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Recent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(β-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15–20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR–Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.

Current efforts to find specific genodermatoses treatments and define precise pathogenesis mechanisms require appropriate surrogate models with human cells. Although transgenic and gene knockout mouse models for several of these disorders exist, they often fail to faithfully replicate the clinical and histopathological features of the human skin condition. We have established a highly efficient method for precise deletion of critical gene sequences in primary human keratinocytes, based on CRISPR-Cas9-mediated gene editing. Using this methodology, in the present study we generated a model of Netherton syndrome by disruption of SPINK5. Gene-edited cells showed absence of LEKTI expression and were able to recapitulate a hyperkeratotic phenotype with most of the molecular hallmarks of Netherton syndrome, after grafting to immunodeficient mice and in organotypic cultures. To validate the model as a platform for therapeutic intervention, we tested an ex vivo gene therapy approach using a lentiviral vector expressing SPINK5. Re-expression of SPINK5 in an immortalized clone of SPINK5-knockout keratinocytes was capable of reverting from Netherton syndrome to a normal skin phenotype in vivo and in vitro. Our results demonstrate the feasibility of modeling genodermatoses, such as Netherton syndrome, by efficiently disrupting the causative gene to better understand its pathogenesis and to develop novel therapeutic approaches., Graphical Abstract, Exploiting the advantages of their highly efficient CRISPR-Cas9 gene editing strategy, in this study, Gálvez et al. abrogate SPINK5, the Netherton syndrome-causing gene, in primary human keratinocytes. SPINK5-knockout cells show disease hallmarks in vitro and in vivo, enabling the development of trustworthy models suitable for the evaluation of advanced therapies for genodermatoses.

Genome-editing technologies that enable the introduction of precise changes in DNA sequences have the potential to lead to a new class of treatments for genetic diseases. Epidermolysis bullosa (EB) is a group of rare genetic disorders characterized by extreme skin fragility. The recessive dystrophic subtype of EB (RDEB), which has one of the most severe phenotypes, is caused by mutations in COL7A1. In this study, we report a gene-editing approach for ex vivo homology-directed repair (HDR)-based gene correction that uses the CRISPR-Cas9 system delivered as a ribonucleoprotein (RNP) complex in combination with donor DNA templates delivered by adeno-associated viral vectors (AAVs). We demonstrate sufficient mutation correction frequencies to achieve therapeutic benefit in primary RDEB keratinocytes containing different COL7A1 mutations as well as efficient HDR-mediated COL7A1 modification in healthy cord blood-derived CD34+ cells and mesenchymal stem cells (MSCs). These results are a proof of concept for HDR-mediated gene correction in different cell types with therapeutic potential for RDEB. This work was supported by Spanish grants PI17/01747, PI20/00615, AC17/00054 (MutaEB-E-rare), and CIBERER ER18TRL714 from the Instituto de Salud Carlos III and grant SAF2017-86810-R from the Ministry of Economy and Competitiveness , all co-funded with European Regional Development Funds , and Avancell-CM grant ( S2017/BMD-3692 ). Authors are indebted to Almudena Holguín and Nuria Illera for grafting experiments, and to Jesus Martínez and Edilia De Almeida for animal maintenance and care.

Gene editing constitutes a novel approach for precisely correcting disease-causing gene mutations. Frameshift mutations in COL7A1 causing recessive dystrophic epidermolysis bullosa are amenable to open reading frame restoration by non-homologous end joining repair-based approaches. Efficient targeted deletion of faulty COL7A1 exons in polyclonal patient keratinocytes would enable the translation of this therapeutic strategy to the clinic. In this study, using a dual single-guide RNA (sgRNA)-guided Cas9 nuclease delivered as a ribonucleoprotein complex through electroporation, we have achieved very efficient targeted deletion of COL7A1 exon 80 in recessive dystrophic epidermolysis bullosa (RDEB) patient keratinocytes carrying a highly prevalent frameshift mutation. This ex vivo non-viral approach rendered a large proportion of corrected cells producing a functional collagen VII variant. The effective targeting of the epidermal stem cell population enabled long-term regeneration of a properly adhesive skin upon grafting onto immunodeficient mice. A safety assessment by next-generation sequencing (NGS) analysis of potential off-target sites did not reveal any unintended nuclease activity. Our strategy could potentially be extended to a large number of COL7A1 mutation-bearing exons within the long collagenous domain of this gene, opening the way to precision medicine for RDEB., Highly efficient and safe approaches for precise correction of pathogenic mutations are required to realize the full potential of gene-editing protocols for clinical practice. Here we describe a single-step NHEJ-based CRISPR/Cas9 strategy of COL7A1 mutation-bearing exon deletion with unprecedented efficacy for ex vivo correction of recessive dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa is a rare blistering condition caused by mutations in the COL7A1 gene. Different clinical variants have been described, with dominant and recessive inheritance, but no consistent findings have been elucidated to establish a genotype-phenotype correlation. We present three unrelated patients with two identical pathogenic compound heterozygous mutations in the COL7A1 gene that developed different clinical forms of dystrophic epidermolysis bullosa-epidermolysis bullosa pruriginosa and mild recessive non-Hallopeau-Siemens-raising the possibility of other genetic or environmental modifying factors responsible for the phenotype of the disease.

Here we report the functional assessment of two novel deafness-associated gamma-actin mutants, K118N and E241K, in a spectrum of different situations with increasing biological complexity by combining biochemical and cell biological analysis in yeast and mammalian cells. Our in vivo experiments showed that while the K118N had a very mild effect on yeast behaviour, the phenotype caused by the E241K mutation was very severe and characterized by a highly compromised ability to grow on glycerol as a carbon source, an aberrant multi-vacuolar pattern and the deposition of thick F-actin bundles randomly in the cell. The latter feature is consistent with the highly unusual spontaneous tendency of the E241K mutant to form bundles in vitro, although this propensity to bundle was neutralized by tropomyosin and the E241K filament bundles were hypersensitive to severing in the presence of cofilin. In transiently transfected NIH3T3 cells both mutant actins were normally incorporated into cytoskeleton structures, although cytoplasmic aggregates were also observed indicating an element of abnormality caused by the mutations in vivo. Interestingly, gene-gun mediated expression of these mutants in cochlear hair cells results in no gross alteration in cytoskeletal structures or the morphology of stereocilia. Our results provide a more complete picture of the biological consequences of deafness-associated gamma-actin mutants and support the hypothesis that the post-lingual and progressive nature of the DFNA20/26 hearing loss is the result of a progressive deterioration of the hair cell cytoskeleton over time.

Miguel Moreno-Pelayo and colleagues report mutations in the seed region of human miR-96 segregating with progressive hearing loss in two families. In an accompanying paper, Karen Steel and colleagues show that the mouse diminuendo mutant, which also shows progressive hearing loss, carries a similar mutation in the seed region of mouse miR-96. MicroRNAs (miRNAs) bind to complementary sites in their target mRNAs to mediate post-transcriptional repression1,2, with the specificity of target recognition being crucially dependent on the miRNA seed region3. Impaired miRNA target binding resulting from SNPs within mRNA target sites has been shown to lead to pathologies associated with dysregulated gene expression4,5,6,7. However, no pathogenic mutations within the mature sequence of a miRNA have been reported so far. Here we show that point mutations in the seed region of miR-96, a miRNA expressed in hair cells of the inner ear8, result in autosomal dominant, progressive hearing loss. This is the first study implicating a miRNA in a mendelian disorder. The identified mutations have a strong impact on miR-96 biogenesis and result in a significant reduction of mRNA targeting. We propose that these mutations alter the regulatory role of miR-96 in maintaining gene expression profiles in hair cells required for their normal function.

The TECTA gene encodes alpha-tectorin (TECTA), a major noncollagenous component of the tectorial membrane (TM). In humans, mutations in TECTA lead to either dominant (DFNA8/A12) or recessive (DFNB21) forms of nonsyndromic hearing loss. All missense mutations in TECTA that have been reported thus far are associated with the dominant subtype, whereas those leading to recessive deafness are all inactivating mutations. In this paper, we characterize a spontaneous missense mutation (c.1046C > A, p.A349D) arising in the mouse Tecta gene that is, unlike all previously reported missense mutations in TECTA, recessive. The morphological phenotype of the TectaA349D/A349D mouse resembles but is not identical to that previously described for the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$Tecta^{{{\Delta {\text{ENT}}} \mathord{\left/ {\vphantom {{\Delta {\text{ENT}}} {\Delta {\text{ENT}}}}} \right. \kern-\nulldelimiterspace} {\Delta {\text{ENT}}}}} $$\end{document} mouse. As in the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$Tecta^{{{\Delta {\text{ENT}}} \mathord{\left/ {\vphantom {{\Delta {\text{ENT}}} {\Delta {\text{ENT}}}}} \right. \kern-\nulldelimiterspace} {\Delta {\text{ENT}}}}} $$\end{document} mouse, the TM is completely detached from the surface of the organ of Corti and spiral limbus, lacks a striated-sheet matrix, and is deficient in both beta-tectorin (Tectb) and otogelin. A significant amount of Tecta is, however, detected in the TM of the TectaA349D/A349D mouse, and numerous, electron-dense matrix granules are seen interspersed among the disorganized collagen fibrils. Mutated TectaA349D is therefore incorporated into the TM but presumably unable to interact with either Tectb or otogelin. The TectaA349D/A349D mouse reveals that missense mutations in Tecta can be recessive and lead to TM detachment and suggests that should similar mutations arise in the human population, they would likely cause deafness.

Mutations in the potassium channel gene KCNQ4 underlie DFNA2, a subtype of autosomal dominant progressive, high-frequency hearing loss. Based on a phenotype-guided mutational screening we have identified a novel mutation c.886G>A, leading to the p.G296S substitution in the pore region of KCNQ4 channel. The possible impact of this mutation on total KCNQ4 protein expression, relative surface expression and channel function was investigated. When the G296S mutant was expressed in Xenopus oocytes, electrophysiological recordings did not show voltage-activated K+ currents. The p.G296S mutation impaired KCNQ4 channel activity in two manners. It greatly reduced surface expression and, secondarily, abolished channel function. The deficient expression at the cell surface membrane was further confirmed in non-permeabilized NIH-3T3 cells transfected with the mutant KCNQ4 tagged with the hemagglutinin epitope in the extracellular S1-S2 linker. Co-expression of mutant and wild type KCNQ4 in oocytes was performed to mimic the heterozygous condition of the p.G296S mutation in the patients. The results showed that the G296S mutant exerts a strong dominant-negative effect on potassium currents by reducing the wild type KCNQ4 channel expression at the cell surface. This is the first study to identify a trafficking-dependent dominant mechanism for the loss of KCNQ4 channel function in DFNA2. © Springer-Verlag 2007.

Epidermolysis bullosa with pyloric atresia (EB-PA) is a rare autosomal recessive hereditary disease with a variable prognosis from lethal to very mild. EB-PA is classified into Simplex form (EBS-PA: OMIM #612138) and Junctional form (JEB-PA: OMIM #226730), and it is caused by mutations in ITGA6, ITGB4 and PLEC genes. We report the analysis of six patients with EB-PA, including two dizygotic twins. Skin immunofluorescence epitope mapping was performed followed by PCR and direct sequencing of the ITGB4 gene. Two of the patients presented with non-lethal EB-PA associated with missense ITGB4 gene mutations. For the other four, early postnatal demise was associated with complete lack of β4 integrin due to a variety of ITGB4 novel mutations (2 large deletions, 1 splice-site mutation and 3 missense mutations). One of the deletions spanned 278 bp, being one of the largest reported to date for this gene. Remarkably, we also found for the first time a founder effect for one novel mutation in the ITGB4 gene. We have identified 6 novel mutations in the ITGB4 gene to be added to the mutation database. Our results reveal genotype-phenotype correlations that contribute to the molecular understanding of this heterogeneous disease, a pivotal issue for prognosis and for the development of novel evidence-based therapeutic options for EB management.

Hereditary non-syndromic sensorineural hearing loss (NSSHL) is a genetically highly heterogeneous group of disorders. Autosomal dominant forms account for up to 20% of cases. To date, 39 loci have been identified by linkage analysis of affected families that segregate NSSHL forms in the autosomal dominant mode (DFNA). Investigation of a large Spanish pedigree with autosomal dominant inheritance of bilateral and progressive NSSHL of postlingual onset excluded linkage to known DFNA loci and, in a subsequent genome-wide scan, the disorder locus was mapped to 3q28-29. A maximum two-point LOD score of 4.36 at theta=0 was obtained for marker D3S1601. Haplotype analysis placed the novel locus, DFNA44, within a 3-cM genetic interval defined by markers D3S1314 and D3S2418. Heteroduplex analysis and DNA sequencing of coding regions and exon/intron boundaries of two genes (CLDN16 and FGF12) in this interval did not reveal disease-causing mutations.

Item does not contain fulltext The prevalence of DFNA8/DFNA12 (DFNA8/12), a type of autosomal dominant nonsyndromic hearing loss (ADNSHL), is unknown as comprehensive population-based genetic screening has not been conducted. We therefore completed unbiased screening for TECTA mutations in a Spanish cohort of 372 probands from ADNSHL families. Three additional families (Spanish, Belgian, and English) known to be linked to DFNA8/12 were also included in the screening. In an additional cohort of 835 American ADNSHL families, we preselected 73 probands for TECTA screening based on audiometric data. In aggregate, we identified 23 TECTA mutations in this process. Remarkably, 20 of these mutations are novel, more than doubling the number of reported TECTA ADNSHL mutations from 13 to 33. Mutations lie in all domains of the alpha-tectorin protein, including those for the first time identified in the entactin domain, as well as the vWFD1, vWFD2, and vWFD3 repeats, and the D1-D2 and TIL2 connectors. Although the majority are private mutations, four of them-p.Cys1036Tyr, p.Cys1837Gly, p.Thr1866Met, and p.Arg1890Cys-were observed in more than one unrelated family. For two of these mutations founder effects were also confirmed. Our data validate previously observed genotype-phenotype correlations in DFNA8/12 and introduce new correlations. Specifically, mutations in the N-terminal region of alpha-tectorin (entactin domain, vWFD1, and vWFD2) lead to mid-frequency NSHL, a phenotype previously associated only with mutations in the ZP domain. Collectively, our results indicate that DFNA8/12 hearing loss is a frequent type of ADNSHL.

We previously mapped a novel autosomal dominant deafness locus, DFNA44, by studying a family with postlingual, progressive, nonsyndromic hearing loss. We report here on the identification of a mutation in CCDC50 as the cause of hearing loss in the family. CCDC50 encodes Ymer, an effector of epidermal growth factor (EGF)–mediated cell signaling that is ubiquitously expressed in different organs and has been suggested to inhibit down-regulation of the EGF receptor. We have examined its expression pattern in mouse inner ear. Western blotting and cell transfection results indicate that Ymer is a soluble, cytoplasmic protein, and immunostaining shows that Ymer is expressed in a complex spatiotemporal pattern during inner ear development. In adult inner ear, the expression of Ymer is restricted to the pillar cells of the cochlea, the stria vascularis, and the vestibular sensory epithelia, where it shows spatial overlap with the microtubule-based cytoskeleton. In dividing cells, Ymer colocalizes with microtubules of the mitotic apparatus. We suggest that DFNA44 hearing loss may result from a time-dependent disorganization of the microtubule-based cytoskeleton in the pillar cells and stria vascularis of the adult auditory system.

Approximately 1 in 1000 children is born with a serious permanent hearing impairment (pre-lingual deafness), and it is estimated that more than half of these cases in developed countries are due to genetic factors.1–3 The prevalence of hearing loss increases dramatically with age; it is estimated that approximately 5% of people under 45 years of age have a significant loss of hearing, increasing to approximately 50% by 80 years of age.1 Age related late onset hearing loss (presbycusis) is a heterogeneous trait with many suspected causes.4 Genetic or environmental factors such as diabetes, mitochondrial mutations, or environmental noise exposure may contribute to the trait. In addition, hearing loss beginning at late childhood, youth, or later, clearly segregates as a monogenic autosomal dominant Mendelian trait in many families. The hearing loss phenotype in these families is usually non-syndromic—that is, the hearing loss is not associated with other anomalies, and it accounts for up to 20% of the cases of non-syndromic sensorineural inherited deafness. This type of hearing loss is usually progressive, affecting a particular range of frequencies in each case.5 To date, 36 loci for autosomal dominant non-syndromic sensorineural hearing loss (ADNSSHL) have been mapped, and 17 deafness genes from these loci have been identified.6 These genes encode a wide variety of proteins; some have known function but for most, the underlying mechanisms leading to hearing impairment are uncertain. Given the few described mouse models for progressive hearing loss,7 our current understanding of post-lingual and progressive deafness relies on the identification of genes through human mapping studies. In this work we describe the mapping of a novel DFNA locus on chromosome 1q21–q23 segregating in a Spanish family with post-lingual and progressive hearing loss. ### Family data We ascertained a four generation family (S277) segregating ADNSSHL, through the …

Progressive hearing loss is a significant problem in all ageing populations. By the age of 80 years, nearly 50% of individuals have hearing loss that impairs their ability to communicate easily, leading to increasing social isolation.1 Progressive hearing loss in middle and late adulthood is considered multifactorial, with involvement of both genetic and environmental factors.2 In contrast, childhood or adolescent hearing loss is often inherited as an autosomal dominant Mendelian trait, representing about 20% of all cases of hereditary nonsyndromic sensorineural hearing impairment (NSSHI). Postlingual inherited deafness is usually moderate to severe and progressive, and it often affects a particular range of frequencies.3 Hereditary deafness has proved extremely heterogeneous genetically; more than 40 loci have been mapped for autosomal dominant (AD) NSSHI, and 17 deafness genes from these loci have been identified to date.4 Linkage analysis using large pedigrees is a useful tool for mapping and identifying novel deafness genes, a key step for improving our current understanding of auditory function. Here we describe the localisation of a novel DFNA locus on chromosome 7q32, involving the study of a Spanish family with postlingual and progressive hearing loss affecting all frequencies. ### Family data A five generation family (S403) with a history of ADNSSHI was identified through the Hospital Universitario de Valladolid, Spain. The pedigree consisted of 74 members, 37 of whom were affected (fig 1). Appropriate informed consent was obtained from all participants of the study and from parents of subjects younger than 18 years. Clinical evaluation was carried out, blood samples were collected from 55 family members, and DNA was extracted by standard techniques. Environmental factors were excluded as causes of hearing impairment. No syndromic features were present. Tympanometry with acoustic reflex testing indicated proper functioning of the middle ear, and pure tone audiometry was performed to test …