Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma.

Mutations in myocilin (MYOC) are the leading known genetic cause of primary open-angle glaucoma, responsible for about 4% of all cases. Mutations in MYOC cause a gain-of-function phenotype in which mutant myocilin accumulates in the endoplasmic reticulum (ER) leading to ER stress and trabecular meshwork (TM) cell death. Therefore, knocking out myocilin at the genome level is an ideal strategy to permanently cure the disease. We have previously utilized CRISPR/Cas9 genome editing successfully to target MYOC using adenovirus 5 (Ad5). However, Ad5 is not a suitable vector for clinical use. Here, we sought to determine the efficacy of adeno-associated viruses (AAVs) and lentiviruses (LVs) to target the TM. First, we examined the TM tropism of single-stranded (ss) and self-complimentary (sc) AAV serotypes as well as LV expressing GFP via intravitreal (IVT) and intracameral (IC) injections. We observed that LV_GFP expression was more specific to the TM injected via the IVT route. IC injections of Trp-mutant scAAV2 showed a prominent expression of GFP in the TM. However, robust GFP expression was also observed in the ciliary body and retina. We next constructed lentiviral particles expressing Cas9 and guide RNA (gRNA) targeting MYOC (crMYOC) and transduction of TM cells stably expressing mutant myocilin with LV_crMYOC significantly reduced myocilin accumulation and its associated chronic ER stress. A single IVT injection of LV_crMYOC in Tg-MYOCY437H mice decreased myocilin accumulation in TM and reduced elevated IOP significantly. Together, our data indicates, LV_crMYOC targets MYOC gene editing in TM and rescues a mouse model of myocilin-associated glaucoma.

POMC Neuron BBSome Regulation of Body Weight is Independent of its Ciliary Function.

The BBSome, a complex of several Bardet-Biedl syndrome (BBS) proteins including BBS1, has emerged as a critical regulator of energy homeostasis. Although the BBSome is best known for its involvement in cilia trafficking, through a process that involve BBS3, it also regulates the localization of cell membrane receptors underlying metabolic regulation. Here, we show that inducible Bbs1 gene deletion selectively in proopiomelanocortin (POMC) neurons cause a gradual increase in body weight, which was associated with higher fat mass. In contrast, inducible deletion of Bbs3 gene in POMC neurons failed to affect body weight and adiposity. Interestingly, loss of BBS1 in POMC neurons led to glucose intolerance and insulin insensitivity, whereas BBS3 deficiency in these neurons is associated with slight impairment in glucose handling, but normal insulin sensitivity. BBS1 deficiency altered the plasma membrane localization of serotonin 5-HT2C receptor (5-HT2CR) and ciliary trafficking of neuropeptide Y2 receptor (NPY2R).In contrast, BBS3 deficiency, which disrupted the ciliary localization of the BBSome, did not interfere with plasma membrane expression of 5-HT2CR, but reduced the trafficking of NPY2R to cilia. We also show that deficiency in BBS1, but not BBS3, alters mitochondria dynamics and decreased total and phosphorylated levels of dynamin-like protein 1 (DRP1) protein. Importantly, rescuing DRP1 activity restored mitochondria dynamics and localization of 5-HT2CR and NPY2R in BBS1-deficient cells. The contrasting effects on energy and glucose homeostasis evoked by POMC neuron deletion of BBS1 versus BBS3 indicate that BBSome regulation of metabolism is not related to its ciliary function in these neurons.

Subretinal gene therapy delays vision loss in a Bardet-Biedl Syndrome type 10 mouse model.

Blindness in Bardet-Biedl syndrome (BBS) is caused by dysfunction and loss of photoreceptor cells in the retina. BBS10, mutations of which account for approximately 21% of all BBS cases, encodes a chaperonin protein indispensable for the assembly of the BBSome, a cargo adaptor important for ciliary trafficking. The loss of BBSome function in the eye causes a reduced light sensitivity of photoreceptor cells, photoreceptor ciliary malformation, dysfunctional ciliary trafficking, and photoreceptor cell death. Cone photoreceptors lacking BBS10 have congenitally low electrical function in electroretinography. In this study, we performed gene augmentation therapy by injecting a viral construct subretinally to deliver the coding sequence of the mouse Bbs10 gene to treat retinal degeneration in a BBS10 mouse model. Long-term efficacy was assessed by measuring the electrical functions of the retina over time, imaging of the treated regions to visualize cell survival, conducting visually guided swim assays to measure functional vision, and performing retinal histology. We show that subretinal gene therapy slowed photoreceptor cell death and preserved retinal function in treated eyes. Notably, cone photoreceptors regained their electrical function after gene augmentation. Measurement of functional vision showed that subretinal gene therapy provided a significant benefit in delaying vision loss.

The BBSome regulates mitochondria dynamics and function.

OBJECTIVE: The essential role of mitochondria in regulation of metabolic function and other physiological processes has garnered enormous interest in understanding the mechanisms controlling the function of this organelle. We assessed the role of the BBSome, a protein complex composed of eight Bardet-Biedl syndrome (BBS) proteins, in the control of mitochondria dynamic and function. METHODS: We used a multidisciplinary approach that include CRISPR/Cas9 technology-mediated generation of a stable Bbs1 gene knockout hypothalamic N39 neuronal cell line. We also analyzed the phenotype of BBSome deficient mice in presence or absence of the gene encoding A-kinase anchoring protein 1 (AKAP1). RESULTS: Our data show that the BBSome play an important role in the regulation of mitochondria dynamics and function. Disruption of the BBSome cause mitochondria hyperfusion in cell lines, fibroblasts derived from patients as well as in hypothalamic neurons and brown adipocytes of mice. The morphological changes in mitochondria translate into functional abnormalities as indicated by the reduced oxygen consumption rate and altered mitochondrial distribution and calcium handling. Mechanistically, we demonstrate that the BBSome modulates the activity of dynamin-like protein 1 (DRP1), a key regulator of mitochondrial fission, by regulating its phosphorylation and translocation to the mitochondria. Notably, rescuing the decrease in DRP1 activity through deletion of one copy of the gene encoding AKAP1 was effective to normalize the defects in mitochondrial morphology and activity induced by BBSome deficiency. Importantly, this was associated with improvement in several of the phenotypes caused by loss of the BBSome such as the neuroanatomical abnormalities, metabolic alterations and obesity highlighting the importance of mitochondria defects in the pathophysiology of BBS. CONCLUSIONS: These findings demonstrate a critical role of the BBSome in the modulation of mitochondria function and point to mitochondrial defects as a key disease mechanism in BBS.

Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma.

Mutations in myocilin (MYOC) are the leading known genetic cause of primary open-angle glaucoma, responsible for about 4% of all cases. Mutations in MYOC cause a gain-of-function phenotype in which mutant myocilin accumulates in the endoplasmic reticulum (ER) leading to ER stress and trabecular meshwork (TM) cell death. Therefore, knocking out myocilin at the genome level is an ideal strategy to permanently cure the disease. We have previously utilized CRISPR/Cas9 genome editing successfully to target MYOC using adenovirus 5 (Ad5). However, Ad5 is not a suitable vector for clinical use. Here, we sought to determine the efficacy of adeno-associated viruses (AAVs) and lentiviruses (LVs) to target the TM. First, we examined the TM tropism of single-stranded (ss) and self-complimentary (sc) AAV serotypes as well as LV expressing GFP via intravitreal (IVT) and intracameral (IC) injections. We observed that LV_GFP expression was more specific to the TM injected via the IVT route. IC injections of Trp-mutant scAAV2 showed a prominent expression of GFP in the TM. However, robust GFP expression was also observed in the ciliary body and retina. We next constructed lentiviral particles expressing Cas9 and guide RNA (gRNA) targeting MYOC (crMYOC) and transduction of TM cells stably expressing mutant myocilin with LV_crMYOC significantly reduced myocilin accumulation and its associated chronic ER stress. A single IVT injection of LV_crMYOC in Tg-MYOCY437H mice decreased myocilin accumulation in TM and reduced elevated IOP significantly. Together, our data indicates, LV_crMYOC targets MYOC gene editing in TM and rescues a mouse model of myocilin-associated glaucoma.

Progressive retinal degeneration of rods and cones in a Bardet-Biedl syndrome type 10 mouse model.

Bardet-Biedl syndrome (BBS) is a multi-organ autosomal-recessive disorder caused by mutations in at least 22 different genes. A constant feature is early-onset retinal degeneration leading to blindness. Among the most common forms is BBS type 10 (BBS10), which is caused by mutations in a gene encoding a chaperonin-like protein. To aid in developing treatments, we phenotyped a Bbs10 knockout (Bbs10-/-) mouse model. Analysis by optical coherence tomography (OCT), electroretinography (ERG) and a visually guided swim assay (VGSA) revealed a progressive degeneration (from P19 to 8 months of age) of the outer nuclear layer that is visible by OCT and histology. Cone ERG was absent from at least P30, at which time rod ERG was reduced to 74.4% of control levels; at 8 months, rod ERG was 2.3% of that of controls. VGSA demonstrated loss of functional vision at 9 months. These phenotypes progressed more rapidly than retinal degeneration in the Bbs1M390R/M390R knock-in mouse. This study defines endpoints for preclinical trials that can be utilized to detect a treatment effect in the Bbs10-/- mouse and extrapolated to human clinical trials.

Disruption of dopamine receptor 1 localization to primary cilia impairs signaling in striatal neurons.

A rod-shaped appendage called a primary cilium projects from the soma of most central neurons in the mammalian brain. The importance of cilia within the nervous system is highlighted by the fact that human syndromes linked to primary cilia dysfunction, collectively termed ciliopathies, are associated with numerous neuropathologies, including hyperphagia-induced obesity, neuropsychiatric disorders, and learning and memory deficits. Neuronal cilia are enriched with signaling molecules, including specific G protein-coupled receptors (GPCRs) and their downstream effectors, suggesting they act as sensory organelles that respond to neuromodulators in the extracellular space. We previously showed that GPCR ciliary localization is disrupted in neurons from mouse models of the ciliopathy Bardet-Biedl syndrome (BBS). Based on this finding we hypothesized that mislocalization of ciliary GPCRs may impact receptor signaling and contribute to the BBS phenotypes. Here, we show that disrupting localization of the ciliary GPCR dopamine receptor 1 (D1) in male and female mice, either by loss of a BBS protein or loss of the cilium itself, specifically in D1-expressing neurons, results in obesity. Interestingly, the weight gain is associated with reduced locomotor activity, rather than increased food intake. Moreover, loss of a BBS protein or cilia on D1-expressing neurons leads to a reduction in D1-mediated signaling. Together, these results indicate that cilia impact D1 activity in the nervous system and underscore the importance of neuronal cilia for proper GPCR signaling.SIGNIFICANCE STATEMENT:Most mammalian neurons possess solitary appendages called primary cilia. These rod-shaped structures are enriched with signaling proteins, such as G protein-coupled receptors (GPCRs), suggesting they respond to neuromodulators. This study examines the consequences of disrupting ciliary localization of the GPCR dopamine receptor 1 (D1) in D1-expressing neurons. Remarkably, mice that have either abnormal accumulation of D1 in cilia or loss of D1 ciliary localization become obese. In both cases the obesity is associated with lower locomotor activity rather than overeating. As D1 activation increases locomotor activity, these results are consistent with a reduction in D1 signaling. Indeed, we found that D1-mediated signaling is reduced in brain slices from both mouse models. Thus, cilia impact D1 signaling in the brain.

An open source and convenient method for the wide-spread testing of COVID-19 using deep throat sputum samples.

Importance: The rise of novel, more infectious SARS-CoV-2 variants has made clear the need to rapidly deploy large-scale testing for COVID-19 to protect public health. However, testing remains limited due to shortages of personal protective equipment (PPE), naso- and oropharyngeal swabs, and healthcare workers. Simple test methods are needed to enhance COVID-19 screening. Here, we describe a simple, and inexpensive spit-test for COVID-19 screening called Patient Self-Collection of Sample-CoV2 (PSCS-CoV2). Objective: To evaluate an affordable and convenient test for COVID-19. Methods: The collection method relies on deep throat sputum (DTS) self-collected by the subject without the use of swabs, and was hence termed the Self-Collection of Sample for SARS-CoV-2 (abbreviated PSCS-CoV2). We used a phenol-chloroform extraction method for the viral RNA. We then tested for SARS-CoV-2 using real-time reverse transcription polymerase chain reaction with primers against at least two coding regions of the viral nucleocapsid protein (N1 and N2 or E) of SARS-CoV-2. We evaluted the sensitivity and specificity of our protocol. In addition we assess the limit of detection, and efficacy of our Viral Inactivating Solution. We also evaluated our protocol, and pooling strategy from volunteers on a local college campus. Results: We show that the PSCS-CoV2 method accurately identified 42 confirmed COVID-19 positives, which were confirmed through the nasopharyngeal swabbing method of an FDA approved testing facility. For samples negative for COVID-19, we show that the cycle threshold for N1, N2, and RP are similar between the PSCS-CoV2 and nasopharynx swab collection method (n = 30). We found a sensitivity of 100% (95% Confidence Interval [CI], 92-100) and specifity of 100% (95% CI, 89-100) for our PSCS-CoV2 method. We determined our protocol has a limit of detection of 1/10,000 for DTS from a COVID-19 patient. In addition, we show field data of the PSCS-CoV2 method on a college campus. Ten of the twelve volunteers (N1 < 30) that we tested as positive were subsequently tested positive by an independent laboratory. Finally, we show proof of concept of a pooling strategy to test for COVID-19, and recommend pool sizes of four if the positivity rate is less than 15%. Conclusion and Relevance: We developed a DTS-based protocol for COVID-19 testing with high sensitivity and specificity. This protocol can be used by non-debilitated adults without the assistance of another adult, or by non-debilitated children with the assistance of a parent or guardian. We also discuss pooling strategies based on estimated positivity rates to help conserve resources, time, and increase throughput. The PSCS-CoV2 method can be a key component of community-wide efforts to slow the spread of COVID-19.

Retinal ciliopathies through the lens of Bardet-Biedl Syndrome: Past, present and future.

The primary cilium is a highly specialized and evolutionary conserved organelle in eukaryotes that plays a significant role in cell signaling and trafficking. Over the past few decades tremendous progress has been made in understanding the physiology of cilia and the underlying pathomechanisms of various ciliopathies. Syndromic ciliopathies consist of a group of disorders caused by ciliary dysfunction or abnormal ciliogenesis. These disorders have multiorgan involvement in addition to retinal degeneration underscoring the ubiquitous distribution of primary cilia in different cell types. Genotype-phenotype correlation is often challenging due to the allelic heterogeneity and pleiotropy of these disorders. In this review, we discuss the clinical and genetic features of syndromic ciliopathies with a focus on Bardet-Biedl syndrome (BBS) as a representative disorder. We discuss the structure and function of primary cilia and their role in retinal photoreceptors. We describe the progress made thus far in understanding the functional and genetic characterization including expression quantitative trait locus (eQTL) analysis of BBS genes. In the future directions section, we discuss the emerging technologies, such as gene therapy, as well as anticipated challenges and their implications in therapeutic development for ciliopathies.

Ectopic expression of BBS1 rescues male infertility, but not retinal degeneration, in a BBS1 mouse model.

Bardet-Biedl syndrome (BBS) is a rare ciliopathy for which there are no current effective treatments. BBS is a genetically heterogeneous disease, though the M390R mutation in BBS1 is involved in ~25% of all genetic diagnoses of BBS. The principle features of BBS include retinal degeneration, obesity, male infertility, polydactyly, intellectual disability, and renal abnormalities. Patients with mutations in BBS genes often present with night blindness within the first decade of life, which progresses to complete blindness. This is due to progressive loss of photoreceptor cells. Male infertility is caused by a lack of spermatozoa flagella, rendering them immobile. In this study, we have crossed the wild-type human BBS1 gene, driven by the CAG promoter, onto the Bbs1M390R/M390R mouse model to determine if ectopic expression of BBS1 rescues male infertility and retinal degeneration. qRT-PCR indicates that the BBS1 transgene is expressed in multiple tissues throughout the mouse, with the highest expression seen in the testes, and much lower expression in the eye and hypothalamus. Immunohistochemistry of the transgene in the eye showed little if any expression in the photoreceptor outer nuclear layer. When male Bbs1M30R/M390R;BBS1TG+ mice are housed with WT females, they are able to sire offspring, indicating that the male infertility phenotype of BBS is rescued by the transgene. Using electroretinography (ERGs) to measure retinal function and optical coherence tomography to measure retinal thickness, we show that the transgene does not confer protection against retinal degeneration in Bbs1M300R/M390R;BBS1TG+ mice. The results of this study indicate that the male infertility aspect of BBS is an attractive target for gene therapy.

Gene therapy and gene correction: targets, progress, and challenges for treating human diseases.

The field of gene therapy has made significant strides over the last several decades toward the treatment of previously untreatable genetic disease. Gene therapy techniques have been aimed at mitigating disease features of recessive and dominant disorders, as well as several cancers and other diseases. While there have been numerous disease targets of gene therapy trials, only four therapies have reached FDA and/or EMA approval for clinical use. Gene correction using CRISPR-Cas9 is an extension of gene therapy that has received considerable attention in recent years and boasts many possible uses beyond classical gene therapy approaches. While there is significant therapeutic potential using gene therapy and gene correction strategies, a number of hurdles remain to be overcome before they become more common in clinical use, particularly with regards to safety and efficacy. As research progresses in this exciting field, it is likely that these therapies will become first-line treatments and will have tremendous positive impacts on the lives of patients with genetic disorders.

Gene therapy rescues olfactory perception in a clinically relevant ciliopathy model of Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is a hereditary genetic disorder that results in numerous clinical manifestations including olfactory dysfunction. Of at least 21 BBS-related genes that can carry multiple mutations, a pathogenic mutation, BBS1M390R, is the single most common mutation of clinically diagnosed BBS outcomes. While the deletion of BBS-related genes in mice can cause variable penetrance in different organ systems, the impact of the Bbs1M390R mutation in the olfactory system remains unclear. Using a clinically relevant knock-in mouse model homozygous for Bbs1M390R, we investigated the impact of the mutation on the olfactory system and tested the potential of viral-mediated, wildtype gene replacement therapy to rescue smell loss. The cilia of olfactory sensory neurons (OSNs) in Bbs1M390R/M390R mice were significantly shorter and fewer than those of wild-type mice. Also, both peripheral cellular odor detection and synaptic-dependent activity in the olfactory bulb were significantly decreased in the mutant mice. Furthermore, to gain insight into the degree to which perceptual features are impaired in the mutant mice, we used whole-body plethysmography to quantitatively measure odor-evoked sniffing. The Bbs1M390R/M390R mice showed significantly higher odor detection thresholds (reduced odor sensitivity) compared to wild-type mice; however, their odor discrimination acuity was still well maintained. Importantly, adenoviral expression of Bbs1 in OSNs restored cilia length and re-established both peripheral odorant detection and odor perception. Together, our findings further expand our understanding for the development of gene therapeutic treatment for congenital ciliopathies in the olfactory system.

Exome-based investigation of the genetic basis of human pigmentary glaucoma.

BACKGROUND: Glaucoma is a leading cause of visual disability and blindness. Release of iris pigment within the eye, pigment dispersion syndrome (PDS), can lead to one type of glaucoma known as pigmentary glaucoma. PDS has a genetic component, however, the genes involved with this condition are largely unknown. We sought to discover genes that cause PDS by testing cohorts of patients and controls for mutations using a tiered analysis of exome data. RESULTS: Our primary analysis evaluated melanosome-related genes that cause dispersion of iris pigment in mice (TYRP1, GPNMB, LYST, DCT, and MITF). We identified rare mutations, but they were not statistically enriched in PDS patients. Our secondary analyses examined PMEL (previously linked with PDS), MRAP, and 19 other genes. Four MRAP mutations were identified in PDS cases but not in controls (p = 0.016). Immunohistochemical analysis of human donor eyes revealed abundant MRAP protein in the iris, the source of pigment in PDS. However, analysis of MRAP in additional cohorts (415 cases and 1645 controls) did not support an association with PDS. We also did not confirm a link between PMEL and PDS in our cohorts due to lack of reported mutations and similar frequency of the variants in PDS patients as in control subjects. CONCLUSIONS: We did not detect a statistical enrichment of mutations in melanosome-related genes in human PDS patients and we found conflicting data about the likely pathogenicity of MRAP mutations. PDS may have a complex genetic basis that is not easily unraveled with exome analyses.

A mouse model of Bardet-Biedl Syndrome has impaired fear memory, which is rescued by lithium treatment.

Primary cilia are microtubule-based organelles present on most cells that regulate many physiological processes, ranging from maintaining energy homeostasis to renal function. However, the role of these structures in the regulation of behavior remains unknown. To study the role of cilia in behavior, we employ mouse models of the human ciliopathy, Bardet-Biedl Syndrome (BBS). Here, we demonstrate that BBS mice have significant impairments in context fear conditioning, a form of associative learning. Moreover, we show that postnatal deletion of BBS gene function, as well as congenital deletion, specifically in the forebrain, impairs context fear conditioning. Analyses indicated that these behavioral impairments are not the result of impaired hippocampal long-term potentiation. However, our results indicate that these behavioral impairments are the result of impaired hippocampal neurogenesis. Two-week treatment with lithium chloride partially restores the proliferation of hippocampal neurons which leads to a rescue of context fear conditioning. Overall, our results identify a novel role of cilia genes in hippocampal neurogenesis and long-term context fear conditioning.

Autophagy stimulation reduces ocular hypertension in a murine glaucoma model via autophagic degradation of mutant myocilin.

Elevation of intraocular pressure (IOP) due to trabecular meshwork (TM) damage is associated with primary open-angle glaucoma (POAG). Myocilin mutations resulting in elevated IOP are the most common genetic causes of POAG. We have previously shown that mutant myocilin accumulates in the ER and induces chronic ER stress, leading to TM damage and IOP elevation. However, it is not understood how chronic ER stress leads to TM dysfunction and loss. Here, we report that mutant myocilin activated autophagy but was functionally impaired in cultured human TM cells and in a mouse model of myocilin-associated POAG (Tg-MYOCY437H). Genetic and pharmacological inhibition of autophagy worsened mutant myocilin accumulation and exacerbated IOP elevation in Tg-MYOCY437H mice. Remarkably, impaired autophagy was associated with chronic ER stress-induced transcriptional factor CHOP. Deletion of CHOP corrected impaired autophagy, enhanced recognition and degradation of mutant myocilin by autophagy, and reduced glaucoma in Tg-MYOCY437H mice. Stimulating autophagic flux via tat-beclin 1 peptide or torin 2 promoted autophagic degradation of mutant myocilin and reduced elevated IOP in Tg-MYOCY437H mice. Our study provides an alternate treatment strategy for myocilin-associated POAG by correcting impaired autophagy in the TM.

Photoreceptor cilia, in contrast to primary cilia, grant entry to a partially assembled BBSome.

The BBSome is a protein complex consisting of BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9 and BBS18 that associates with intraflagellar transport complexes and specializes in ciliary trafficking. In primary cilia, ciliary entry requires the fully assembled BBSome as well as the small GTPase, ARL6 (BBS3). Retinal photoreceptors possess specialized cilia. In light of key structural and functional differences between primary and specialized cilia, we examined the principles of BBSome recruitment to photoreceptor cilia. We performed sucrose gradient fractionation using retinal lysates of Bbs2-/-, Bbs7-/-, Bbs8-/- and Bbs3-/- mice to determine the status of BBSome assembly, then determined localization of BBSome components using immunohistochemistry. Surprisingly, we found that a subcomplex of the BBSome containing at least BBS1, BBS5, BBS8 and BBS9 is recruited to cilia in the absence of BBS2 or BBS7. In contrast, a BBSome subcomplex consisting of BBS1, BBS2, BBS5, BBS7 and BBS9 is found in Bbs8-/- retinas and is denied ciliary entry in photoreceptor cells. In addition, the BBSome remains fully assembled in Bbs3-/- retinas and can be recruited to photoreceptor cilia in the absence of BBS3. We compared phenotypic severity of their retinal degeneration phenotypes. These findings demonstrate that unlike primary cilia, photoreceptor cilia admit a partially assembled BBSome meeting specific requirements. In addition, the recruitment of the BBSome to photoreceptor cilia does not require BBS3. These findings indicate that the ciliary entry of the BBSome is subjected to cell-specific regulation, particularly in cells with highly adapted forms of cilia such as photoreceptors.