Evaluating precision medicine approaches for gene therapy in patient-specific cellular models of Bietti crystalline dystrophy.

Patient-specific induced pluripotent stem cell-derived (iPSC-derived) cell lines allow for therapies to be tailored to individual patients, increasing therapeutic precision and efficiency. Bietti crystalline dystrophy (BCD) is a rare blinding disease estimated to affect about 67,000 individuals worldwide. Here, we used iPSC-derived retinal pigment epithelium (iRPE) cells from patients with BCD to evaluate adeno-associated virus-mediated (AAV-mediated) gene augmentation therapy strategies. We found that BCD iRPE cells were vulnerable to blue light-induced oxidative stress and that cellular phenotype can be quantified using 3 robust biomarkers: reactive oxygen species (ROS), 4-hydroxy 2-nonenal (4-HNE) levels, and cell death rate. Additionally, we demonstrated that AAV-mediated gene therapy can significantly reduce light-induced cell death in BCD iRPE cells. This is the first proof-of-concept study to our knowledge to show that AAV-CYP4V2 gene therapy can be used to treat light-induced RPE damage in BCD. Furthermore, we observed significant variability in cellular phenotypes among iRPE from patients with BCD of divergent mutations, which outlined genotype-phenotype correlations in BCD patient-specific cell disease models. Our results reveal that patient-specific iRPE cells retained personalized responses to AAV-mediated gene therapy. Therefore, this approach can advance BCD therapy and set a precedent for precision medicine in other diseases, emphasizing the necessity for personalization in healthcare to accommodate individual diversity.

In vivo genome editing via CRISPR/Cas9-mediated homology-independent targeted integration for Bietti crystalline corneoretinal dystrophy treatment.

Bietti crystalline corneoretinal dystrophy (BCD) is an autosomal recessive chorioretinal degenerative disease without approved therapeutic drugs. It is caused by mutations in CYP4V2 gene, and about 80% of BCD patients carry mutations in exon 7 to 11. Here, we apply CRISPR/Cas9 mediated homology-independent targeted integration (HITI)-based gene editing therapy in HEK293T cells, BCD patient derived iPSCs, and humanized Cyp4v3 mouse model (h-Cyp4v3mut/mut) using two rAAV2/8 vectors via sub-retinal administration. We find that sgRNA-guided Cas9 generates double-strand cleavage on intron 6 of the CYP4V2 gene, and the HITI donor inserts the carried sequence, part of intron 6, exon 7-11, and a stop codon into the DNA break, achieving precise integration, effective transcription and translation both in vitro and in vivo. HITI-based editing restores the viability of iPSC-RPE cells from BCD patient, improves the morphology, number and metabolism of RPE and photoreceptors in h-Cyp4v3mut/mut mice. These results suggest that HITI-based editing could be a promising therapeutic strategy for those BCD patients carrying mutations in exon 7 to 11, and one injection will achieve lifelong effectiveness.

Gene replacement therapy in Bietti crystalline corneoretinal dystrophy: an open-label, single-arm, exploratory trial.

Bietti crystalline corneoretinal dystrophy is an inherited retinal disease caused by mutations in CYP4V2, which results in blindness in the working-age population, and there is currently no available treatment. Here, we report the results of the first-in-human clinical trial (NCT04722107) of gene therapy for Bietti crystalline corneoretinal dystrophy, including 12 participants who were followed up for 180-365 days. This open-label, single-arm exploratory trial aimed to assess the safety and efficacy of a recombinant adeno-associated-virus-serotype-2/8 vector encoding the human CYP4V2 protein (rAAV2/8-hCYP4V2). Participants received a single unilateral subretinal injection of 7.5 × 1010 vector genomes of rAAV2/8-hCYP4V2. Overall, 73 treatment-emergent adverse events were reported, with the majority (98.6%) being of mild or moderate intensity and considered to be procedure- or corticosteroid-related; no treatment-related serious adverse events or local/systemic immune toxicities were observed. Compared with that measured at baseline, 77.8% of the treated eyes showed improvement in best-corrected visual acuity (BCVA) on day 180, with a mean ± standard deviation increase of 9.0 ± 10.8 letters in the 9 eyes analyzed (p = 0.021). By day 365, 80% of the treated eyes showed an increase in BCVA, with a mean increase of 11.0 ± 10.6 letters in the 5 eyes assessed (p = 0.125). Importantly, the patients' improvement observed using multifocal electroretinogram, microperimetry, and Visual Function Questionnaire-25 further supported the beneficial effects of the treatment. We conclude that the favorable safety profile and visual improvements identified in this trial encourage the continued development of rAAV2/8-hCYP4V2 (named ZVS101e).

Mini-Review: Clinical Features and Management of Granular Corneal Dystrophy Type 2.

Granular corneal dystrophy type 2 (GCD2) is an autosomal dominant corneal stromal dystrophy that is caused by p.Arg124His mutation of transforming growth factor ß induced (TGFBI) gene. It is characterized by well demarcated granular shaped opacities in central anterior stroma and as the disease progresses, extrusion of the deposits results in ocular pain due to corneal epithelial erosion. Also, diffuse corneal haze which appears late, causes decrease in visual acuity. The prevalence of GCD2 is high in East Asia including Korea. Homozygous patients show a severe phenotype from an early age, and the heterozygote phenotype varies among patients, depending on several types of compound heterozygous TGFBI mutations. In the initial stage, conservative treatments such as artificial tears, antibiotic eye drops, and bandage contact lenses are used to treat corneal erosion. Different surgical methods are used depending on the depth and extent of the stromal deposits. Phototherapeutic keratectomy removes anterior opacities and is advantageous in terms of its applicability and repeatability. For deeper lesions, deep anterior lamellar keratoplasty can be used as the endothelial layer is not always affected. Recurrence following these treatments are reported within a wide range of rates in different studies due to varying definition of recurrence and follow-up period. In patients who have undergone corneal laser vision-correction surgeries such as photorefractive keratectomy, LASEK, or LASIK including SMILE surgery, corneal opacity exacerbates rapidly with severe deterioration of visual acuity. Further investigations on new treatments of GCD2 are necessary.

[Salzmann nodular degeneration: pathogenesis, clinical characteristics and treatment]. / Uzelkovaya degeneratsiya Zal'tsmana: osobennosti patogeneza, klinicheskoi kartiny i lecheniya.

Salzmann nodular degeneration (SND) is a rare non-inflammatory disease observed primarily in middle-aged women. The disease generally occurs in patients with chronic inflammation of the anterior ocular surface. Its etiopathogenesis remains poorly investigated. This literature review describes clinical manifestations, risk factors and diagnostic methods, evaluates the effectiveness of different therapeutic and surgical treatment methods. Understanding of the pathogenetic mechanisms, precise diagnosis and identification of the risk factors can help clinical physicians make the optimal choice of treatment strategy and achieve the best clinical outcomes.

Identifying and categorizing compounds that reduce corneal transforming growth factor beta induced protein levels: a scoping review.

INTRODUCTION: Transforming growth factor beta induced (TGFBI) gene mutations have been reported as the cause of a group of genetically inherited, visually debilitating, corneal dystrophies (CD). A scoping literature review to identify and categorize compounds that inhibit corneal TGFBI expression and/or promote TGFBIp degradation was performed. Emphasis was given to their potential to be used as a cost-effective approach via drug repurposing. AREAS COVERED: We performed a thorough search of original peer-reviewed literature using electronic bibliographic databases and selected articles according to a set of criteria. The total number of articles retrieved from the search terms applied to the databases was 2344. The number of relevant full-text articles included added up to 19. We identified 16 compounds that can theoretically reduce the levels of mutant TGFBIp in human corneal cells. EXPERT OPINION: Currently, the only temporary treatments available for this condition are lubricant drops and surgery. Here, we explored the crosstalk between cascades that regulate TGFBI expression and identified compounds that target these pathways. Compounds that inhibit DNA synthesis and function, increase elimination of TGFBIp or bind to mutant TGFBIp were also explored with the aim of highlighting promising compounds that can be used in future cost-effective drug-repurposing studies.

Update on the Corneal Dystrophies-Genetic Testing and Therapy.

ABSTRACT: One major purpose of the IC3D Corneal Dystrophy Nomenclature Revision was to include genetic information with a goal of facilitating investigation into the pathogenesis, treatment, and perhaps even prevention of the corneal dystrophies, an ambitious goal. Over a decade has passed since the first publication of the IC3D Corneal Dystrophy Nomenclature Revision. Gene therapy is available for an early-onset form of inherited retinal degeneration called Leber congenital amaurosis, but not yet for corneal degenerations. We review the current state of affairs regarding our original ambitious goal. We discuss genetic testing, gene therapy [RNA interference (RNAi) and genome editing], and ocular delivery of corneal gene therapy for the corneal dystrophies. Why have gene therapy techniques not yet been introduced for the corneal dystrophies?

New Frontier in the Management of Corneal Dystrophies: Basics, Development, and Challenges in Corneal Gene Therapy and Gene Editing.

ABSTRACT: Corneal dystrophies represent a group of heterogeneous hereditary disorders causing progressive corneal opacification and blindness. Current corneal transplant management for corneal dystrophies faces the challenges of repeated treatments, complex surgical procedures, shortage of appropriate donor cornea, and, more importantly, graft rejection. Genetic medicine could be an alternative treatment regime to overcome such challenges. Cornea carries promising scope for a gene-based therapy involving gene supplementation, gene silencing, and gene editing in both ex vivo and in vivo platforms. In the cornea, ex vivo gene therapeutic strategies were attempted for corneal graft survival, and in vivo gene augmentation therapies aimed to prevent herpes stromal keratitis, neovascularization, corneal clouding, and wound healing. However, none of these studies followed a clinical trial-based successful outcome. CRISPR/Cas system offers a broad scope of gene editing and engineering to correct underlying genetic causes in corneal dystrophies. Corneal tissue--specific gene correction in vitro with minimal off-target effects and optimal gene correction efficiency followed by their successful surgical implantation, or in vivo CRISPR administration targeting pathogenic genes finds a way to explore therapeutic intervention for corneal dystrophies. However, there are many limitations associated with such CRISPR-based corneal treatment management. This review will look into the development of corneal gene therapy and CRISPR-based study in corneal dystrophies, associated challenges, potential approaches, and future directions.

AAV2-mediated gene therapy for Bietti crystalline dystrophy provides functional CYP4V2 in multiple relevant cell models.

Bietti crystalline dystrophy (BCD) is an inherited retinal disease (IRD) caused by mutations in the CYP4V2 gene. It is a relatively common cause of IRD in east Asia. A number of features of this disease make it highly amenable to gene supplementation therapy. This study aims to validate a series of essential precursor in vitro experiments prior to developing a clinical gene therapy for BCD. We demonstrated that HEK293, ARPE19, and patient induced pluripotent stem cell (iPSC)-derived RPE cells transduced with AAV2 vectors encoding codon optimization of CYP4V2 (AAV2.coCYP4V2) resulted in elevated protein expression levels of CYP4V2 compared to those transduced with AAV2 vectors encoding wild type CYP4V2 (AAV2.wtCYP4V2), as assessed by immunocytochemistry and western blot. Similarly, we observed significantly increased CYP4V2 enzyme activity in cells transduced with AAV2.coCYP4V2 compared to those transduced with AAV2.wtCYP4V2. We also showed CYP4V2 expression in human RPE/choroid explants transduced with AAV2.coCYP4V2 compared to those transduced with AAV2.wtCYP4V2. These preclinical data support the further development of a gene supplementation therapy for a currently untreatable blinding condition-BCD. Codon-optimized CYP4V2 transgene was superior to wild type in terms of protein expression and enzyme activity. Ex vivo culture of human RPE cells provided an effective approach to test AAV-mediated transgene delivery.

Corneal dystrophies: pathophysiological, genetic, clinical, and therapeutic considerations.

Corneal dystrophies represent a group of progressive, genetically transmitted disorders with variable pathological, histological, and clinical manifestations. Recent ophthalmological examination techniques and genetic investigation methods have brought benefits in the deepening of these conditions. However, many aspects remain unknown, so the results of treatments are unsatisfactory. The aim of this article was to recall the clinical, genetic, histological and treatment types of these conditions.

The role of the extracellular matrix protein TGFBI in cancer.

The secreted extracellular protein, transforming growth factor beta induced (TGFBI or ßIGH3), has roles in regulating numerous biological functions, including cell adhesion and bone formation, both during embryonic development and during the pathogenesis of human disease. TGFBI has been most studied in the context of hereditary corneal dystrophies, where mutations in TGFBI result in accumulation of TGFBI in the cornea. In cancer, early studies focused on TGFBI as a tumor suppressor, in part by promoting chemotherapy sensitivity. However, in established tumors, TGFBI largely has a role in promoting tumor progression, with elevated levels correlating to poorer clinical outcomes. As an important regulator of cancer progression, TGFBI expression and function is tightly regulated by numerous mechanisms including epigenetic silencing through promoter methylation and microRNAs. Mechanisms to target TGFBI have potential clinical utility in treating advanced cancers, while assessing TGFBI levels could be a biomarker for chemotherapy resistance and tumor progression.

Clinical Evaluation of Electrolysis for Reis-Bücklers Corneal Dystrophies and In Vivo Histological Analysis Using Anterior Segment Optical Coherence Tomography.

PURPOSE: To report the effectiveness and clinical outcomes of corneal electrolysis for Reis-Bücklers corneal dystrophies (RBCDs) and provide in vivo histological analysis using anterior segment optical coherence tomography (AS-OCT). METHODS: A total of 7 patients with RBCD (2 male patients, 5 female patients; mean age, 50.0 years) from 3 pedigrees were enrolled in this prospective study. All patients underwent corneal electrolysis for decreased visual acuity or blurred vision. Changes in visual acuity, refraction, and corneal thickness were evaluated 1 month after electrolysis and statistically analyzed. Changes observed in AS-OCT images before and after electrolysis were qualitatively and quantitatively assessed. RESULTS: A total of 22 electrolysis procedures were performed on 7 patients during the median follow-up period of 10.7 years. Mean visual acuity significantly improved postoperatively, from 0.59 to 0.31 logarithm of the minimum angle of resolution (P = 0.013). Changes in refraction (from -2.42 to -2.12 D) and corneal thickness (from 570.8 to 577.6 µm) were not significant (P = 0.77 and P = 0.80, respectively). In all cases, assessment of AS-OCT images showed that the band lesion in Bowman's layer became less reflective after electrolysis. The mean thickness of the band lesion decreased from 101.5 to 88.3 µm (P = 0.002). CONCLUSIONS: Corneal electrolysis is an excellent treatment option for corneal opacities caused by RBCD. Corneal electrolysis improves visual acuity without changing corneal thickness or refraction. Therefore, repeat corneal electrolysis over the years might be a preferred long-term treatment strategy for RBCD. Moreover, AS-OCT is suitable for evaluating the effectiveness of this treatment and might be useful during postoperative follow-up.

Atypical Case of Bilateral Chandler Syndrome With Recurrent Band Keratopathy.

PURPOSE: To report a unique case of bilateral Chandler syndrome with recurrent band keratopathy. METHODS: This is a retrospective observational case report. RESULTS: A 39-year-old Asian man presented with progressive painless diminution of vision in both eyes for 6 years. Examination revealed diffuse corneal edema, hammered silver appearance of endothelium with guttae-like lesions, and corectopia in the right eye and mild corneal edema, central band keratopathy, and guttae-like lesions on the endothelium and peripheral anterior synechiae in the left eye. Routine specular microscopy, confocal microscopy, and pachymetry were performed. A clinical diagnosis of bilateral Chandler syndrome with band keratopathy was made. Superficial epithelial keratectomy with ethylenediaminetetraacetic acid (EDTA) chelation was performed in the left eye first, followed by Descemet-stripping automated endothelial keratoplasty in the right eye. Histopathological examination of the surgically excised Descemet membrane in the right eye showed multilayered endothelium with adhered epithelial cells consistent with Chandler syndrome. At 9-month follow-up, the right eye showed a clear cornea with an attached graft and the left eye revealed recurrence of central band keratopathy for which repeat EDTA chelation was successfully performed. CONCLUSIONS: Recurrent band keratopathy coincident with endothelial dysfunction in iridocorneal endothelial syndrome can be repeatedly treated with EDTA chelation, whereas endothelial keratoplasty might be delayed until the time point of corneal decompensation.

HIV and antiretroviral therapy-related fat alterations.

Early in the HIV epidemic, lipodystrophy, characterized by subcutaneous fat loss (lipoatrophy), with or without central fat accumulation (lipohypertrophy), was recognized as a frequent condition among people living with HIV (PLWH) receiving combination antiretroviral therapy. The subsequent identification of thymidine analogue nucleoside reverse transcriptase inhibitors as the cause of lipoatrophy led to the development of newer antiretroviral agents; however, studies have demonstrated continued abnormalities in fat and/or lipid storage in PLWH treated with newer drugs (including integrase inhibitor-based regimens), with fat gain due to restoration to health in antiretroviral therapy-naive PLWH, which is compounded by the rising rates of obesity. The mechanisms of fat alterations in PLWH are complex, multifactorial and not fully understood, although they are known to result in part from the direct effects of HIV proteins and antiretroviral agents on adipocyte health, genetic factors, increased microbial translocation, changes in the adaptive immune milieu after infection, increased tissue inflammation and accelerated fibrosis. Management includes classical lifestyle alterations with a role for pharmacological therapies and surgery in some patients. Continued fat alterations in PLWH will have an important effect on lifespan, healthspan and quality of life as patients age worldwide, highlighting the need to investigate the critical uncertainties regarding pathophysiology, risk factors and management.

Treating Bietti crystalline dystrophy in a high-fat diet-exacerbated murine model using gene therapy.

Lipid metabolic deficiencies are associated with many genetic disorders. Bietti crystalline dystrophy (BCD), a blindness-causing inherited disorder with changed lipid profiles, is more common in Chinese and Japanese than other populations. Our results reveal that mouse models lacking Cyp4v3 have less physiological and functional changes than those of BCD patients with this gene defect. After the administration of a high-fat diet (HFD), the occurrence of retinal lesions were both accelerated and aggregated in the Cyp4v3-/- mouse models, implying that changed lipid levels were not only associated factors but also risk factors to BCD patients. Facilitated by the results, we found that the reduced electroretinography waveforms and retinal thickness observed in the HFD-induced mouse models were effectively recovered after subretinal delivery of a human CYP4V2 gene carried by an adeno-associated virus vector, which demonstrates the potential curability of BCD by gene therapy.

Biochemical mechanisms of aggregation in TGFBI-linked corneal dystrophies.

Transforming growth factor-ß-induced protein (TGFBIp), an extracellular matrix protein, is the second most abundant protein in the corneal stroma. In this review, we summarize the current knowledge concerning the expression, molecular structure, binding partners, and functions of human TGFBIp. To date, 74 mutations in the transforming growth factor-ß-induced gene (TGFBI) are associated with amyloid and amorphous protein deposition in TGFBI-linked corneal dystrophies. We discuss the current understanding of the biochemical mechanisms of TGFBI-linked corneal dystrophies and propose that mutations leading to granular corneal dystrophy (GCD) decrease the solubility of TGFBIp and affect the interactions between TGFBIp and components of the corneal stroma, whereas mutations associated with lattice corneal dystrophy (LCD) lead to a destabilization of the protein that disrupts proteolytic turnover, especially by the serine protease HtrA1. Future research should focus on TGFBIp function in the cornea, confirmation of the biochemical mechanisms in vivo, and the development of disease models. Future therapies for TGFBI-linked corneal dystrophies might include topical agents that regulate protein aggregation or gene therapy that targets the mutant allele by CRISPR/Cas9 technology.

Harboyan Syndrome.

Harboyan syndrome is a degenerative corneal disorder characterized by congenital hereditary endothelial dystrophy along with progressive, post lingual sensorineural hearing loss. We present the case of a 16-year-old female, who came to us for her follow up after surgical correction for bilateral corneal opacities (bilateral keratplasy) and use of hearing aid for SNHL. Her symptoms resolved significantly. Currently, she's living a healthy life after being treated for Harboyan syndrome. Physical examination and laboratory investigations ruled out other causes (eg: Congenital cataracts, Peters anomaly, Sclerocornea) and the diagnosis of Harboyan syndrome was established.