Trans-Ocular Electric Current In Vivo Enhances AAV-Mediated Retinal Transduction in Large Animal Eye After Intravitreal Vector Administration.

Purpose: Electric micro-current has been shown to enhance penetration and transduction of adeno-associated viral (AAV) vectors in mouse retina after intravitreal administration. We termed this: "electric-current vector mobility (ECVM)." The present study considered whether ECVM could augment retinal transduction efficiency of intravitreal AAV8-CMV-EGFP in normal rabbit and nonhuman primate (NHP) macaque.

Cone ERG Changes During Light Adaptation in Two All-Cone Mutant Mice: Implications for Rod-Cone Pathway Interactions.

Purpose: The b-wave of the cone ERG increases in amplitude and speed during the first few minutes of adaptation to a rod-suppressing background light. Earlier studies implicate rod pathway input to the cone pathway in these changes.

Methods: The timing and amplitude of the cone b-wave and isolated oscillatory potentials (OP) during the first 10 minutes of light adaptation in wild-type (WT) mice and two mutant lines without functional rods was examined: rhodopsin knockout (Rho-/-), lacking rod outer segments, and NRL knockout (Nrl-/-), in which rods are replaced by S-cones.

AAVrh-10 transduces outer retinal cells in rodents and rabbits following intravitreal administration.

Recombinant adeno-associated virus (rAAV) has been widely used for gene delivery in animal models and successfully applied in clinical trials for treating inherited retinal disease. Although subretinal delivery of AAVs can effectively transduce photoreceptors and/or retinal pigmental epithelium (RPE), cells most affected by inherited retinal diseases, the procedure is invasive and complicated, and only delivers the gene to a limited retinal area. AAVs can also be delivered intravitreally to the retina, a much less invasive nonsurgical procedure.

Trans-ocular Electric Current Enhances AAV-Mediated Retinal Gene Transduction after Intravitreal Vector Administration.

Adeno-associated virus (AAV) vector-mediated gene delivery is a promising approach for therapy, but implementation in the eye currently is hampered by the need for delivering the vector underneath the retina, using surgical application into the subretinal space. This limits the extent of the retina that is treated and may cause surgical injury. Vector delivery into the vitreous cavity would be preferable because it is surgically less invasive and would reach more of the retina.

Retinal AAV8-RS1 Gene Therapy for X-Linked Retinoschisis: Initial Findings from a Phase I/IIa Trial by Intravitreal Delivery.

This study evaluated the safety and tolerability of ocular RS1 adeno-associated virus (AAV8-RS1) gene augmentation therapy to the retina of participants with X-linked retinoschisis (XLRS). XLRS is a monogenic trait affecting only males, caused by mutations in the RS1 gene. Retinoschisin protein is secreted principally in the outer retina, and its absence results in retinal cavities, synaptic dysfunction, reduced visual acuity, and susceptibility to retinal detachment.

Rearing Light Intensity Affects Inner Retinal Pathology in a Mouse Model of X-Linked Retinoschisis but Does Not Alter Gene Therapy Outcome.

Purpose: To test the effects of rearing light intensity on retinal function and morphology in the retinoschisis knockout (Rs1-KO) mouse model of X-linked retinoschisis, and whether it affects functional outcome of RS1 gene replacement.

Methods: Seventy-six Rs1-KO mice were reared in either cyclic low light (LL, 20 lux) or moderate light (ML, 300 lux) and analyzed at 1 and 4 months. Retinal function was assessed by electroretinogram and cavity size by optical coherence tomography.

Ocular and systemic safety of a recombinant AAV8 vector for X-linked retinoschisis gene therapy: GLP studies in rabbits and Rs1-KO mice.

X-linked retinoschisis (XLRS) is a retinal disease caused by mutations in the gene encoding the protein retinoschisin (RS1) and is one of the most common causes of macular degeneration in young men. Our therapeutic approach for XLRS is based on the administration of AAV8-scRS/IRBPhRS, an adeno-associated viral vector coding the human RS1 protein, via the intravitreal (IVT) route. Two Good Laboratory Practice studies, a 9-month study in New Zealand White rabbits (n = 124) injected with AAV8-scRS/IRBPhRS at doses of 2E9, 2E10, 2E11, and 1.

Retinal Structure and Gene Therapy Outcome in Retinoschisin-Deficient Mice Assessed by Spectral-Domain Optical Coherence Tomography.

Purpose: Spectral-domain optical coherence tomography (SD-OCT) was used to characterize the retinal phenotype, natural history, and treatment responses in a mouse model of X-linked retinoschisis (Rs1-KO) and to identify new structural markers of AAV8-mediated gene therapy outcome.

Methods: Optical coherence tomography scans were performed on wild-type and Rs1-KO mouse retinas between 1 and 12 months of age and on Rs1-KO mice after intravitreal injection of AAV8-scRS/IRBPhRS (AAV8-RS1). Cavities and photoreceptor outer nuclear layer (ONL) thickness were measured, and outer retina reflective band (ORRB) morphology was examined with age and after AAV8-RS1 treatment.

NADPH Oxidase Contributes to Photoreceptor Degeneration in Constitutively Active RAC1 Mice.

Purpose: The active form of small GTPase RAC1 is required for activation of NADPH oxidase (NOX), which in turn generates reactive oxygen species (ROS) in nonphagocytic cells. We explored whether NOX-induced oxidative stress contributes to rod degeneration in retinas expressing constitutively active (CA) RAC1.

Methods: Transgenic (Tg)-CA-RAC1 mice were given apocynin (10 mg/kg, intraperitoneal), a NOX inhibitor, or vehicle daily for up to 13 weeks.

Preclinical Dose-Escalation Study of Intravitreal AAV-RS1 Gene Therapy in a Mouse Model of X-linked Retinoschisis: Dose-Dependent Expression and Improved Retinal Structure and Function.

Gene therapy for inherited retinal diseases has been shown to ameliorate functional and structural defects in both animal models and in human clinical trials. X-linked retinoschisis (XLRS) is an early-age onset macular dystrophy resulting from loss of an extracellular matrix protein (RS1). In preparation for a human clinical gene therapy trial, we conducted a dose-range efficacy study of the clinical vector, a self-complementary AAV delivering a human retinoschisin (RS1) gene under control of the RS1 promoter and an interphotoreceptor binding protein enhancer (AAV8-scRS/IRBPhRS), in the retinoschisin knockout (Rs1-KO) mouse.

Intravitreal Ciliary Neurotrophic Factor Transiently Improves Cone-Mediated Function in a CNGB3-/- Mouse Model of Achromatopsia.

Purpose: Ciliary neurotrophic factor (CNTF) was recently shown to augment cone function in CNGB3 mutant achromat dogs. However, testing CNTF-releasing implant in human CNGB3 achromats failed to show benefit. We evaluated the effects of CNTF protein on the retinal function in an additional achromatopsia model, the CNGB3-/- mouse.

Convergence of Human Genetics and Animal Studies: Gene Therapy for X-Linked Retinoschisis.

Retinoschisis is an X-linked recessive genetic disease that leads to vision loss in males. X-linked retinoschisis (XLRS) typically affects young males; however, progressive vision loss continues throughout life. Although discovered in 1898 by Haas in two brothers, the underlying biology leading to blindness has become apparent only in the last 15 years with the advancement of human genetic analyses, generation of XLRS animal models, and the development of ocular monitoring methods such as the electroretinogram and optical coherence tomography.

Synaptic pathology and therapeutic repair in adult retinoschisis mouse by AAV-RS1 transfer.

Strategies aimed at invoking synaptic plasticity have therapeutic potential for several neurological conditions. The human retinal synaptic disease X-linked retinoschisis (XLRS) is characterized by impaired visual signal transmission through the retina and progressive visual acuity loss, and mice lacking retinoschisin (RS1) recapitulate human disease. Here, we demonstrate that restoration of RS1 via retina-specific delivery of adeno-associated virus type 8-RS1 (AAV8-RS1) vector rescues molecular pathology at the photoreceptor-depolarizing bipolar cell (photoreceptor-DBC) synapse and restores function in adult Rs1-KO animals.

Preclinical safety evaluation of a recombinant AAV8 vector for X-linked retinoschisis after intravitreal administration in rabbits.

X-linked retinoschisis (XLRS) is a retinal disease caused by mutations in the gene encoding the protein retinoschisin (RS1) and one of the most common causes of macular degeneration in young men. Currently, no FDA-approved treatments are available for XLRS and a replacement gene therapy could provide a promising strategy. We have developed a novel gene therapy approach for XLRS, based on the administration of AAV8-scRS/IRBPhRS, an adeno-associated viral vector coding the human RS1 protein, via the intravitreal route.

CNGB3-achromatopsia clinical trial with CNTF: diminished rod pathway responses with no evidence of improvement in cone function.

Purpose: Ciliary neurotrophic factor (CNTF) protects rod photoreceptors from retinal degenerative disease in multiple nonhuman models. Thus far, CNTF has failed to demonstrate rod protection in trials for human retinitis pigmentosa. Recently, CNTF was found to improve cone photoreceptor function in a canine CNGB3 achromatopsia model.

Photoreceptor pathology in the X-linked retinoschisis (XLRS) mouse results in delayed rod maturation and impaired light driven transducin translocation.

Light-activated movement of transducin-α (Gαt1) from rod photoreceptor outer segments (ROS) into inner segments (IS) enables rods to rapidly adapt to changes in light intensity. The threshold light intensity at which Gαt1 translocates from ROS into IS is primarily determined by the rates of activation and inactivation of Gαt1. Loss- of- expression of the retina specific cell surface protein, retinoschsin (Rs1-KO), led to a dramatic 3-10 fold increase, depending on age, in the luminance threshold for transducin translocation from ROS into IS compared with wild-type control.

Transgenic expression of constitutively active RAC1 disrupts mouse rod morphogenesis.

Purpose: Dominant-active RAC1 rescues photoreceptor structure in Drosophila rhodopsin-null mutants, indicating an important role in morphogenesis. This report assesses the morphogenetic effect of activated RAC1 during mammalian rod photoreceptor development using transgenic mice that express constitutively active (CA) RAC1.

Methods: Transgenic mice were generated by expressing CA RAC1 under control of the Rhodopsin promoter, and morphological features of the photoreceptors were evaluated by histology, immunohistochemistry, and transmission electron microscopy.

Loss of retinoschisin (RS1) cell surface protein in maturing mouse rod photoreceptors elevates the luminance threshold for light-driven translocation of transducin but not arrestin.

Loss of retinoschisin (RS1) in Rs1 knock-out (Rs1-KO) retina produces a post-photoreceptor phenotype similar to X-linked retinoschisis in young males. However, Rs1 is expressed strongly in photoreceptors, and Rs1-KO mice have early reduction in the electroretinogram a-wave. We examined light-activated transducin and arrestin translocation in young Rs1-KO mice as a marker for functional abnormalities in maturing rod photoreceptors.

X-linked retinoschisis: RS1 mutation severity and age affect the ERG phenotype in a cohort of 68 affected male subjects.

Purpose: To assess the effect of age and RS1 mutation on the phenotype of X-linked retinoschisis (XLRS) subjects using the clinical electroretinogram (ERG) in a cross-sectional analysis.

Methods: Sixty-eight XLRS males 4.5 to 55 years of age underwent genotyping, and the retinoschisis (RS1) mutations were classified as less severe (27 subjects) or more severe (41 subjects) based on the putative impact on the protein.

Probing potassium channel function in vivo by intracellular delivery of antibodies in a rat model of retinal neurodegeneration.

Inward rectifying potassium (Kir) channels participate in regulating potassium concentration (K(+)) in the central nervous system (CNS), including in the retina. We explored the contribution of Kir channels to retinal function by delivering Kir antibodies (Kir-Abs) into the rat eye in vivo to interrupt channel activity. Kir-Abs were coupled to a peptide carrier to reach intracellular epitopes.

Depleting Rac1 in mouse rod photoreceptors protects them from photo-oxidative stress without affecting their structure or function.

In nonphagocytic cells, Rac1 is a component of NADPH oxidase that produces reactive oxygen species [Ushio-Fukai M (2006) Sci STKE 2006:re8]. Rac1 is expressed abundantly in mammalian retinal photoreceptors, where it is activated in response to light stimuli [Balasubramanian N, Slepak VZ (2003) Curr Biol 13:1306-1310]. We used Cre-LoxP conditional gene targeting to knock down Rac1 expression in mouse rod photoreceptors and found protection against light-induced photoreceptor death compared with WT litter-mates.

Photoreceptor protection by adeno-associated virus-mediated LEDGF expression in the RCS rat model of retinal degeneration: probing the mechanism.

Purpose: Lens epithelium-derived growth factor (LEDGF) is upregulated in response to stress and enhances the survival of neurons in the retina and optic nerve, as well as a wide range of other cells, such as fibroblasts and keratinocytes. Photoreceptor protection was investigated in the RCS rat retinal degeneration model after Ledgf delivery with an adeno-associated virus (AAV) and the mechanism of protection explored.

Methods: Thirty-six RCS and nine P23H rats had bilateral subretinal injections of AAV-Ledgf in one eye and buffer in the contralateral eye as the control.

Double homozygous waltzer and Ames waltzer mice provide no evidence of retinal degeneration.

Purpose: To determine whether cadherin 23 and protocadherin 15 can substitute for one another in the maintenance of the retina and other tissues in the mouse. Does homozygosity for both v and av mutant alleles (i.e.

Synaptic pathology in retinoschisis knockout (Rs1-/y) mouse retina and modification by rAAV-Rs1 gene delivery.

Purpose: At an early age, the retinoschisin knockout (Rs1-KO) mouse retina has progressive photoreceptor degeneration with severe disruption of the outer plexiform layer (OPL) that decreases at older ages. The electroretinogram (ERG) undergoes parallel changes. The b-wave amplitude from bipolar cells is reduced disproportionately to the photoreceptor a-wave at young but not at older ages.