EIAV-based retinal gene therapy in the shaker1 mouse model for usher syndrome type 1B: development of UshStat.

Usher syndrome type 1B is a combined deaf-blindness condition caused by mutations in the MYO7A gene. Loss of functional myosin VIIa in the retinal pigment epithelia (RPE) and/or photoreceptors leads to blindness. We evaluated the impact of subretinally delivered UshStat, a recombinant EIAV-based lentiviral vector expressing human MYO7A, on photoreceptor function in the shaker1 mouse model for Usher type 1B that lacks a functional Myo7A gene. Subretinal injections of EIAV-CMV-GFP, EIAV-RK-GFP (photoreceptor specific), EIAV-CMV-MYO7A (UshStat) or EIAV-CMV-Null (control) vectors were performed in shaker1 mice. GFP and myosin VIIa expression was evaluated histologically. Photoreceptor function in EIAV-CMV-MYO7A treated eyes was determined by evaluating α-transducin translocation in photoreceptors in response to low light intensity levels, and protection from light induced photoreceptor degeneration was measured. The safety and tolerability of subretinally delivered UshStat was evaluated in macaques. Expression of GFP and myosin VIIa was confirmed in the RPE and photoreceptors in shaker1 mice following subretinal delivery of the EIAV-CMV-GFP/MYO7A vectors. The EIAV-CMV-MYO7A vector protected the shaker1 mouse photoreceptors from acute and chronic intensity light damage, indicated by a significant reduction in photoreceptor cell loss, and restoration of the α-transducin translocation threshold in the photoreceptors. Safety studies in the macaques demonstrated that subretinal delivery of UshStat is safe and well-tolerated. Subretinal delivery of EIAV-CMV-MYO7A (UshStat) rescues photoreceptor phenotypes in the shaker1 mouse. In addition, subretinally delivered UshStat is safe and well-tolerated in macaque safety studies These data support the clinical development of UshStat to treat Usher type 1B syndrome.

Photoreceptors in whirler mice show defective transducin translocation and are susceptible to short-term light/dark changes-induced degeneration.

Usher syndrome combines congenital hearing loss and retinitis pigmentosa (RP). Mutations in the whirlin gene (DFNB31/WHRN) cause a subtype of Usher syndrome (USH2D). Whirler mice have a defective whirlin gene. They have inner ear defects but usually do not develop retinal degeneration. Here we report that, in whirler mouse photoreceptors, the light-activated rod transducin translocation is delayed and its activation threshold is shifted to a higher level. Rhodopsin mis-localization is observed in rod inner segments. Continuous moderate light exposure can induce significant rod photoreceptor degeneration. Whirler mice reared under a 1500 lux light/dark cycle also develop severe photoreceptor degeneration. Previously, we have reported that shaker1 mice, a USH1B model, show moderate light-induced photoreceptor degeneration with delayed transducin translocation. Here, we further show that, in both whirler and shaker1 mice, short-term moderate light/dark changes can induce rod degeneration as severe as that induced by continuous light exposure. The results from shaker1 and whirler mice suggest that defective transducin translocation may be functionally related to light-induced degeneration, and these two symptoms may be caused by defects in Usher protein function in rods. Furthermore, these results indicate that both Usher syndrome mouse models possess a light-induced retinal phenotype and may share a closely related pathobiological mechanism.

Light-induced translocation of RGS9-1 and Gß5L in mouse rod photoreceptors.

The transducin GTPase-accelerating protein complex, which determines the photoresponse duration of photoreceptors, is composed of RGS9-1, Gß5L and R9AP. Here we report that RGS9-1 and Gß5L change their distribution in rods during light/dark adaptation. Upon prolonged dark adaptation, RGS9-1 and Gß5L are primarily located in rod inner segments. But very dim-light exposure quickly translocates them to the outer segments. In contrast, their anchor protein R9AP remains in the outer segment at all times. In the dark, Gß5L's interaction with R9AP decreases significantly and RGS9-1 is phosphorylated at S(475) to a significant degree. Dim light exposure leads to quick de-phosphorylation of RGS9-1. Furthermore, after prolonged dark adaptation, RGS9-1 and transducin Gα are located in different cellular compartments. These results suggest a previously unappreciated mechanism by which prolonged dark adaptation leads to increased light sensitivity in rods by dissociating RGS9-1 from R9AP and redistributing it to rod inner segments.

Moderate light-induced degeneration of rod photoreceptors with delayed transducin translocation in shaker1 mice.

PURPOSE. Usher syndrome is characterized by congenital deafness associated with retinitis pigmentosa (RP). Mutations in the myosin VIIa gene (MYO7A) cause a common and severe subtype of Usher syndrome (USH1B). Shaker1 mice have mutant MYO7A. They are deaf and have vestibular dysfunction but do not develop photoreceptor degeneration. The goal of this study was to investigate abnormalities of photoreceptors in shaker1 mice. METHODS. Immunocytochemistry and hydroethidine-based detection of intracellular superoxide production were used. Photoreceptor cell densities under various conditions of light/dark exposures were evaluated. RESULTS. In shaker1 mice, the rod transducin translocation is delayed because of a shift of its light activation threshold to a higher level. Even moderate light exposure can induce oxidative damage and significant rod degeneration in shaker1 mice. Shaker1 mice reared under a moderate light/dark cycle develop severe retinal degeneration in less than 6 months. CONCLUSIONS. These findings show that, contrary to earlier studies, shaker1 mice possess a robust retinal phenotype that may link to defective rod protein translocation. Importantly, USH1B animal models are likely vulnerable to light-induced photoreceptor damage, even under moderate light.

Progressive morphological and functional defects in retinas from alpha1 integrin-null mice.

PURPOSE: The role of integrin/cell matrix interactions between the RPE and the basement membrane in retinal maintenance and function is not well characterized. In this study the functional importance of alpha1beta1 integrin for retinal pigment epithelial cell homeostasis and retinal health was assessed by comparing alpha1 integrin knockout mice with strain- and age-matched wild-type mice. METHODS: Immunolocalization and Western blot analysis of retinas and ARPE19 cells were performed to examine the expression of alpha1beta1 integrin in the RPE. Retinal abnormality was assessed by funduscopy, histology, and transmission electron microscopy. Progressive retinal damage was quantified by direct counting of rod photoreceptors. Light-induced translocation of arrestin and alpha-transducin was documented by immunohistochemical analysis of retinal cryosections. RESULTS: Integrin alpha1beta1 localizes to the basal aspect of retinal pigment epithelial cells colocalizing with the basal lamina of the RPE. Integrin alpha1-null mice have delayed-onset progressive retinal degeneration associated with thickening of the basement membrane, dysmorphology of basal processes, synaptic malformations, and funduscopic abnormalities. Integrin alpha1-null mice display marked delays in transducin translocation compared with dark-adapted wild-type mice after exposure to light. CONCLUSIONS: Collectively, these data suggest an essential role for alpha1beta1 integrin/basement membrane interactions in the RPE in basement membrane metabolism and translocation of transducin in photoreceptors. This is the first report describing evidence supporting an essential role for integrin/basement membrane interaction in the RPE. Further, this report demonstrates a direct link between integrin alpha1beta1 function in retinal pigment epithelial and molecular defects in photoreceptor cell function before retinal abnormality is apparent.

Oxidative damage is a potential cause of cone cell death in retinitis pigmentosa.

Retinitis pigmentosa (RP) is a prevalent cause of blindness caused by a large number of different mutations in many different genes. The mutations result in rod photoreceptor cell death, but it is unknown why cones die. In this study, we tested the hypothesis that cones die from oxidative damage by performing immunohistochemical staining for biomarkers of oxidative damage in a transgenic pig model of RP. The presence of acrolein- and 4-hydroxynonenal-adducts on proteins is a specific indicator that lipid peroxidation has occurred, and there was strong immunofluorescent staining for both in cone inner segments (IS) of two 10-month-old transgenic pigs in which almost all rods had died, compared to faint staining in two 10-month-old control pig retinas. In 22- and 24-month-old transgenic pigs in which all rods and many cones had died, staining was strong in cone axons and some cell bodies as well as IS indicating progression in oxidative damage between 10 and 22 months. Biomarkers for oxidative damage to proteins and DNA also showed progressive oxidative damage to those macromolecules in cones during the course of RP. These data support the hypothesis that the death of rods results in decreased oxygen consumption and hyperoxia in the outer retina resulting in gradual cone cell death from oxidative damage. This hypothesis has important therapeutic implications and deserves rapid evaluation.

Altered light responses of single rod photoreceptors in transgenic pigs expressing P347L or P347S rhodopsin.

PURPOSE: Numerous mutations of rhodopsin lead to rod cell death and ultimately to complete blindness, yet little is known about the alterations in the physiology of the light sensors containing the aberrant protein, the rod photoreceptors. METHODS: Suction pipettes were used to record the light responses from single rod photoreceptors isolated from the retinas of transgenic pigs of various ages and at progressive stages of retinal degeneration. RESULTS: We have observed changes in the photoresponse of transgenic porcine rods containing both wild type and mutant rhodospin. Our findings are consistent with the idea that substitutions at position proline 347 of rhodopsin interfere with the inactivation of R*. In addition the level of photoreceptor degeneration is positively correlated with an acceleration and desensitization of the photoresponse to dim flashes. CONCLUSIONS: It appears that the phototransduction cascade, even when initiated by wild type rhodopsin molecules is altered in a way that is progressive with the level of retinal degeneration. A model consistent with our observations introduces the idea of a binding site for the carboxy terminus of rhodopsin on rhodopsin kinase.