[Gene therapy in ophthalmology]. / Gentherapie in der Augenheilkunde.

In 2017 the gene therapy medication voretigene neparvovec-rzyl was approved by the U.S. Food and Drug Administration (FDA) for retinal gene therapy of hereditary retinal dystrophies caused by mutations in the RPE65 gene. Voretigene neparvovec-rzyl is a gene augmentation therapy using an  adeno-associated virus-based vector to express a healthy copy of the human RPE65 gene in the patient's retinal pigment epithelial (RPE) cells. The success of gene augmentation therapy in RPE65-linked retinal dystrophy encouraged research activities on the concept of gene supplementation to be extended to nongenetic diseases, such as age-related macular degeneration; however, it also showed that the principle of success cannot be easily extended to other retinal dystrophies. This review article presents the most commonly used principles and technologies of gene therapy and provides an overview of the current challenges and limitations. Furthermore, practice-relevant aspects of the indications and the treatment procedure are discussed. Particular attention is paid to the consideration of disease stages, especially with respect to patient's expectations and the evaluation of treatment success.

CNG channel-related retinitis pigmentosa.

The genes CNGA1 and CNGB1 encode the alpha and beta subunits of the rod CNG channel, a ligand-gated cation channel whose activity is controlled by cyclic guanosine monophosphate (cGMP). Autosomal inherited mutations in either of the genes lead to a progressive rod-cone retinopathy known as retinitis pigmentosa (RP). The rod CNG channel is expressed in the plasma membrane of the outer segment and functions as a molecular switch that converts light-mediated changes in cGMP into a voltage and Ca2+ signal. Here, we will first review the molecular properties and physiological role of the rod CNG channel and then discuss the characteristics of CNG-related RP. Finally, we will summarize recent activities in the field of gene therapy aimed at developing therapies for CNG-related RP.

Biology, Pathobiology and Gene Therapy of CNG Channel-Related Retinopathies.

The visual process begins with the absorption of photons by photopigments of cone and rod photoreceptors in the retina. In this process, the signal is first amplified by a cyclic guanosine monophosphate (cGMP)-based signaling cascade and then converted into an electrical signal by cyclic nucleotide-gated (CNG) channels. CNG channels are purely ligand-gated channels whose activity can be controlled by cGMP, which induces a depolarizing Na+/Ca2+ current upon binding to the channel. Structurally, CNG channels belong to the superfamily of pore-loop cation channels and share structural similarities with hyperpolarization-activated cyclic nucleotide (HCN) and voltage-gated potassium (KCN) channels. Cone and rod photoreceptors express distinct CNG channels encoded by homologous genes. Mutations in the genes encoding the rod CNG channel (CNGA1 and CNGB1) result in retinitis-pigmentosa-type blindness. Mutations in the genes encoding the cone CNG channel (CNGA3 and CNGB3) lead to achromatopsia. Here, we review the molecular properties of CNG channels and describe their physiological and pathophysiological roles in the retina. Moreover, we summarize recent activities in the field of gene therapy aimed at developing the first gene therapies for CNG channelopathies.

[Phototherapeutic keratectomy for recurrent corneal erosion of various epithelial origins: influence of depth of ablation on pachymetry and refraction]. / Phototherapeutische Keratektomie bei rezidivierenden Hornhauterosionen verschiedener epithelialer Genese: Einfluss der Ablationstiefe auf Pachymetrie und Refraktion.

BACKGROUND: Phototherapeutic keratectomy (PTK) is an established treatment method for patients suffering from either genetic corneal dystrophy or recurrent corneal erosion (RCE) without underlying basement membrane dystrophy, often caused by trauma. OBJECTIVE: This study aimed to describe the changes in manifest subjective refraction and pachymetry after PTK treatment in patients suffering from epithelial basement membrane dystrophy (EBMD) and traumatic or atraumatic RCE without underlying EBMD. MATERIAL AND METHODS: This was a retrospective, single-center study performed at the Department of Ophthalmology of the Ludwig-Maximilians University (Munich). Patient data were retrospective collected using the smart-eye database of the Department of Ophthalmology including diagnostic data from an autorefractometer and from the Pentacam HR. All laser treatments were performed with an ablation depth of 10 µm for EBMD patients and 6 µm for RCE patients without EBMD. RESULTS: Both collectives showed a decrease in pachymetry larger than the calculated ablation depths after a follow-up interval of 126 days (95% CI 104-147 days). While the EBMD collective receiving an ablation of 10 µm showed a decrease of 25.8 µm (N = 74; 95% CI 21.2-30.3; p < 0.001), the non-EBMD collective receiving an ablation of 6 µm showed a decrease of 12.3 µm (N = 44, 95% CI 7.0-17.7; p < 0.001). Both, total corneal refractive power (TCRP) as well as spherical equivalent (SE) offered no significant change in preoperative and postoperative comparison for the EBMD collective. On the other hand, patients without underlying EBMD showed a significant myopic decrease in SE of 0.4 dpt (±0.7 dpt SD, p < 0.05). The mean follow-up interval was 126 days (95% CI 104-147 days). CONCLUSION: PTK treatment offers an effective method for patients suffering from either EBMD dystrophy or RCE syndrome without underlying EBMD. The final ablation based on pachymetry at the apex can be estimated at 2.3 to 2.6 times higher compared to the original ablation depth. The reasons for this are on the one hand the laser ablation itself and the influence of the reactive wound healing of the corneal epithelium.

Gene Therapy with Voretigene Neparvovec Improves Vision and Partially Restores Electrophysiological Function in Pre-School Children with Leber Congenital Amaurosis.

Leber congenital amaurosis caused by mutations in the RPE65 gene belongs to the most severe early-onset hereditary childhood retinopathies naturally progressing to legal blindness. The novel gene therapy voretigene neparvovec is the first approved causative treatment option for this devastating eye disease and is specifically designed to treat RPE65-mediated retinal dystrophies. Herein, we present a follow-up of the youngest treated patients in Germany so far, including four pre-school children who received treatment with voretigene neparvovec at a single treatment center between January 2020 and May 2022. All patients underwent pars plana vitrectomy with circumferential peeling of the internal limiting membrane at the injection site and subretinal injection of voretigene neparvovec. Pre- and postoperative diagnostics included imaging (spectral domain optical coherence tomography, fundus autofluorescence, fundus wide-angle imaging), electrophysiologic examination (ERG), retinal light sensitivity measurements (FST) and visual acuity testing. Behavioral changes were assessed using a questionnaire and by observing the children's vision-guided behavior in different levels of illumination. All children showed marked increase in vision-guided behavior shortly after therapy, as well as marked increase in visual acuity in the postoperative course up to full visual acuity in one child. Two eyes showed partial electrophysiological recovery of an ERG that was undetectable before treatment-a finding that has not been described in humans before.

In Vivo Potency Testing of Subretinal rAAV5.hCNGB1 Gene Therapy in the Cngb1 Knockout Mouse Model of Retinitis Pigmentosa.

Retinitis pigmentosa type 45 (RP45) is an autosomal-recessively inherited blinding disease caused by mutations in the cyclic nucleotide-gated channel subunit beta 1 (CNGB1) gene. In this study, we developed and tested a novel gene supplementation therapy suitable for clinical translation. To this end, we designed a recombinant adeno-associated virus (rAAV) vector carrying a genome that features a novel human rhodopsin promoter (hRHO194) driving rod-specific expression of full-length human CNGB1 (rAAV5.hCNGB1). rAAV5.hCNGB1 was evaluated for efficacy in the Cngb1 knockout (Cngb1-/-) mouse model of RP45. In particular, increasing doses of rAAV5.hCNGB1 were delivered through single subretinal injection in 4-week-old Cngb1-/- mice and the treatment effect was assessed over a follow-up period of 9 months at the level of (1) retinal morphology, (2) retinal function, (3) vision-guided behavior, and (4) transgene expression. We found that subretinal treatment with rAAV5.hCNGB1 resulted in efficient expression of the human CNGB1 protein in mouse rods and was able to normalize the expression of the endogenous mouse CNGA1 subunit, which together with CNGB1 forms the native heterotetrameric cyclic guanosine monophosphate-gated cation channel in rod photoreceptors. The treatment led to a dose-dependent recovery of rod photoreceptor-driven function and preservation of retinal morphology in Cngb1-/- mice. In summary, these results demonstrate the efficacy of hCNGB1 gene supplementation therapy in the Cngb1-/- mouse model of RP45 and support the translation of this approach toward future clinical application.

Novel AAV capsids for intravitreal gene therapy of photoreceptor disorders.

Gene therapy using recombinant adeno-associated virus (rAAV) vectors to treat blinding retinal dystrophies has become clinical reality. Therapeutically impactful targeting of photoreceptors still relies on subretinal vector delivery, which detaches the retina and harbours substantial risks of collateral damage, often without achieving widespread photoreceptor transduction. Herein, we report the development of novel engineered rAAV vectors that enable efficient targeting of photoreceptors via less invasive intravitreal administration. A unique in vivo selection procedure was performed, where an AAV2-based peptide-display library was intravenously administered in mice, followed by isolation of vector DNA from target cells after only 24 h. This stringent selection yielded novel vectors, termed AAV2.GL and AAV2.NN, which mediate widespread and high-level retinal transduction after intravitreal injection in mice, dogs and non-human primates. Importantly, both vectors efficiently transduce photoreceptors in human retinal explant cultures. As proof-of-concept, intravitreal Cnga3 delivery using AAV2.GL lead to cone-specific expression of Cnga3 protein and rescued photopic cone responses in the Cnga3-/- mouse model of achromatopsia. These novel rAAV vectors expand the clinical applicability of gene therapy for blinding human retinal dystrophies.

ER stress-induced aggresome trafficking of HtrA1 protects against proteotoxicity.

High temperature requirement A1 (HtrA1) belongs to an ancient protein family that is linked to various human disorders. The precise role of exon 1-encoded N-terminal domains and how these influence the biological functions of human HtrA1 remain elusive. In this study, we traced the evolutionary origins of these N-terminal domains to a single gene fusion event in the most recent common ancestor of vertebrates. We hypothesized that human HtrA1 is implicated in unfolded protein response. In highly secretory cells of the retinal pigmented epithelia, endoplasmic reticulum (ER) stress upregulated HtrA1. HtrA1 co-localized with vimentin intermediate filaments in highly arborized fashion. Upon ER stress, HtrA1 tracked along intermediate filaments, which collapsed and bundled in an aggresome at the microtubule organizing center. Gene silencing of HtrA1 altered the schedule and amplitude of adaptive signaling and concomitantly resulted in apoptosis. Restoration of wild-type HtrA1, but not its protease inactive mutant, was necessary and sufficient to protect from apoptosis. A variant of HtrA1 that harbored exon 1 substitutions displayed reduced efficacy in rescuing cells from proteotoxicity. Our results illuminate the integration of HtrA1 in the toolkit of mammalian cells against protein misfolding and the implications of defects in HtrA1 in proteostasis.