Thiago Cabral

Gene and cell‐based therapies for inherited retinal disorders: An update

Retinal degenerations present a unique challenge as disease progression is irreversible and the retina has little regenerative potential. No current treatments for inherited retinal disease have the ability to reverse blindness, and current dietary supplement recommendations only delay disease progression with varied results. However, the retina is anatomically accessible and capable of being monitored at high resolution in vivo. This, in addition to the immune‐privileged status of the eye, has put ocular disease at the forefront of advances in gene‐ and cell‐based therapies. This review provides an update on gene therapies and randomized control trials for inherited retinal disease, including Leber congenital amaurosis, choroideremia, retinitis pigmentosa, Usher syndrome, X‐linked retinoschisis, Leber hereditary optic neuropathy, and achromatopsia. New gene‐modifying and cell‐based strategies are also discussed.

Correction of Monogenic and Common Retinal Disorders with Gene Therapy

The past decade has seen major advances in gene-based therapies, many of which show promise for translation to human disease. At the forefront of research in this field is ocular disease, as the eye lends itself to gene-based interventions due to its accessibility, relatively immune-privileged status, and ability to be non-invasively monitored. A landmark study in 2001 demonstrating successful gene therapy in a large-animal model for Leber congenital amaurosis set the stage for translation of these strategies from the bench to the bedside. Multiple clinical trials have since initiated for various retinal diseases, and further improvements in gene therapy techniques have engendered optimism for alleviating inherited blinding disorders. This article provides an overview of gene-based strategies for retinal disease, current clinical trials that engage these strategies, and the latest techniques in genome engineering, which could serve as the next frontline of therapeutic interventions.

Crispr applications in ophthalmologic genome surgery

Purpose of review The present review seeks to summarize and discuss the application of clustered regularly interspaced short palindromic repeats (CRISPR)-associated systems (Cas) for genome editing, also called genome surgery, in the field of ophthalmology. Recent findings Precision medicine is an emerging approach for disease treatment and prevention that takes into account the variability of an individuals genetic sequence. Various groups have used CRISPR-Cas genome editing to make significant progress in mammalian preclinical models of eye disease, the basic science of eye development in zebrafish, the in vivo modification of ocular tissue, and the correction of stem cells with therapeutic applications. In addition, investigators have creatively used the targeted mutagenic potential of CRISPR-Cas systems to target pathogenic alleles in vitro. Summary Over the past year, CRISPR-Cas genome editing has been used to correct pathogenic mutations in vivo and in transplantable stem cells. Although off-target mutagenesis remains a concern, improvement in CRISPR-Cas technology and careful screening for undesired mutations will likely lead to clinical eye therapeutics employing CRISPR-Cas systems in the near future.

Retrospective Analysis of Structural Disease Progression in Retinitis Pigmentosa Utilizing Multimodal Imaging

In this report, we assess the natural progression rate of retinitis pigmentosa (RP) over an average of three years using spectral-domain optical coherence tomography (SD-OCT) and short wavelength fundus autofluorescence (SW-AF). Measurement of the ellipsoid zone (EZ) line width and hyperautofluorescent ring diameters was performed in 81 patients with RP in a retrospective, longitudinal fashion. Rate of structural disease progression, symmetry between eyes, and test-retest variability were quantified. We observed on average, EZ-line widths decreased by 140 µm (5.2%, p < 0.001) per year, and average horizontal and vertical hyperautofluorescent ring diameters decreased by 149 µm (3.6%, p < 0.001) and 120 µm (3.9%, p < 0.001) per year, respectively. The 95th percentile of this cohort had differences in progression slopes between eyes that were less than 154 µm, 118 µm, and 132 µm for EZ-line width and horizontal and vertical ring diameters, respectively. For all measures except horizontal ring diameter, progression rates were significantly slower at end-stage disease. From our data, we observed a statistically significant progression rate in EZ line width and SW-AF ring diameters over time, verifying the utility of these measurements for disease monitoring purposes. Additionally, calculated differences in progression slopes between eyes may prove useful for investigators evaluating the efficacy of unilateral treatments for RP in clinical trials.

Ocular Biocompatibility of Poly-N-Isopropylacrylamide (pNIPAM)

Purpose. To study the safety of intravitreal injections of poly-N-isopropylacrylamide (pNIPAM) tissue adhesive in rabbit eyes. Methods. Twelve study rabbits received an intravitreal injection of 0.1 mL 50% pNIPAM in the right eye. Follow-up examinations included color fundus photography, fundus fluorescein angiography (FA), optical coherence tomography (OCT), and electroretinography (ERG). Subsequent to the last follow-up assessment, the rabbits were sacrificed and histopathological study on the scleral incision sites was performed. Results. All study animals developed mild to moderate levels of inflammatory reaction in the conjunctiva, anterior chamber, and the anterior vitreous during the first month of follow-up. After this period, the level of the inflammatory reaction progressively decreased and completely disappeared after the third month of follow-up. The lens and cornea remained clear during the entire follow-up period. OCT and FA did not show areas of retinal damage or neovascularization. Histological and ERG studies of eyes injected with pNIPAM demonstrated absence of retinal toxicity. Conclusion. Intravitreal injections of pNIPAM were nontoxic in this animal study, and pNIPAM may be safe to be used as a bioadhesive in certain retinal diseases.

Retinal and choroidal angiogenesis: a review of new targets

Retinal and choroidal neovascularization are a major cause of significant visual impairment, worldwide. Understanding the various factors involved in the accompanying physiopathology is vital for development of novel treatments, and most important, for preserving patient vision. The intraocular use of anti-vascular endothelial growth factor therapeutics has improved management of the retinal and choroidal neovascularization but some patients do not respond, suggesting other vascular mediators may also contribute to ocular angiogenesis. Several recent studies examined possible new targets for future anti-angiogenic therapies. Potential targets of retinal and choroidal neovascularization therapy include members of the platelet-derived growth factor family, vascular endothelial growth factor sub-family, epidermal growth factor family, fibroblast growth factor family, transforming growth factor-β superfamily (TGF-β1, activins, follistatin and bone morphogenetic proteins), angiopoietin-like family, galectins family, integrin superfamily, as well as pigment epithelium derived factor, hepatocyte growth factor, angiopoietins, endothelins, hypoxia-inducible factors, insulin-like growth factors, cytokines, matrix metalloproteinases and their inhibitors and glycosylation proteins. This review highlights current antiangiogenic therapies under development, and discusses future retinal and choroidal pro- and anti-angiogenic targets as wells as the importance of developing of new drugs.

Bevacizumab Injection in Patients with Neovascular Age-Related Macular Degeneration Increases Angiogenic Biomarkers

Purpose To evaluate the expression of 19 angiogenic biomarkers in the aqueous humor before and after intravitreal bevacizumab injection (IVB) in eyes with neovascular age-related macular degeneration (AMD). Design Prospective, noncomparative, interventional case series. Participants Twenty-three eyes of 23 treatment-naïve patients with choroidal neovascularization (CNV) secondary to neovascular AMD. Methods Eyes were diagnosed with CNV secondary to neovascular AMD and were treated with 3 monthly IVBs. Aqueous humor samples were obtained by anterior chamber paracentesis at baseline and immediately before each intravitreal bevacizumab injection. Main Outcome Measures Aqueous humor levels of 19 angiogenic biomarkers (angiopoietin 2, bone morphogenetic protein 9 [BMP-9], epidermal growth factor [EGF], endoglin, endothelin 1, fibroblast growth factor [FGF]-1 and FGF-2, follistatin, granulocyte colony-stimulating factor [GCSF], heparin-binding EGF-like growth factor [HB-EGF], hepatocyte growth factor [HGF], interleukin 8, leptin, placental growth factor [PLGF], vascular endothelial growth factor [VEGF]-A, VEGF-C, VEGF-D, and tissue inhibitor of metalloproteinases [TIMP]-1 and TIMP-2) were measured. Best-corrected visual acuity (BCVA), spectral-domain OCT parameters, and intraocular pressure also were evaluated. Results Baseline aqueous VEGF-A expression was elevated in all study eyes before treatment initiation. A statistically significant decrease of VEGF-A was observed at the 1- and 2-month follow-ups. A statistically significant increased concentration was observed in 7 biomarkers: VEGF-C, angiopoietin 2, endothelin 1, follistatin, HB-EGF, HGF, and interleukin 8. The other 11 study biomarker levels (VEGF-D, BMP-9, EGF, endoglin, FGF-1, FGF-2, GCSF, leptin, PLGF, TIMP-1, and TIMP-2) did not show any significant difference during follow-up. The BCVA statistically improved significantly at 2 months. Spectral-domain OCT parameters improved significantly at all follow-ups. Mean intraocular pressure values were not statistically different during the study period. Conclusions Despite a decrease in VEGF-A, the aqueous levels of VEGF-C, angiopoietin 2, endothelin 1, follistatin, HB-EGF, HGF, and interleukin 8 increased significantly after intravitreal injection of bevacizumab. These upregulated angiogenic biomarkers may represent new therapeutic targets in exudative AMD.

Electroretinography Reveals Difference in Cone Function between Syndromic and Nonsyndromic USH2A Patients

Usher syndrome is an inherited and irreversible disease that manifests as retinitis pigmentosa (RP) and bilateral neurosensory hearing loss. Mutations in Usherin 2A (USH2A) are not only a frequent cause of Usher syndrome, but also nonsyndromic RP. Although gene- and cell-based therapies are on the horizon for RP and Usher syndrome, studies characterizing natural disease are lacking. In this retrospective analysis, retinal function of USH2A patients was quantified with electroretinography. Both groups had markedly reduced rod and cone responses, but nonsyndromic USH2A patients had 30 Hz-flicker electroretinogram amplitudes that were significantly higher than syndromic patients, suggesting superior residual cone function. There was a tendency for Usher syndrome patients to have a higher distribution of severe mutations, and alleles in this group had a higher odds of containing nonsense or frame-shift mutations. These data suggest that the previously reported severe visual phenotype seen in syndromic USH2A patients could relate to a greater extent of cone dysfunction. Additionally, a genetic threshold may exist where mutation burden relates to visual phenotype and the presence of hearing deficits. The auditory phenotype and allelic hierarchy observed among patients should be considered in prospective studies of disease progression and during enrollment for future clinical trials.

CRISPR Repair Reveals Causative Mutation in a Preclinical Model of Retinitis Pigmentosa: A Brief Methodology

CRISPR/Cas9 genome engineering is currently the leading genome surgery technology in most genetics laboratories. Combined with other complementary techniques, it serves as a powerful tool for uncovering genotype-phenotype correlations. Here, we describe a simplified protocol that was used in our publication, CRISPR Repair Reveals Causative Mutation in a Preclinical Model of Retinitis Pigmentosa, providing an overview of each section of the experimental process.

CRISPR-Cas Genome Surgery in Ophthalmology

Genetic disease affecting vision can significantly impact patient quality of life. Gene therapy seeks to slow the progression of these diseases by treating the underlying etiology at the level of the genome. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated systems (Cas) represent powerful tools for studying diseases through the creation of model organisms generated by targeted modification and by the correction of disease mutations for therapeutic purposes. CRISPR-Cas systems have been applied successfully to the visual sciences and study of ophthalmic disease – from the modification of zebrafish and mammalian models of eye development and disease, to the correction of pathogenic mutations in patient-derived stem cells. Recent advances in CRISPR-Cas delivery and optimization boast improved functionality that continues to enhance genome-engineering applications in the eye. This review provides a synopsis of the recent implementations of CRISPR-Cas tools in the field of ophthalmology.