Dario Marangoni

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. Initial development of the photoreceptor-DBC synapse was normal in the Rs1-KO retina; however, the metabotropic glutamate receptor 6/transient receptor potential melastatin subfamily M member 1-signaling (mGluR6/TRPM1-signaling) cascade was not properly maintained. Specifically, the TRPM1 channel and G proteins Gαo, Gβ5, and RGS11 were progressively lost from postsynaptic DBC dendritic tips, whereas the mGluR6 receptor and RGS7 maintained proper synaptic position. This postsynaptic disruption differed from other murine night-blindness models with an electronegative electroretinogram response, which is also characteristic of murine and human XLRS disease. Upon AAV8-RS1 gene transfer to the retina of adult XLRS mice, TRPM1 and the signaling molecules returned to their proper dendritic tip location, and the DBC resting membrane potential was restored. These findings provide insight into the molecular plasticity of a critical synapse in the visual system and demonstrate potential therapeutic avenues for some diseases involving synaptic pathology.

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. This study explores whether this finding translates to humans with CNGB3 achromatopsia. METHODS A five-subject, open-label Phase I/II study was initiated by implanting intraocular microcapsules releasing CNTF (nominally 20 ng/d) into one eye each of CNGB3 achromat participants. Fellow eyes served as untreated controls. Subjects were followed for 1 year. RESULTS Pupil constriction in treated eyes gave evidence of intraocular CNTF release. Additionally, scotopic ERG responses were reduced, and dark-adapted psychophysical absolute thresholds were increased, attributable to diminished rod or rod pathway activity. Optical coherence tomography revealed that the cone-rich fovea underwent structural changes as the foveal hyporeflective zone (HRZ) became diminished in CNTF-treated eyes. No objectively measurable enhancement of cone function was found by assessments of visual acuity, mesopic increment sensitivity threshold, or the photopic ERG. Careful measurements of color hue discrimination showed no change. Nonetheless, subjects reported beneficial changes of visual function in the treated eyes, including reduced light sensitivity and aversion to bright light, which may trace to decreased effective ambient light from the pupillary constriction; further they noted slowed adaptation to darkness, consistent with CNTF action on rod photoreceptors. CONCLUSIONS Ciliary neurotrophic factor did not measurably enhance cone function, which reveals a species difference between human and canine CNGB3 cones in response to CNTF. (ClinicalTrials.gov number, NCT01648452.).

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. On the basis of our prior study in an Rs1-KO mouse, this construct transduces efficiently all the retinal layers, resulting in an RS1 expression similar to that observed in the wild-type and improving retinal structure and function. In support of a clinical trial, we carried out a study to evaluate the ocular safety of intravitreal administration of AAV8-scRS/IRBPhRS into 39 New Zealand White rabbits. Two dose levels of vector, 2e(10) and 2e(11) vector genomes per eye (vg/eye), were tested and ocular inflammation was monitored over a 12-week period by serial ophthalmological and histopathological analysis. A mild ocular inflammatory reaction, consisting mainly of vitreous infiltrates, was observed within 4 weeks from injection, in both 2e(10) and 2e(11) vg/eye groups and was likely driven by the AAV8 capsid. At 12-week follow-up, ophthalmological examination revealed no clinical signs of vitreitis in either of the dose groups. However, while vitreous inflammatory infiltrate was significantly reduced in the 2e(10) vg/eye group at 12 weeks, some rabbits in the higher dose group still showed persistence of inflammatory cells, histologically. In conclusion, intravitreal administration of AAV8-scRS/IRBPhRS into the rabbit eye produces a mild and transient intraocular inflammation that resolves, at a 2e(10) vg/eye dose, within 3 months, and does not cause irreversible tissue damages. These data support the initiation of a clinical trial of intravitreal administration of AAV8-scRS/IRBPhRS in XLRS patients.

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 1.5E12 vector genomes/eye (vg/eye), and a 6-month study in Rs1-KO mice (n = 162) dosed with 2E9 and 2E10 vg/eye of the same vector were conducted to assess ocular and systemic safety. A self-resolving, dose-dependent vitreal inflammation was the main ocular finding, and except for a single rabbit dosed with 1.5E12 vg/eye, which showed a retinal detachment, no other ocular adverse event was reported. Systemic toxicity was not identified in either species. Biodistribution analysis in Rs1-KO mice detected spread of vector genome in extraocular tissues, but no evidence of organ or tissues damage was found. These studies indicate that IVT administration of AAV8-scRS/IRBPhRS is safe and well tolerated and support its advancement into a phase 1/2a clinical trial for XLRS.

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. METHODS Fifty-nine CNGB3-/- mice (postnatal day [PD] ± SD = 30 ± 7) received a unilateral intravitreal injection of 1 or 2 μg CNTF protein, and 15 wild-type (WT) mice (PD = 34 ± 3) received 1 μg CNTF. Retinal function was evaluated by flash ERG and photopic flicker ERG (fERG) at 7 and 14 days after treatment. RESULTS Seven days post CNTF, the photopic b-wave Vmax was significantly increased in CNGB3-/- mice (P < 0.01), whereas it was reduced in WT mice (P < 0.05). Ciliary neurotrophic factor significantly increased the amplitude of photopic fERG and the photopic oscillatory potentials (OPs) in CNGB3-/- mice. Ciliary neurotrophic factor did not alter the scotopic a-wave in either CNGB3-/- or WT mice, but it increased the scotopic b-wave k (P < 0.01) in CNGB3-/- mice, indicating diminished scotopic sensitivity, and reduced the scotopic b-wave Vmax in WT mice (P < 0.05). No difference was found in ERG parameters between 1 or 2 μg CNTF. Fourteen days after CNTF injection the ERG changes in CNGB3-/- mice were lost. CONCLUSIONS Intravitreal bolus CNTF protein caused a small and transient improvement of cone-mediated function in CNGB3-/- mice, whereas it reduced rod-mediated function. The increase in photopic OPs and the lack of changes in scotopic a-wave suggest a CNTF effect on the inner retina.

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. Superoxide production and oxidative damage were assessed by dihydroethidium staining and by protein carbonyls and malondialdehyde levels, respectively. Outer nuclear layer (ONL) cells were counted and electroretinogram (ERG) amplitudes measured in Tg-CA-RAC1 mice. Outer nuclear layer cells were counted in wild-type (WT) mice after transfer of CA-Rac1 gene by subretinal injection of AAV8-pOpsin-CA Rac1-GFP. Results Transgenic-CA-RAC1 retinas had significantly fewer photoreceptor cells and more apoptotic ONL cells than WT controls from postnatal week (Pw) 3 to Pw13. Superoxide accumulation and protein and lipid oxidation were increased in Tg-CA-RAC1 retinas and were reduced in mice treated with apocynin. Apocynin reduced the loss of photoreceptors and increased the rod ERG a- and b-wave amplitudes when compared with vehicle-injected transgenic controls. Photoreceptor loss was also observed in regions of adult WT retina transduced with AAV8-pOpsin-CA Rac1-GFP but not in neighboring regions that were not transduced or in AAV8-pOpsin-GFP–transduced retinas. Conclusions Constitutively active RAC1 promotes photoreceptor cell death by oxidative damage that occurs, at least partially, through NOX-induced ROS. Reactive oxygen species are likely involved in multiple forms of retinal degenerations, and our results support investigating RAC1 inhibition as a therapeutic approach that targets this disease pathway.

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. This phase I/IIa single-center, prospective, open-label, three-dose-escalation clinical trial administered vector to nine participants with pathogenic RS1 mutations. The eye of each participant with worse acuity (≤63 letters; Snellen 20/63) received the AAV8-RS1 gene vector by intravitreal injection. Three participants were assigned to each of three dosage groups: 1e9 vector genomes (vg)/eye, 1e10 vg/eye, and 1e11 vg/eye. The investigational product was generally well tolerated in all but one individual. Ocular events included dose-related inflammation that resolved with topical and oral corticosteroids. Systemic antibodies against AAV8 increased in a dose-related fashion, but no antibodies against RS1 were observed. Retinal cavities closed transiently in one participant. Additional doses and immunosuppressive regimens are being explored to pursue evidence of safety and efficacy (ClinicalTrials.gov: NCT02317887).

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. Expression of inward-rectifier K+ channel (Kir4.1), water channel aquaporin-4 (AQP4), and glial fibrillary acidic protein (GFAP) were analyzed by Western blotting. In a separate study, Rs1-KO mice reared in LL (n = 29) or ML (n = 27) received a unilateral intravitreal injection of scAAV8-hRs-IRBP at 21 days, and functional outcome was evaluated at 4 months by electroretinogram. Results At 1 month, no functional or structural differences were found between LL- or ML-reared Rs1-KO mice. At 4 months, ML-reared Rs1-KO mice showed significant reduction of b-wave amplitude and b-/a-wave ratio with no changes in a-wave, and a significant increase in cavity size, compared to LL-reared animals. Moderate light rearing increased Kir4.1 expression in Rs1-KO mice by 4 months, but not AQP4 and GFAP levels. Administration of scAAV8-hRS1-IRBP to Rs1-KO mice showed similar improvement of inner retinal ERG function independent of LL or ML rearing. Conclusions Rearing light conditions affect the development of retinal cavities and post-photoreceptor function in Rs1-KO mice. However, the effect of rearing light intensity does not interact with the efficacy of RS1 gene replacement in Rs1-KO mice.

600. Human Gene Therapy for a Synaptic Disease: X-Linked Retinoschisis (XLRS)

We have initiated a clinical trial for an ocular condition that has prominent synaptic dysfunction caused by mutations in an extracellular matrix protein, RS1 (retinoschisin). This report describes our findings on dissection of the molecular pathology at the synapse and reversal of the structural and functional defects when RS1 is introduced into mature Rs1-KO mouse retina using an AAV8 vector. We have started a human trial (NCT02317887) using the same vector, which will probe the synaptic plasticity in XLRS patients. RS1 mutations result in XLRS, a rare but recognized ocular condition that causes splitting of the neural retinal layers and results in intraretinal cysts. Medical attention generally was focused on the structural abnormalities of XLRS. However, visual function testing shows a selective depression of the electroretinogram (ERG) b-wave despite a normal a-wave, indicating intact photoreceptor responses but deficient post-synaptic signaling.The AAV8-RS1 vector for the murine studies and the clinical trial contains a self-complementary vector genome with the human RS1 promoter and the RS1 cDNA. For synaptic structural studies in adult Rs1-KO retina, 2.5e9 viral vector genomes (vg)/eye were administered by intravitreal injection at postnatal day 30. Retinal morphology, function and pathology were evaluated 2 months post injection, with contralateral untreated eyes as controls.The photoreceptor-depolarizing bipolar cell (DBC) synapse initially develops normally in Rs1 -KO mice, but the mGluR6-TRPM1 signaling cascade elements in the post-synaptic structure are progressively delocalized to the dendritic shaft and soma of the DBC. Concomitantly, the DBC resting membrane potential is altered. This molecular pattern of post-synaptic disruption is different from several other murine night-blindness models which share a “negative-going” ERG response that characterizes murine and human XLRS. Following AAV8-RS1 gene transfer to the XLRS mouse eye, TRPM1 and the signaling molecules return to their proper location at the dendritic tips, and the proper DBC resting membrane potential is restored, along with the ERG b-wave.The clinical trial utilizing the same vector is a prospective, dose-escalation, single-center study conducted at the National Eye Institute. One eye of each participant receives the RS1 gene vector given by intravitreal injection. While the primary outcome is safety, the dose range is scaled to probe possible efficacy.Invoking synaptic plasticity for therapeutic repair would be a desirable treatment strategy for a number of neurological conditions. These findings in Rs1 -KO mice provide insight into the molecular pathology of XLRS disease and demonstrate remarkable plasticity of a critical synapse in the visual system. Reversal of synaptic pathology by an AAV8-RS1 gene construct demonstrates novel possible therapeutic avenues for this and other diseases involving synaptic pathology.