484. Preclinical Proof of Concept of Transcriptional Silencing and Replacement Strategy for Treatment of Retinitis Pigmentosa Due To RHODOPSIN Mutations

Silencing and replacement strategy is a promising approach to overcome mutational heterogeneity of genetic defects. In autosomal dominant retinitis pigmentosa (adRP) due to rhodopsin gene (RHO) approximately 200 different mutations have been described, posing a challenge for the design of effective therapeutics.We designed a silencing and replacement strategy based on transcriptional silencing through an artificial zinc finger DNA-binding protein lacking effector domains (ZF6DBD), and tested both efficacy and safety in two animal models.In a murine model of adRP, we show that AAV-mediate retinal delivery (AAV2/8-CMV-ZF6-DBD) is associated with selective transcriptional silencing of the human mutated allele resulting in morphological and functional (Electroretinography, ERG a-wave and b-wave responses) rescue. We then tested the effect of transcriptional silencing in the porcine large pre-clinical model. Delivery of a low dose (AAV2/8-CMV-ZF6-DBD, 1×10e10 vector genomes, vg) of the ZF6 transcriptional silencer to the porcine retina resulted in robust transcriptional silencing of the endogenous porcine RHO transcript. Cell sorting of transduced photoreceptors showed an almost complete RHO transcriptional silencing effect (90% RHO transcriptional repression), underscoring the potency of the system. To determine the safety of the zinc-finger silencer we performed extensive RNA-seq analysis on treated and control retinae. The data sets generated demonstrate selective RHO gene transcriptional repression and a remarkably low number of differential expressed genes (DEGs), supporting specificity and thus, safety. The co-administration to the porcine retina of the AAV-ZF6 silencer (AAV2/8-CMV-ZF6-DBD) and the AAV-RHO replacement (5×10e11 vg, AAV2/8-GNAT1-HumanRHO) constructs resulted in a balanced silencing and replacement effect. This data support the use of zinc-finger based RHO transcriptional silencing for the development of a clinical trial for adRP patients

The Role of Optical Coherence Tomography in an Atypical Case of Oculocutaneous Albinism: A Case Report

Background: Oculocutaneous albinism is a group of autosomal recessive disorders featuring hypopigmentation of the hair, skin and eyes. Ocular signs associated with the disease are nystagmus, decreased visual acuity, hypopigmentation of the retina, foveal hypoplasia, translucency of the iris, macular transparency, photophobia and abnormal decussation of nerve fibers at the chiasm. Case Report: An 8-year-old Caucasian girl presented to our clinic ‘Referral Center for Hereditary Retinopathies’ of the Second University of Naples with a diagnosis of Stargardt disease and a progressive reduction in visual acuity in both eyes. She underwent a complete ophthalmic examination including standard electroretinography and optical coherence tomography (OCT). A molecular analysis was also performed. Best-corrected visual acuity was 20/30 in the right eye and 20/40 in the left eye. Biomicroscopy of the anterior segment revealed a transparent cornea, in situ and transparent lens and normally pigmented iris. A mild diffuse depigmentation and macular dystrophy were observed at fundus examination. Standard electroretinography showed normal scotopic and photopic responses. OCT revealed high reflectivity across the fovea without depression. The typical OCT pattern led us to direct the molecular analysis towards the genes involved in oculocutaneous albinism. The molecular analysis identified mutations in the TYR gene. Conclusion: In this case, the role of OCT was crucial in guiding the molecular analysis for the diagnosis of albinism. OCT is therefore instrumental in similar cases that do not present typical characteristics of a disease. The case also proves the relevance of molecular analysis to confirm clinical diagnoses in hereditary retinal diseases

320 transcriptional silencing via synthetic dna binding protein lacking canonical repressor domains as a potent tool to generate therapeutics

Transcription factors (TFs) function by the combined activity of their DNA-binding domains (DBDs) and effector domains (EDs). Here we show that in vivo delivery of an engineered DNA-binding protein uncoupled from the repressor domain entails complete and gene-specific transcriptional silencing. To silence RHODOPSIN (RHO) gain-of-function mutations, we engineered a synthetic DNA-binding protein lacking canonical repressor domains and targeted to the regulatory region of the RHO gene. AAV-mediate retinal delivery at a low dose (AAV2/8-CMV-ZF6-DBD, 1×10e10 vector genomes, vg) in the porcine retina resulted in selective transcriptional silencing of RHO expression. The rod photoreceptors (the RHO expressing cells) transduced cells when isolated by FACS-sorting showed the remarkable 90% RHO transcriptional repression. To evaluate genome-wide transcriptional specificity, we analyzed the porcine retina transcriptome by RNA sequencing (RNA-Seq). The differentially expressed genes (DEGs) analysis showed that only 19 genes were perturbed. In this study, we describe a system based on a synthetic DNA binding protein enabling targeted transcriptional silencing of the RHO gene by in vivo gene transfer. The high rate of transcriptional silencing occurring in transduced cells supports applications of this regulatory genomic interference with a synthetic trans-acting factor for diseases requiring gene silencing in a large number of affected cells, including for instance a number of neurodegeneration disorders. The result support a novel mode of gene targeted silencing with a DNA-binding protein lacking intrinsic activity

Zinc-finger-based transcriptional repression of rhodopsin in a model of dominant retinitis pigmentosa

Despite the recent success of gene-based complementation approaches for genetic recessive traits, the development of therapeutic strategies for gain-of-function mutations poses great challenges. General therapeutic principles to correct these genetic defects mostly rely on post-transcriptional gene regulation (RNA silencing). Engineered zinc-finger (ZF) protein-based repression of transcription may represent a novel approach for treating gain-of-function mutations, although proof-of-concept of this use is still lacking. Here, we generated a series of transcriptional repressors to silence human rhodopsin (hRHO), the gene most abundantly expressed in retinal photoreceptors. The strategy was designed to suppress both the mutated and the wild-type hRHO allele in a mutational-independent fashion, to overcome mutational heterogeneity of autosomal dominant retinitis pigmentosa due to hRHO mutations. Here we demonstrate that ZF proteins promote a robust transcriptional repression of hRHO in a transgenic mouse model of autosomal dominant retinitis pigmentosa. Furthermore, we show that specifically decreasing the mutated human RHO transcript in conjunction with unaltered expression of the endogenous murine Rho gene results in amelioration of disease progression, as demonstrated by significant improvements in retinal morphology and function. This zinc-finger-based mutation-independent approach paves the way towards a ‘repression–replacement’ strategy, which is expected to facilitate widespread applications in the development of novel therapeutics for a variety of disorders that are due to gain-of-function mutations

Amelioration of both Functional and Morphological Abnormalities in the Retina of a Mouse Model of Ocular Albinism Following AAV-Mediated Gene Transfer

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A comprehensive map of CNS transduction by eight recombinant adeno-associated virus serotypes upon cerebrospinal fluid administration in pigs

Cerebrospinal fluid administration of recombinant adeno-associated viral (rAAV) vectors has been demonstrated to be effective in delivering therapeutic genes to the central nervous system (CNS) in different disease animal models. However, a quantitative and qualitative analysis of transduction patterns of the most promising rAAV serotypes for brain targeting in large animal models is missing. Here, we characterize distribution, transduction efficiency, and cellular targeting of rAAV serotypes 1, 2, 5, 7, 9, rh.10, rh.39, and rh.43 delivered into the cisterna magna of wild-type pigs. rAAV9 showed the highest transduction efficiency and the widest distribution capability among the vectors tested. Moreover, rAAV9 robustly transduced both glia and neurons, including the motor neurons of the spinal cord. Relevant cell transduction specificity of the glia was observed after rAAV1 and rAAV7 delivery. rAAV7 also displayed a specific tropism to Purkinje cells. Evaluation of biochemical and hematological markers suggested that all rAAV serotypes tested were well tolerated. This study provides a comprehensive CNS transduction map in a useful preclinical large animal model enabling the selection of potentially clinically transferable rAAV serotypes based on disease specificity. Therefore, our data are instrumental for the clinical evaluation of these rAAV vectors in human neurodegenerative diseases

MiR-211 is essential for adult cone photoreceptor maintenance and visual function.

MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression that play an important role in the control of fundamental biological processes in both physiological and pathological conditions. Their function in retinal cells is just beginning to be elucidated, and a few have been found to play a role in photoreceptor maintenance and function. MiR-211 is one of the most abundant miRNAs in the developing and adult eye. However, its role in controlling vertebrate visual system development, maintenance and function so far remain incompletely unexplored. Here, by targeted inactivation in a mouse model, we identify a critical role of miR-211 in cone photoreceptor function and survival. MiR-211 knockout (-/-) mice exhibited a progressive cone dystrophy accompanied by significant alterations in visual function. Transcriptome analysis of the retina from miR-211-/- mice during cone degeneration revealed significant alteration of pathways related to cell metabolism. Collectively, this study highlights for the first time the impact of miR-211 function in the retina and significantly contributes to unravelling the role of specific miRNAs in cone photoreceptor function and survival

Mouse Embryonic Retina Delivers Information Controlling Cortical Neurogenesis

The relative contribution of extrinsic and intrinsic mechanisms to cortical development is an intensely debated issue and an outstanding question in neurobiology. Currently, the emerging view is that interplay between intrinsic genetic mechanisms and extrinsic information shape different stages of cortical development [1]. Yet, whereas the intrinsic program of early neocortical developmental events has been at least in part decoded [2], the exact nature and impact of extrinsic signaling are still elusive and controversial. We found that in the mouse developing visual system, acute pharmacological inhibition of spontaneous retinal activity (retinal waves-RWs) during embryonic stages increase the rate of corticogenesis (cell cycle withdrawal). Furthermore, early perturbation of retinal spontaneous activity leads to changes of cortical layer structure at a later time point. These data suggest that mouse embryonic retina delivers long-distance information capable of modulating cell genesis in the developing visual cortex and that spontaneous activity is the candidate long-distance acting extrinsic cue mediating this process. In addition, these data may support spontaneous activity to be a general signal coordinating neurogenesis in other developing sensory pathways or areas of the central nervous system

Therapeutic homology-independent targeted integration in retina and liver

Challenges to the widespread application of gene therapy with adeno-associated viral (AAV) vectors include dominant conditions due to gain-of-function mutations which require allele-specific knockout, as well as long-term transgene expression from proliferating tissues, which is hampered by AAV DNA episomal status. To overcome these challenges, we used CRISPR/Cas9-mediated homology-independent targeted integration (HITI) in retina and liver as paradigmatic target tissues. We show that AAV-HITI targets photoreceptors of both mouse and pig retina, and this results in significant improvements to retinal morphology and function in mice with autosomal dominant retinitis pigmentosa. In addition, we show that neonatal systemic AAV-HITI delivery achieves stable liver transgene expression and phenotypic improvement in a mouse model of a severe lysosomal storage disease. We also show that HITI applications predominantly result in on-target editing. These results lay the groundwork for the application of AAV-HITI for the treatment of diseases affecting various organs