Biology subjects

Cohn, R. D.

Publications and source records attributed to Cohn, R. D..

7 recordsLinked to original sources

bioRxiv2023

Generation and characterization of a novel mouse model of Becker Muscular Dystrophy with a deletion of exons 52 to 55

Becker Muscular Dystrophy (BMD) is a rare X-linked recessive neuromuscular disorder caused by in-frame deletions in the DMD gene that result in the production of a truncated, yet functional, dystrophin protein. BMD is often considered a milder form of Duchenne Muscular Dystrophy, in which mutations typically result in the disruption of the reading frame and the malfunction or loss of dystrophin. The consequences of BMD-causing in-frame deletions on the organism are more difficult to predict, especially in regard to long-term prognosis. Here, we employed CRISPR-Cas9 technology to generate a new Dmd del52-55 mouse model by deleting exons 52-55, resulting in a typical BMD-like in-frame deletion. To delineate the long-term effects of this deletion, we studied these mice over 52 weeks. Our results suggest that a truncated dystrophin is sufficient to maintain wildtype-like muscle and heart functions in young mice. However, the truncated protein appears insufficient to maintain normal muscle homeostasis and protect against exercise-induced damage at 52 weeks. To further delineate the effects of the exons 52-55 in-frame deletion, we performed RNA-Seq pre- and post-exercise and identified several differentially expressed pathways that could explain the abnormal muscle phenotype observed at 52 weeks in the BMD model. Summary StatementWe generated and characterized the long-term effects of a Becker Muscular Dystrophy-like in-frame deletion of exon 52 to 55 in mice.

genetics↗

bioRxiv2023

A Cas9-fusion proximity-based approach generates an Irak1-Mecp2 tandem duplication mouse model for the study of MeCP2 duplication syndrome.

MECP2 duplication syndrome (MDS) is a neurodevelopmental disorder caused by tandem duplication of the MECP2 locus and its surrounding genes, including IRAK1. Current MDS mouse models involve transgenic expression of MECP2 only, limiting their applicability to the study of the disease. Herein, we show that an efficient and precise CRISPR/Cas9 fusion proximity-based approach can be utilized to generate an Irak1-Mecp2 tandem duplication mouse model. The Mecp2 Dup model displays a neurological phenotype in keeping with MDS and demonstrates an abnormal immune response to infection not previously observed in other mouse models, possibly stemming from concurrent Irak1 overexpression. The Mecp2 Dup mouse line thus provides an innovative tool to investigate disease mechanisms and potential therapeutic development.

genetics↗

bioRxiv2021

Saturation variant interpretation using CRISPR prime editing

Over the last decade, next generation sequencing has become widely implemented in clinical practice. However, as genetic variants of uncertain significance (VUS) are frequently identified, the need for scaled functional interpretation of such variants has become increasingly apparent. One method to address this is saturation genome editing (SGE), which allows for scaled multiplexed functional assessment of single nucleotide variants. The current applications of SGE, however, rely on homology-directed repair (HDR) to introduce variants of interest, which is limited by low editing efficiencies and low product purity. Here, we have adapted CRISPR prime editing for SGE and demonstrated its utility in understanding the functional significance of variants in the NPC1 gene underlying the lysosomal storage disorder Niemann-Pick disease type C1 (NPC). Additionally, we have designed a genome editing strategy that allows for the haploidization of gene loci, which permits isolated variant interpretation in virtually any cell type. By combining saturation prime editing (SPE) with a clinically relevant assay, we have functionally scored and interpreted 256 variants in NPC1 haploidized HEK293T cells. To further demonstrate the applicability of this strategy, we used SPE and cell model haploidization to functionally score 465 variants in the BRCA2 gene. We anticipate that our work will be translatable to any gene with an appropriate cellular assay, allowing for more rapid and accurate diagnosis and improved genetic counselling and ultimately precise patient care.

genetics↗

bioRxiv2020

Heterozygous Cell Models of STAT1 Gain-of-Function Reveal a Broad Spectrum of Interferon-Signature Gene Transcriptional Responses

Signal Transducer and Activator of Transcription 1 (STAT1) gain-of-function (GOF) is an autosomal dominant immune disorder marked by wide infectious predisposition, autoimmunity, vascular disease and malignancy. Its molecular hallmark, elevated phospho-STAT1 (pSTAT1) following interferon (IFN) stimulation, is seen consistently in all patients and may not fully account for the broad phenotypic spectrum associated with this disorder. While over 100 mutations have been implicated in STAT1 GOF, genotype-phenotype correlation remains limited, and current overexpression models may be of limited use in gene expression studies. We generated heterozygous mutants in diploid HAP1 cells using CRISPR/Cas9 base-editing, targeting the endogenous STAT1 gene. Our models recapitulated the molecular phenotype of elevated pSTAT1, and were used to characterize the expression of five IFN-stimulated genes under a number of conditions. At baseline, transcriptional polarization was evident among mutants compared with wild type, and this was maintained following prolonged serum starvation. This suggests a possible role for unphosphorylated STAT1 in the pathogenesis of STAT1 GOF. Following stimulation with IFN or IFN{gamma}, differential patterns of gene expression emerged among mutants, including both gain and loss of transcriptional function. This work highlights the importance of modelling heterozygous conditions, and in particular transcription factor-related disorders, in a manner which accurately reflects patient genotype and molecular signature. Furthermore, we propose a complex and multifactorial transcriptional profile associated with various STAT1 mutations, adding to global efforts in establishing STAT1 GOF genotype-phenotype correlation and enhancing our understanding of disease pathogenesis.

immunology↗

bioRxiv2020

Cell-based analysis of CAD variants identifies individuals likely to benefit from uridine therapy

PurposePathogenic autosomal recessive variants in CAD, encoding the multienzymatic protein initiating pyrimidine de novo biosynthesis, cause a severe inborn metabolic disorder treatable with a dietary supplement of uridine. This condition is difficult to diagnose given the large size of CAD with over 1000 missense variants and the non-specific clinical presentation. We aimed to develop a reliable and discerning assay to assess the pathogenicity of CAD variants and to select affected individuals that might benefit from uridine therapy. MethodsUsing CRISPR/Cas9, we generated a human CAD-knockout cell line that requires uridine supplements for survival. Transient transfection of the knockout cells with recombinant CAD restores growth in absence of uridine. This system determines missense variants that inactivate CAD and do not rescue the growth phenotype. ResultsWe identified 25 individuals with biallelic variants in CAD and a phenotype consistent with a CAD deficit. We used the CAD-knockout complementation assay to test a total of 34 variants, identifying 16 as deleterious for CAD activity. Combination of these pathogenic variants confirmed 11 subjects with a CAD deficit, for whom we describe the clinical phenotype. ConclusionsWe designed a cell-based assay to test the pathogenicity of CAD variants, identifying 11 CAD deficient individuals, who could benefit from uridine therapy.

genetics↗

bioRxiv2019

Modelling Niemann-Pick disease type C in a human haploid cell line allows for patient variant characterization and clinical interpretation.

The accurate clinical interpretation of human sequence variation is foundational to personalized medicine. This remains a pressing challenge, however, as genome sequencing becomes routine and new functionally undefined variants rapidly accumulate. Here, we describe a platform for the rapid generation, characterization and interpretation of genomic variants in haploid cells focusing on Niemann-Pick disease type C (NPC) as an example. NPC is a fatal neurodegenerative disorder characterized by a lysosomal accumulation of unesterified cholesterol and glycolipids. In 95% of cases, NPC is caused by mutations in the NPC1 gene, where over 200 unique disease-causing variants have been reported to date. Furthermore, the majority of patients with NPC are compound heterozygotes that often carry at least one private mutation, presenting a challenge for the characterization and classification of individual variants. Here, we have developed the first haploid cell model of NPC. This haploid cell model recapitulates the primary biochemical and molecular phenotypes typically found in patient-derived fibroblasts, illustrating its utility in modelling NPC. Additionally, we demonstrate the power of CRISPR-Cas9-mediated base editing in quickly and efficiently generating haploid cell models of individual patient variants in NPC. These models provide a platform for understanding the disease mechanisms underlying individual NPC1 variants while allowing for definitive clinical variant interpretation for NPC. While this study has focused on modelling NPC, the outlined approach could be translated widely and applied to a variety of genetic disorders or understanding the pathogenicity of somatic mutations in cancer.

genetics↗

bioRxiv2018

A mutation-independent approach via transcriptional upregulation of a disease modifier gene rescues muscular dystrophy in vivo.

Introductory paragraphIdentification of protective and/or pathogenic genetic modifiers provides important insight into the heterogeneity of disease presentations in individuals affected by neuromuscular disorders (NMDs), despite having well-defined pathogenic variants. Targeting modifier genes to improve disease phenotypes could be especially beneficial in cases where the causative genes are large, structurally complex and the mutations are heterogeneous. Here, we report a mutation-independent strategy to upregulate expression of a compensatory disease-modifying gene in Congenital Muscular Dystrophy type 1A (MDC1A) using a CRISPR/dCas9-based transcriptional activation system. MDC1A is caused by nonfunctional Laminin 2, which compromises muscle fibers stability and axon myelination in peripheral nerves 1. Transgenic overexpression of Lama1, encoding a structurally similar protein Laminin 1, ameliorates muscle wasting and paralysis in the MDC1A mouse models, demonstrating its role as a protective disease modifier 2. Yet, upregulation of Lama1 as a postnatal gene therapy is hampered by its large size, which exceeds the current genome packaging capacity of clinically relevant delivery vehicles such as adeno-associated viral vectors (AAVs). In this study, we sought to upregulate Lama1 using CRISPR/dCas9-based transcriptional activation system, comprised of catalytically inactive S. aureus Cas9 (dCas9) fused to VP64 transactivation domains and sgRNAs targeting the Lama1 promoter. We first demonstrated robust upregulation of Lama1 in myoblasts, and following AAV9-mediated intramuscular delivery, in skeletal muscles of dy2j/dy2j mouse model of MDC1A.\n\nWe therefore assessed whether systemic upregulation of Lama1 would yield therapeutic benefits in dy2j/dy2j mice. When the intervention was started early in pre-symptomatic dy2j/dy2j mice, Lama1 upregulation prevented muscle fibrosis and hindlimb paralysis. An important question for future therapeutic approaches for a variety of disorders concerns the therapeutic window and phenotypic reversibility. This is particularly true for muscular dystrophies as it has long been hypothesized that fibrotic changes in skeletal muscle represent an irreversible disease state that would impair any therapeutic intervention at advanced stages of the disease. Here, we demonstrate that dystrophic features and disease progression were significantly improved and partially reversed when the treatment was initiated in symptomatic 3-week old dy2j/dy2j mice with already-apparent hind limb paralysis and significant muscle fibrosis.\n\nCollectively, our data demonstrate the feasibility and therapeutic benefit of CRISPR/dCas9-mediated modulation of a disease modifier gene, which opens up an entirely new and mutation-independent treatment approach for all MDC1A patients. Moreover, this treatment strategy provides evidence that muscle fibrosis can be reversible, thus extending the therapeutic window for this disorder. Our data provide a proof-of-concept strategy that can be applied to a variety of disease modifier genes and a powerful therapeutic approach for various inherited and acquired diseases.

genetics↗