combination with aminoglycoside G418, it enhanced readthrough up to 180-fold over G418 alone. The combination also increased readthrough at all three nonsense codons in cancer cells with other TP53 nonsense mutations, as well as in cells from rare genetic disease patients with nonsense mutations in the CLN2, SMARCAL1 and DMD genes. These findings open up the possibility of treating patients across a spectrum of genetic diseases caused by nonsense mutations.

Genome- and Cell-Based Strategies in Therapy of Muscular Dystrophies.

Muscular dystrophies are a group of heterogeneous genetic disorders characterized by progressive loss of skeletal muscle mass. Depending on the muscular dystrophy, the muscle weakness varies in degree of severity. The majority of myopathies are due to genetic events leading to a loss of function of key genes involved in muscle function. Although there is until now no curative treatment to stop the progression of most myopathies, a significant number of experimental gene- and cell-based strategies and approaches have been and are being tested in vitro and in animal models, aiming to restore gene function. Genome editing using programmable endonucleases is a powerful tool for modifying target genome sequences and has been extensively used over the last decade to correct in vitro genetic defects of many single-gene diseases. By inducing double-strand breaks (DSBs), the engineered endonucleases specifically target chosen sequences. These DSBs are spontaneously repaired either by homologous recombination in the presence of a sequence template, or by nonhomologous-end joining error prone repair. In this review, we highlight recent developments and challenges for genome-editing based strategies that hold great promise for muscular dystrophies and regenerative medicine.

Gene editing of DNAH11 restores normal cilia motility in primary ciliary dyskinesia

BackgroundPrimary ciliary dyskinesia (PCD) is a rare autosomal recessive genetic disorder characterised by dysfunction of motile cilia. Ciliary dysmotility causes poor mucociliary clearance and leads to impairment of pulmonary function...