A mutation-independent approach for muscular dystrophy via upregulation of a modifier gene.

Neuromuscular disorders are often caused by heterogeneous mutations in large, structurally complex genes. Targeting compensatory modifier genes could be beneficial to improve disease phenotypes. Here we report a mutation-independent strategy to upregulate the expression of a disease-modifying gene associated with congenital muscular dystrophy type 1A (MDC1A) using the CRISPR activation system in mice. MDC1A is caused by mutations in LAMA2 that lead to nonfunctional laminin-α2, which compromises the stability of muscle fibres and the myelination of peripheral nerves. Transgenic overexpression of Lama1, which encodes a structurally similar protein called laminin-α1, ameliorates muscle wasting and paralysis in mouse models of MDC1A, demonstrating its importance as a compensatory modifier of the disease1. However, postnatal upregulation of Lama1 is hampered by its large size, which exceeds the packaging capacity of vehicles that are clinically relevant for gene therapy. We modulate expression of Lama1 in the dy2j/dy2j mouse model of MDC1A using an adeno-associated virus (AAV9) carrying a catalytically inactive Cas9 (dCas9), VP64 transactivators and single-guide RNAs that target the Lama1 promoter. When pre-symptomatic mice were treated, Lama1 was upregulated in skeletal muscles and peripheral nerves, which prevented muscle fibrosis and paralysis. However, for many disorders it is important to investigate the therapeutic window and reversibility of symptoms. In muscular dystrophies, it has been hypothesized that fibrotic changes in skeletal muscle are irreversible. However, we show that dystrophic features and disease progression were improved and reversed when the treatment was initiated in symptomatic dy2j/dy2j mice with apparent hindlimb paralysis and muscle fibrosis. Collectively, our data demonstrate the feasibility and therapeutic benefit of CRISPR-dCas9-mediated upregulation of Lama1, which may enable mutation-independent treatment for all patients with MDC1A. This approach has a broad applicability to a variety of disease-modifying genes and could serve as a therapeutic strategy for many inherited and acquired diseases.

Modeling 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, for which more than 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 modeling NPC. Additionally, we show 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.

Hedgehog-GLI signaling in Foxd1-positive stromal cells promotes murine nephrogenesis via TGFß signaling.

Normal kidney function depends on the proper development of the nephron: the functional unit of the kidney. Reciprocal signaling interactions between the stroma and nephron progenitor compartment have been proposed to control nephron development. Here, we show that removal of hedgehog intracellular effector smoothened (Smo-deficient mutants) in the cortical stroma results in an abnormal renal capsule, and an expanded nephron progenitor domain with an accompanying decrease in nephron number via a block in epithelialization. We show that stromal-hedgehog-Smo signaling acts through a GLI3 repressor. Whole-kidney RNA sequencing and analysis of FACS-isolated stromal cells identified impaired TGFß2 signaling in Smo-deficient mutants. We show that neutralization and knockdown of TGFß2 in explants inhibited nephrogenesis. In addition, we demonstrate that concurrent deletion of Tgfbr2 in stromal and nephrogenic cells in vivo results in decreased nephron formation and an expanded nephrogenic precursor domain similar to that observed in Smo-deficient mutant mice. Together, our data suggest a mechanism whereby a stromal hedgehog-TGFß2 signaling axis acts to control nephrogenesis.

Suppressor of fused controls cerebellar neuronal differentiation in a manner modulated by GLI3 repressor and Fgf15.

BACKGROUND: Deficiency of Suppressor of Fused (SuFu), an intracellular mediator of Hedgehog signaling, in the murine mid-hindbrain disrupts cerebellar morphogenesis and cell differentiation in a manner that is rescued by constitutive expression of GLI3 transcriptional repressor (GLI3R). Here, we determined SuFu functions in cerebellar radial precursors following the stage of mid-hindbrain specification using a Blbp-Cre transgene. RESULTS: SuFu-deficient cerebella were severely dysplastic, and characterized by laminar disorganization, and delayed differentiation of ventricular zone-derived precursors. In vitro analysis of cerebellar precursors isolated from control and mutant mice demonstrated an increased proportion of radial glial precursors vs. Tuj1-positive neurons in mutant cultures. Abnormal cell differentiation in SuFu-deficient precursors was rescued by a constitutively expressed GLI3R knock-in allele, albeit with variable penetrance. Using RNA expression analysis in control and SuFu-deficient cerebellar anlage, we identified up-regulation of Fgf15 in mutant tissue. Strikingly, exogenous hFGF19, a mFGF15 ortholog, inhibited neuronal differentiation in cultures of wild-type cerebellar precursors. Moreover, siRNA-mediated knockdown of Fgf15 in SuFu-deficient cerebellar precursors rescued their delayed differentiation to neurons. CONCLUSIONS: Together, our results show that SuFu promotes cerebellar radial precursor differentiation to neurons. SuFu function is mediated in part by GLI3R and down-regulation of Fgf15 expression. Developmental Dynamics 247:156-169, 2018. © 2017 Wiley Periodicals, Inc.

Species-specific differences in NPC1 protein trafficking govern therapeutic response in Niemann-Pick type C disease.

The folding and trafficking of transmembrane glycoproteins are essential for cellular homeostasis and are compromised in many diseases. In Niemann-Pick type C disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, the transmembrane glycoprotein NPC1 misfolds due to disease-causing missense mutations. While mutant NPC1 has emerged as a robust target for proteostasis modulators, drug development efforts have been unsuccessful in mouse models. Here, we demonstrated unexpected differences in trafficking through the medial Golgi between mouse and human I1061T-NPC1, a common disease-causing mutant. We established that these distinctions are governed by differences in the NPC1 protein sequence rather than by variations in the endoplasmic reticulum-folding environment. Moreover, we demonstrated direct effects of mutant protein trafficking on the response to small molecules that modulate the endoplasmic reticulum-folding environment by affecting Ca++ concentration. Finally, we developed a panel of isogenic human NPC1 iNeurons expressing WT, I1061T-, and R934L-NPC1 and demonstrated their utility in testing these candidate therapeutics. Our findings identify important rules governing mutant NPC1's response to proteostatic modulators and highlight the importance of species- and mutation-specific responses for therapy development.

Saturation variant interpretation using CRISPR prime editing.

High-throughput functional characterization of genetic variants in their endogenous locus has so far been possible only with methods that rely on homology-directed repair, which are limited by low editing efficiencies. Here, we adapted CRISPR prime editing for high-throughput variant classification and combined it with a strategy that allows for haploidization of any locus, which simplifies variant interpretation. We demonstrate the utility of saturation prime editing (SPE) by applying it to the NPC intracellular cholesterol transporter 1 gene (NPC1), mutations in which cause the lysosomal storage disorder Niemann-Pick disease type C. Our data suggest that NPC1 is very sensitive to genetic perturbation, with 410 of 706 assayed missense mutations being classified as deleterious, and that the derived function score of variants is reflective of diverse molecular defects. We further applied our approach to the BRCA2 gene, demonstrating that SPE is translatable to other genes with an appropriate cellular assay. In sum, we show that SPE allows for efficient, accurate functional characterization of genetic variants.

STAT1 gain-of-function heterozygous cell models reveal diverse 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 IFNα or IFNγ, differential patterns of gene expression emerged among mutants, including both gain and loss of transcriptional function. This work highlights the importance of modeling 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.

Embryo-Based Large Fragment Knock-in in Mammals: Why, How and What's Next.

Endonuclease-mediated genome editing technologies, most notably CRISPR/Cas9, have revolutionized animal genetics by allowing for precise genome editing directly through embryo manipulations. As endonuclease-mediated model generation became commonplace, large fragment knock-in remained one of the most challenging types of genetic modification. Due to their unique value in biological and biomedical research, however, a diverse range of technological innovations have been developed to achieve efficient large fragment knock-in in mammalian animal model generation, with a particular focus on mice. Here, we first discuss some examples that illustrate the importance of large fragment knock-in animal models and then detail a subset of the recent technological advancements that have allowed for efficient large fragment knock-in. Finally, we envision the future development of even larger fragment knock-ins performed in even larger animal models, the next step in expanding the potential of large fragment knock-in in animal models.

Allele-Specific Prevention of Nonsense-Mediated Decay in Cystic Fibrosis Using Homology-Independent Genome Editing.

Nonsense-mediated decay (NMD) is a major pathogenic mechanism underlying a diversity of genetic disorders. Nonsense variants tend to lead to more severe disease phenotypes and are often difficult targets for small molecule therapeutic development as a result of insufficient protein production. The treatment of cystic fibrosis (CF), an autosomal recessive disease caused by mutations in the CFTR gene, exemplifies the challenge of therapeutically addressing nonsense mutations in human disease. Therapeutic development in CF has led to multiple, highly successful protein modulatory interventions, yet no targeted therapies have been approved for nonsense mutations. Here, we have designed a CRISPR-Cas9-based strategy for the targeted prevention of NMD of CFTR transcripts containing the second most common nonsense variant listed in CFTR2, W1282X. By introducing a deletion of the downstream genic region following the premature stop codon, we demonstrate significantly increased protein expression of this mutant variant. Notably, in combination with protein modulators, genome editing significantly increases the potentiated channel activity of W1282X-CFTR in human bronchial epithelial cells. Furthermore, we show how the outlined approach can be modified to permit allele-specific editing. The described approach can be extended to other late-occurring nonsense mutations in the CFTR gene or applied as a generalized approach for gene-specific prevention of NMD in disorders where a truncated protein product retains full or partial functionality.

Activity of lumacaftor is not conserved in zebrafish Cftr bearing the major cystic fibrosis-causing mutation.

F508del-cystic fibrosis transmembrane conductance regulator (CFTR) is the major mutant responsible for cystic fibrosis (CF). ORKAMBI®, approved for patients bearing this mutant, contains lumacaftor (VX-809) that partially corrects F508del-CFTR's processing defect and ivacaftor (VX-770) that potentiates its defective channel activity. Unfortunately, the clinical efficacy of ORKAMBI® is modest, highlighting the need to understand how the small molecules work so that superior compounds can be developed. Because, human CFTR (hCFTR) and zebrafish Cftr (zCftr) are structurally conserved as determined in recent cryo-EM structural models, we hypothesized that the consequences of the major mutation and small molecule modulators would be similar for the two species of protein. As expected, like the F508del mutation in hCFTR, the homologous mutation in zCftr (F507del) is misprocessed, yet not as severely as the human mutant and this defect was restored by low-temperature (27°C) culture conditions. After rescue to the cell surface, F507del-zCftr exhibited regulated channel activity that was potentiated by ivacaftor. Surprisingly, lumacaftor failed to rescue misprocessing of the F507del-zCftr at either 37 or 27°C suggesting that future comparative studies with F508del-hCFTR would provide insight into its structure: function relationships. Interestingly, the robust rescue of F508del-zCftr at 27°C and availability of methods for in vivo screening in zebrafish present the opportunity to define the cellular pathways underlying rescue.

Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for Genome Editing.

CRISPR-Cas9 systems provide powerful tools for genome editing. However, optimal employment of this technology will require control of Cas9 activity so that the timing, tissue specificity, and accuracy of editing may be precisely modulated. Anti-CRISPR proteins, which are small, naturally occurring inhibitors of CRISPR-Cas systems, are well suited for this purpose. A number of anti-CRISPR proteins have been shown to potently inhibit subgroups of CRISPR-Cas9 systems, but their maximal inhibitory activity is generally restricted to specific Cas9 homologs. Since Cas9 homologs vary in important properties, differing Cas9s may be optimal for particular genome-editing applications. To facilitate the practical exploitation of multiple Cas9 homologs, here we identify one anti-CRISPR, called AcrIIA5, that potently inhibits nine diverse type II-A and type II-C Cas9 homologs, including those currently used for genome editing. We show that the activity of AcrIIA5 results in partial in vivo cleavage of a single-guide RNA (sgRNA), suggesting that its mechanism involves RNA interaction.