Detection of elusive DNA copy-number variations in hereditary disease and cancer through the use of noncoding and off-target sequencing reads.

Copy-number variants (CNVs) play a substantial role in the molecular pathogenesis of hereditary disease and cancer, as well as in normal human interindividual variation. However, they are still rather difficult to identify in mainstream sequencing projects, especially involving exome sequencing, because they often occur in DNA regions that are not targeted for analysis. To overcome this problem, we developed OFF-PEAK, a user-friendly CNV detection tool that builds on a denoising approach and the use of "off-target" DNA reads, which are usually discarded by sequencing pipelines. We benchmarked OFF-PEAK on data from targeted sequencing of 96 cancer samples, as well as 130 exomes of individuals with inherited retinal disease from three different populations. For both sets of data, OFF-PEAK demonstrated excellent performance (>95% sensitivity and >80% specificity vs. experimental validation) in detecting CNVs from in silico data alone, indicating its immediate applicability to molecular diagnosis and genetic research.

PCDH15 Dual-AAV Gene Therapy for Deafness and Blindness in Usher Syndrome Type 1F.

Usher syndrome type 1F (USH1F), resulting from mutations in the protocadherin-15 (PCDH15) gene, is characterized by congenital lack of hearing and balance, and progressive blindness in the form of retinitis pigmentosa. In this study, we explore a novel approach for USH1F gene therapy, exceeding the single AAV packaging limit by employing a dual adeno-associated virus (AAV) strategy to deliver the full-length PCDH15 coding sequence. We demonstrate the efficacy of this strategy in mouse USH1F models, effectively restoring hearing and balance in these mice. Importantly, our approach also proves successful in expressing PCDH15 in clinically relevant retinal models, including human retinal organoids and non-human primate retina, showing efficient targeting of photoreceptors and proper protein expression in the calyceal processes. This research represents a major step toward advancing gene therapy for USH1F and the multiple challenges of hearing, balance, and vision impairment.

Differentially Expressed Inflammation-Regulating MicroRNAs in Oligoarticular Juvenile Idiopathic Arthritis.

OBJECTIVE: To evaluate microRNA expression in synovial fluid (SF), plasma, and leukocytes from patients with juvenile idiopathic arthritis (JIA). METHODS: MicroRNA expression in pooled JIA plasma and SF was assessed by absolute quantitative droplet digital PCR array. The results were validated in individual patient samples. MicroRNA content in leukocytes and extracellular vesicles was evaluated by real-time PCR in JIA blood and SF. Blood microRNA expression was compared with healthy controls (HCs). Principal component analysis was used to profile JIA plasma and SF microRNAs, and the potential biological consequences of microRNA dysregulation were investigated by pathway analysis. RESULTS: MiR-15a-5p and miR-409-3p levels were higher in JIA plasma than in HC plasma. JIA SF contained elevated levels of miR-21-5p, miR-27a-3p, miR-146b-5p, miR-155-5p, and miR-423-5p, and decreased miR-192-5p and miR-451a, compared to JIA plasma. Extracellular vesicle analysis demonstrated variable encapsulation among selected microRNAs, with only miR-155-5p being represented substantially in extracellular vesicles. SF leukocytes also had higher expression of miR-21-5p, miR-27a-3p, miR-146b-5p, and miR-155-5p, and lower expression of miR-409-3p and miR-451a, relative to blood. No differences were observed between JIA and HC blood leukocytes. Clusters of microRNAs were commonly altered in JIA joint fluid and leukocytes compared to JIA blood samples. In silico analysis predicted that differentially expressed microRNAs in JIA target the transforming growth factor (TGF)-ß pathway. CONCLUSION: The expression of multiple microRNAs is dysregulated in JIA both locally and systemically, which may inhibit the TGF-ß pathway. These findings advance our knowledge of JIA immunopathogenesis and may lead to the development of targeted therapies.

CRISPR-Cas9 treatment partially restores amyloid-ß 42/40 in human fibroblasts with the Alzheimer's disease PSEN 1 M146L mutation.

Presenilin 1 (PS1) is a central component of γ-secretase, an enzymatic complex involved in the generation of the amyloid-ß (Aß) peptide that deposits as plaques in the Alzheimer's disease (AD) brain. The M146L mutation in the PS1 gene (PSEN1) leads to an autosomal dominant form of early-onset AD by promoting a relative increase in the generation of the more aggregation-prone Aß42. This change is evident not only in the brain but also in peripheral cells of mutation carriers. In this study we used the CRISPR-Cas9 system from Streptococcus pyogenes to selectively disrupt the PSEN1 M146L allele in human fibroblasts. A disruption of more than 50% of mutant alleles was observed in all CRISPR-Cas9-treated samples, resulting in reduced extracellular Aß42/40 ratios. Fluorescence resonance energy transfer-based conformation and western blot analyses indicated that CRISPR-Cas9 treatment also affects the overall PS1 conformation and reduces PS1 levels. Moreover, our guide RNA did not lead to any detectable editing at the highest-ranking candidate off-target sites identified by ONE-seq and CIRCLE-seq. Overall, our data support the effectiveness of CRISPR-Cas9 in selectively targeting the PSEN1 M146L allele and counteracting the AD-associated phenotype. We believe that this system could be developed into a therapeutic strategy for patients with this and other dominant mutations leading to early-onset AD.

Rare occult macular dystrophy with a pathogenic variant in the RP1L1 gene in a patient of Swiss descent.

Purpose: We report a first case of bilateral occult macular dystrophy (OMD) with a c.133C>T (p.Arg45Trp) pathogenic variant in the retinitis pigmentosa 1-like 1 (RP1L1) gene in a patient of Caucasian Swiss decent. Observations: A 34-year-old man presented with decreased visual acuity known since childhood. Fundus examination of both eyes revealed no pathology other than mildly increased granularity of the foveal retinal pigment epithelium. The full-field electroretinogram (ffERG) presented with normal findings while the multifocal electroretinogram (mfERG) showed severely reduced amplitudes of the foveal response. Optical coherence tomography (OCT) showed foveal outer retinal atrophy. Fundus autofluorescence (FAF) imaging demonstrated near-normal findings with minimal mottling at the posterior pole. The genetic analysis revealed a heterozygous pathogenic variant (c.133C>T, p.Arg45Trp) in the RP1L1 gene. Conclusion and importance: Our present case suggests that OMD shows a wide range of clinical presentations with a variety of ophthalmological findings, age of disease onset, visual acuity, and genetic diversity.

c.-61G>A in OVOL2 is a Pathogenic 5' Untranslated Region Variant Causing Posterior Polymorphous Corneal Dystrophy 1.

PURPOSE: The purpose of this study was to investigate the clinical and genetic features of a man and his daughter with posterior polymorphous corneal dystrophy (PPCD), referred to our clinic for Descemet membrane endothelial keratoplasty. No other known relatives were affected. METHODS: Ophthalmic examination and histology, including electron microscopy, were performed. Genetic testing was conducted by means of whole exome sequencing, and variant analysis was achieved by using an internal in silico pipeline. Molecular tests included a dual-luciferase assay. RESULTS: Slowly progressive blurred vision was reported from childhood by the daughter. The father's symptoms started at age 55. Best-corrected visual acuity was reduced in both patients (0.2-0.4). Slit-lamp examination in both patients revealed bilateral corneal clouding with gray endothelial lesions; other family members had no ophthalmological signs. Descemet membrane endothelial keratoplasty was performed uneventfully in both patients. Histology showed thickened Descemet membrane and abnormal endothelium resembling epithelial-like cells. Both patients carried the OVOL2 5' untranslated region NM_021220.4.c.-61G>A variant in the heterozygous state. This change was associated with increased promoter activity and was not present in the unaffected members of the family. CONCLUSIONS: The 5' untranslated region mutation c.-61G>A in OVOL2 has been previously found in 1 individual with PPCD1 and reported as a variant of unknown significance because of insufficient evidence supporting its pathogenicity. Identification of the second family with 2 individuals affected by PPCD1 carrying this change, together with functional data, provides further proofs that it is disease-causing.

Effect of prolactin on normal and keratoconus human corneal stromal fibroblasts in vitro.

PURPOSE: To examine the effect of prolactin (PRL) on human corneal stromal fibroblasts (CSFs), derived from healthy individuals and from keratoconus (KC) patients, in vitro, specifically assessing physiological and elevated PRL concentrations as apparent during pregnancy. METHODS: Eye bank corneas of 3 female and 3 male healthy individuals as well as the corneal buttons of 3 female and 3 male KC patients were utilized for this study. The endothelium of the cornea was removed with sterile surgical scalpels, the probes were washed repeatedly with Dulbecco's PBS and corneoscleral rims were trimmed off. Subsequently the corneal stroma was digested with collagenase type I and the harvested CSFs were cultured. We then examined (1) cell proliferation, (2) cell viability and (3) cytokine release of CSFs upon exposure to prolactin in vitro. RESULTS: With respect to viability and proliferation our experiments did not show significant differences between CSFs exposed to different PRL concentrations. Our data show a significantly lower IL-8 concentration in normal CSFs exposed to 10ng/ml PRL compared to 0ng/ml and 1000ng/ml at 5 hours post exposition. Moreover, we can report significantly lower secretion of IL-8, IL-6, HGF, VEGF and FGFb in KC CSFs compared to normal CSFs, independent of PRL exposure, as determined by cytokine ELISA. CONCLUSION: Our data in part points towards corneal cytokine secretion as a possible link between altered stromal PRL concentrations and KC progression. However, in our small dataset a significant influence of PRL concentration on cytokine secretion can only be described for IL-8 in normal CSFs. Further our results contribute to existing reports on the importance of cytokines in KC development, with an emphasis on significantly lower cytokine secretion in KC CSFs compared to normal controls.

Gene therapy for tuberous sclerosis complex type 2 in a mouse model by delivery of AAV9 encoding a condensed form of tuberin.

Tuberous sclerosis complex (TSC) results from loss of a tumor suppressor gene - TSC1 or TSC2, encoding hamartin and tuberin, respectively. These proteins formed a complex to inhibit mTORC1-mediated cell growth and proliferation. Loss of either protein leads to overgrowth lesions in many vital organs. Gene therapy was evaluated in a mouse model of TSC2 using an adeno-associated virus (AAV) vector carrying the complementary for a "condensed" form of human tuberin (cTuberin). Functionality of cTuberin was verified in culture. A mouse model of TSC2 was generated by AAV-Cre recombinase disruption of Tsc2-floxed alleles at birth, leading to a shortened lifespan (mean 58 days) and brain pathology consistent with TSC. When these mice were injected intravenously on day 21 with AAV9-cTuberin, the mean survival was extended to 462 days with reduction in brain pathology. This demonstrates the potential of treating life-threatening TSC2 lesions with a single intravenous injection of AAV9-cTuberin.

Mutant Allele-Specific CRISPR Disruption in DYT1 Dystonia Fibroblasts Restores Cell Function.

Most individuals affected with DYT1 dystonia have a heterozygous 3-bp deletion in the TOR1A gene (c.907_909delGAG). The mutation appears to act through a dominant-negative mechanism compromising normal torsinA function, and it is proposed that reducing mutant torsinA may normalize torsinA activity. In this study, we used an engineered Cas9 variant from Streptococcus pyogenes (SpCas9-VRQR) to target the mutation in the TOR1A gene in order to disrupt mutant torsinA in DYT1 patient fibroblasts. Selective targeting of the DYT1 allele was highly efficient with most common non-homologous end joining (NHEJ) edits, leading to a predicted premature stop codon with loss of the torsinA C terminus (delta 302-332 aa). Structural analysis predicted a functionally inactive status of this truncated torsinA due to the loss of residues associated with ATPase activity and binding to LULL1. Immunoblotting showed a reduction of the torsinA protein level in Cas9-edited DYT1 fibroblasts, and a functional assay using HSV infection indicated a phenotypic recovery toward that observed in control fibroblasts. These findings suggest that the selective disruption of the mutant TOR1A allele using CRISPR-Cas9 inactivates mutant torsinA, allowing the remaining wild-type torsinA to exert normal function.

Genome and base editing for genetic hearing loss.

Genome editing opens up a new frontier in developing personalized therapeutic solutions. With the unprecedented advance in the discovery and engineering of gene editing nucleases, it has now become potentially feasible to therapeutically influence up to 90% of all human genetic mutations. Hearing loss is one of the most well studied fields from the genetics perspective, with more than one hundred identified deafness genes. Novel viral and non-viral vectors have been established as safe and efficient modalities to deliver transgenes into cells of the cochlea and to the vestibular system in animal models. Recent studies demonstrated proof-of-concept for therapeutic genome and base editing in the mammalian inner ear and preclinical development is ongoing. This review summarizes important advances and future challenges for this transformative therapeutic modality for genetic and non-genetic hearing loss.

Membrane-bound Gaussia luciferase as a tool to track shedding of membrane proteins from the surface of extracellular vesicles.

Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter and targeting proteins can be modified and exposed on the surface of EVs to investigate their half-life and biodistribution. A characterization of membrane-bound Gaussia luciferase (mbGluc) revealed that its signal was detected also in a form smaller than common EVs (<70 nm). We demonstrated that mbGluc initially exposed on the surface of EVs, likely undergoes proteolytic cleavage and processed fragments of the protein are released into the extracellular space in active form. Based on this observation, we developed a new assay to quantitatively track shedding of membrane proteins from the surface of EVs. We used this assay to show that ectodomain shedding in EVs is continuous and is mediated by specific proteases, e.g. metalloproteinases. Here, we present a novel tool to study membrane protein cleavage and release using both in vitro and in vivo models.

High levels of AAV vector integration into CRISPR-induced DNA breaks.

Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined. In this study, we observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Genome-wide AAV mapping in mouse brain shows no overall increase of AAV integration except at the CRISPR/Cas9 target site. To allow detailed characterization of integration events we engineer a miniature AAV encoding a 465 bp lambda bacteriophage DNA (AAV-λ465), enabling sequencing of the entire integrated vector genome. The integration profile of AAV-465λ in cultured cells display both full-length and fragmented AAV genomes at Cas9 on-target sites. Our data indicate that AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing.

Allele-specific gene editing prevents deafness in a model of dominant progressive hearing loss.

Since most dominant human mutations are single nucleotide substitutions1,2, we explored gene editing strategies to disrupt dominant mutations efficiently and selectively without affecting wild-type alleles. However, single nucleotide discrimination can be difficult to achieve3 because commonly used endonucleases, such as Streptococcus pyogenes Cas9 (SpCas9), can tolerate up to seven mismatches between guide RNA (gRNA) and target DNA. Furthermore, the protospacer-adjacent motif (PAM) in some Cas9 enzymes can tolerate mismatches with the target DNA3,4. To circumvent these limitations, we screened 14 Cas9/gRNA combinations for specific and efficient disruption of a nucleotide substitution that causes the dominant progressive hearing loss, DFNA36. As a model for DFNA36, we used Beethoven mice5, which harbor a point mutation in Tmc1, a gene required for hearing that encodes a pore-forming subunit of mechanosensory transduction channels in inner-ear hair cells6. We identified a PAM variant of Staphylococcus aureus Cas9 (SaCas9-KKH) that selectively and efficiently disrupted the mutant allele, but not the wild-type Tmc1/TMC1 allele, in Beethoven mice and in a DFNA36 human cell line. Adeno-associated virus (AAV)-mediated SaCas9-KKH delivery prevented deafness in Beethoven mice up to one year post injection. Analysis of current ClinVar entries revealed that ~21% of dominant human mutations could be targeted using a similar approach.

Gene Transfer with AAV9-PHP.B Rescues Hearing in a Mouse Model of Usher Syndrome 3A and Transduces Hair Cells in a Non-human Primate.

Hereditary hearing loss often results from mutation of genes expressed by cochlear hair cells. Gene addition using AAV vectors has shown some efficacy in mouse models, but clinical application requires two additional advances. First, new AAV capsids must mediate efficient transgene expression in both inner and outer hair cells of the cochlea. Second, to have the best chance of clinical translation, these new vectors must also transduce hair cells in non-human primates. Here, we show that an AAV9 capsid variant, PHP.B, produces efficient transgene expression of a GFP reporter in both inner and outer hair cells of neonatal mice. We show also that AAV9-PHP.B mediates almost complete transduction of inner and outer HCs in a non-human primate. In a mouse model of Usher syndrome type 3A deafness (gene CLRN1), we use AAV9-PHP.B encoding Clrn1 to partially rescue hearing. Thus, we have identified a vector with promise for clinical treatment of hereditary hearing disorders, and we demonstrate, for the first time, viral transduction of the inner ear of a primate with an AAV vector.

Secretion and Uptake of α-Synuclein Via Extracellular Vesicles in Cultured Cells.

In Parkinson's disease and other Lewy body disorders, the propagation of pathology has been accredited to the spreading of extracellular α-synuclein (α-syn). Although the pathogenic mechanisms are not fully understood, cell-to-cell transfer of α-syn via exosomes and other extracellular vesicles (EVs) has been reported. Here, we investigated whether altered molecular properties of α-syn can influence the distribution and secretion of α-syn in human neuroblastoma cells. Different α-syn variants, including α-syn:hemi-Venus and disease-causing mutants, were overexpressed and EVs were isolated from the conditioned medium. Of the secreted α-syn, 0.1-2% was associated with vesicles. The major part of EV α-syn was attached to the outer membrane of vesicles, whereas a smaller fraction was found in their lumen. For α-syn expressed with N-terminal hemi-Venus, the relative levels associated with EVs were higher than for WT α-syn. Moreover, such EV-associated α-syn:hemi-Venus species were internalized in recipient cells to a higher degree than the corresponding free-floating forms. Among the disease-causing mutants, A53T α-syn displayed an increased association with EVs. Taken together, our data suggest that α-syn species with presumably lost physiological functions or altered aggregation properties may shift the cellular processing towards vesicular secretion. Our findings thus lend further support to the tenet that EVs can mediate spreading of harmful α-syn species and thereby contribute to the pathology in α-synucleinopathies.

TMC1 Forms the Pore of Mechanosensory Transduction Channels in Vertebrate Inner Ear Hair Cells.

The proteins that form the permeation pathway of mechanosensory transduction channels in inner-ear hair cells have not been definitively identified. Genetic, anatomical, and physiological evidence support a role for transmembrane channel-like protein (TMC) 1 in hair cell sensory transduction, yet the molecular function of TMC proteins remains unclear. Here, we provide biochemical evidence suggesting TMC1 assembles as a dimer, along with structural and sequence analyses suggesting similarity to dimeric TMEM16 channels. To identify the pore region of TMC1, we used cysteine mutagenesis and expressed mutant TMC1 in hair cells of Tmc1/2-null mice. Cysteine-modification reagents rapidly and irreversibly altered permeation properties of mechanosensory transduction. We propose that TMC1 is structurally similar to TMEM16 channels and includes ten transmembrane domains with four domains, S4-S7, that line the channel pore. The data provide compelling evidence that TMC1 is a pore-forming component of sensory transduction channels in auditory and vestibular hair cells.

Alzheimer's Disease-Associated ß-Amyloid Is Rapidly Seeded by Herpesviridae to Protect against Brain Infection.

Amyloid-ß peptide (Aß) fibrilization and deposition as ß-amyloid are hallmarks of Alzheimer's disease (AD) pathology. We recently reported Aß is an innate immune protein that protects against fungal and bacterial infections. Fibrilization pathways mediate Aß antimicrobial activities. Thus, infection can seed and dramatically accelerate ß-amyloid deposition. Here, we show Aß oligomers bind herpesvirus surface glycoproteins, accelerating ß-amyloid deposition and leading to protective viral entrapment activity in 5XFAD mouse and 3D human neural cell culture infection models against neurotropic herpes simplex virus 1 (HSV1) and human herpesvirus 6A and B. Herpesviridae are linked to AD, but it has been unclear how viruses may induce ß-amyloidosis in brain. These data support the notion that Aß might play a protective role in CNS innate immunity, and suggest an AD etiological mechanism in which herpesviridae infection may directly promote Aß amyloidosis.

CRISPR/Cas9 Mediated Disruption of the Swedish APP Allele as a Therapeutic Approach for Early-Onset Alzheimer's Disease.

The APPswe (Swedish) mutation in the amyloid precursor protein (APP) gene causes dominantly inherited Alzheimer's disease (AD) as a result of increased ß-secretase cleavage of the amyloid-ß (Aß) precursor protein. This leads to abnormally high Aß levels, not only in brain but also in peripheral tissues of mutation carriers. Here, we selectively disrupted the human mutant APPSW allele using CRISPR. By applying CRISPR/Cas9 from Streptococcus pyogenes, we generated allele-specific deletions of either APPSW or APPWT. As measured by ELISA, conditioned media of targeted patient-derived fibroblasts displayed an approximate 60% reduction in secreted Aß. Next, coding sequences for the APPSW-specific guide RNA (gRNA) and Cas9 were packaged into separate adeno-associated viral (AAV) vectors. Site-specific indel formation was achieved both in primary neurons isolated from APPSW transgenic mouse embryos (Tg2576) and after co-injection of these vectors into hippocampus of adult mice. Taken together, we here present proof-of-concept data that CRISPR/Cas9 can selectively disrupt the APPSW allele both ex vivo and in vivo-and thereby decrease pathogenic Aß. Hence, this system may have the potential to be developed as a tool for gene therapy against AD caused by APPswe and other point mutations associated with increased Aß.

Extracellular RNAs: A New Awareness of Old Perspectives.

Extracellular RNA (exRNA) has recently expanded as a highly important area of study in biomarker discovery and cancer therapeutics. exRNA consists of diverse RNA subpopulations that are normally protected from degradation by incorporation into membranous vesicles or by lipid/protein association. They are found circulating in biofluids, and have proven highly promising for minimally invasive diagnostic and prognostic purposes, particularly in oncology. Recent work has made progress in our understanding of exRNAs-from their biogenesis, compartmentalization, and vesicle packaging to their various applications as biomarkers and therapeutics, as well as the new challenges that arise in isolation and purification for accurate and reproducible analysis. Here we review the most recent advancements in exRNA research.