Rs1-KO Mice Research Articles

OCT Intensity of the Region between Outer Retina Band 2 and Band 3 as a Biomarker for Retinal Degeneration and Therapy.

Optical coherence tomography (OCT) is widely used to probe retinal structure and function. This study investigated the outer retina band (ORB) pattern and reflective intensity for the region between bands 2 and 3 (Dip) in three mouse models of inherited retinal degeneration (Rs1KO, TTLL5KO, RPE65KO) and in human AMD patients from the A2A database. OCT images were manually graded, and reflectivity signals were used to calculate the Dip ratio. Qualitative analyses demonstrated the progressive merging band 2 and band 3 in all three mouse models, leading to a reduction in the Dip ratio compared to wildtype (WT) controls. Gene replacement therapy in Rs1KO mice reverted the ORB pattern to one resembling WT and increased the Dip ratio. The degree of anatomical rescue in these mice was highly correlated with level of transgenic RS1 expression and with the restoration of ERG b-wave amplitudes. While the inner retinal cavity was significantly enlarged in dark-adapted Rs1KO mice, the Dip ratio was not altered. A reduction of the Dip ratio was also detected in AMD patients compared with healthy controls and was also positively correlated with AMD severity on the AMD score. We propose that the ORB and Dip ratio can be used as non-invasive early biomarkers for retina health, which can be used to probe therapeutic gene expression and to evaluate the effectiveness of therapy.

Intravitreal Delivery of rAAV2-hSyn-hRS1 Results in Retinal Ganglion Cell-Specific Gene Expression and Retinal Improvement in the Rs1-KO Mouse.

X-linked retinoschisis (XLRS) is a monogenic recessive inherited retinal disease caused by defects in retinoschisin (RS1). It manifests clinically as retinal schisis cavities and a disproportionate reduction of b-wave amplitude compared with the a-wave amplitude. Currently there is no approved treatment. In the last decade, there has been major progress in the development of gene therapy for XLRS. Previous preclinical studies have demonstrated the treatment benefits of hRS1 gene augmentation therapy in mouse models. However, outcomes in clinical trials have been disappointing, and this might be attributed to dysfunctional assembly of RS1 complexes and/or the impaired targeted cells. In this study, the human synapsin 1 gene promoter (hSyn) was used to control the expression of hRS1 to specifically target retinal ganglion cells and our results confirmed the specific expression and functional assembly of the protein. Moreover, our results demonstrated that a single intravitreal injection of rAAV2-hSyn-hRS1 results in architectural restoration of retinal schisis cavities and improvement in vision in a mouse model of XLRS. In brief, this study not only supports the clinical development of the rAAV2-hSyn-hRS1 vector in XLRS patients but also confirms the therapeutic potential of rAAV-based gene therapy in inherited retinal diseases.

AAV2/4-RS1 gene therapy in the retinoschisin knockout mouse model of X-linked retinoschisis.

To evaluate efficacy of a novel adeno-associated virus (AAV) vector, AAV2/4-RS1, for retinal rescue in the retinoschisin knockout (Rs1-KO) mouse model of X-linked retinoschisis (XLRS). Brinzolamide (Azopt®), a carbonic anhydrase inhibitor, was tested for its ability to potentiate the effects of AAV2/4-RS1. AAV2/4-RS1 with a cytomegalovirus (CMV) promoter (2x1012 viral genomes/mL) was delivered to Rs1-KO mice via intravitreal (N = 5; 1μL) or subretinal (N = 21; 2μL) injections at postnatal day 60-90. Eleven mice treated with subretinal therapy also received topical Azopt® twice a day. Serial full field electroretinography (ERG) was performed starting at day 50-60 post-injection. Mice were evaluated using a visually guided swim assay (VGSA) in light and dark conditions. The experimental groups were compared to untreated Rs1-KO (N = 11), wild-type (N = 12), and Rs1-KO mice receiving only Azopt® (N = 5). Immunofluorescence staining was performed to assess RS1 protein expression following treatment. The ERG b/a ratio was significantly higher in the subretinal plus Azopt® (p<0.0001), subretinal without Azopt® (p = 0.0002), and intravitreal (p = 0.01) treated eyes compared to untreated eyes. There was a highly significant subretinal treatment effect on ERG amplitudes collectively at 7-9 months post-injection (p = 0.0003). Cones showed more effect than rods. The subretinal group showed improved time to platform in the dark VGSA compared to untreated mice (p<0.0001). RS1 protein expression was detected in the outer retina in subretinal treated mice and in the inner retina in intravitreal treated mice. AAV2/4-RS1 shows promise for improving retinal phenotype in the Rs1-KO mouse model. Subretinal delivery was superior to intravitreal. Topical brinzolamide did not improve efficacy. AAV2/4-RS1 may be considered as a potential treatment for XLRS patients.

XLRS Rat with Rs1-/Y Exon-1-Del Shows Failure of Early Postnatal Outer Retina Development.

We generated a Long Evans transgenic rat with targeted deletion of the whole Rs1 exon-1 and evaluated the pathological retinal phenotype of this Rs1-/Y rat model of X-linked retinoschisis (XLRS). The Rs1-/Y rat exhibited very early onset and rapidly progressive photoreceptor degeneration. The outer limiting membrane (OLM) was disrupted and discontinuous by post-natal day (P15) and allowed photoreceptor nuclei to dislocate from the outer nuclear layers (ONL) into the sub-retinal side of the OLM. Dark-adapted electroretinogram (ERG) a-wave and b-wave amplitudes were considerably reduced to only 20-25% of WT by P17. Microglia and Müller glial showed cell marker activation by P7. Intravitreal application of AAV8-RS1 at P5-6 induced RS1 expression by P15 and rescued the inner nuclear layer (INL) and outer plexiform layer (OPL) cavity formation otherwise present at P15, and the outer-retinal structure was less disrupted. This Rs1-/Y exon-1-del rat model displays substantially faster rod cell loss compared to the exon-1-del Rs1-KO mouse. Most unexpected was the rapid appearance of schisis cavities between P7 and P15, and then cavities rapidly disappeared by P21/P30. The rat model provides clues on the molecular and cellular mechanisms underlying XLRS pathology in this model and points to a substantial and early changes to normal retinal development.

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

PurposeTo 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.MethodsSeventy-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.ResultsAt 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.ConclusionsRearing 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.

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

PurposeSpectral-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.MethodsOptical 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. Outer retina reflective band morphology was compared to immunohistochemical staining of the outer limiting membrane (OLM) and photoreceptor inner segment (IS) mitochondria and to electron microscopy (EM) images of IS.ResultsRetinal cavity size in Rs1-KO mice increased between 1 and 4 months and decreased thereafter, while ONL thickness declined steadily, comparable to previous histologic studies. Wild-type retina had four ORRBs. In Rs1-KO, ORRB1was fragmented from 1 month, but was normal after 8 months; ORRB2 and ORRB3 were merged at all ages. Outer retina reflective band morphology returned to normal after AAV-RS1 therapy, paralleling the recovery of the OLM and IS mitochondria as indicated by anti–β-catenin and anti-COX4 labeling, respectively, and EM.ConclusionsSpectral-domain OCT is a sensitive, noninvasive tool to monitor subtle changes in retinal morphology, disease progression, and effects of therapies in mouse models. The ORRBs may be useful to assess the outcome of gene therapy in the treatment of X-linked retinoschisis patients.

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. The therapeutic vector at 1 × 10(6) to 2.5 × 10(9) (1E6-2.5E9) vector genomes (vg)/eye or vehicle was administered to one eye of 229 male Rs1-KO mice by intravitreal injection at 22 ± 3 days postnatal age (PN). Analysis of retinal function (dark-adapted electroretinogram, ERG), structure (cavities and outer nuclear layer thickness) by in vivo retinal imaging using optical coherence tomography, and retinal immunohistochemistry (IHC) for RS1 was done 3-4 months and/or 6-9 months postinjection (PI). RS1 IHC staining was dose dependent across doses ≥1E7 vg/eye, and the threshold for significant improvement in all measures of retinal structure and function was 1E8 vg/eye. Higher doses, however, did not produce additional improvement. At all doses showing efficacy, RS1 staining in Rs1-KO mouse was less than that in wild-type mice. Improvement in the ERG and RS1 staining was unchanged or greater at 6-9 months than at 3-4 months PI. This study demonstrates that vitreal administration of AAV8 scRS/IRBPhRS produces significant improvement in retinal structure and function in the mouse model of XLRS over a vector dose range that can be extended to a human trial. It indicates that a fully normal level of RS1 expression is not necessary for a therapeutic effect.

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.

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.

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.

Erratum: Intravitreal delivery of AAV8 retinoschisin results in cell type-specific gene expression and retinal rescue in the Rs1-KO mouse

Correction to: Gene Therapy (2009) 16, 916–926; doi:10.1038/gt.2009.61 TK Park and Z Wu contributed equally to this work. The authors apologize for this error.

Intravitreal delivery of AAV8 retinoschisin results in cell type-specific gene expression and retinal rescue in the Rs1-KO mouse

X-linked juvenile retinoschisis (XLRS) is a neurodevelopmental abnormality caused by retinoschisin gene mutations. XLRS is characterized by splitting through the retinal layers and impaired synaptic transmission of visual signals resulting in impaired acuity and a propensity to retinal detachment. Several groups have treated murine retinoschisis models successfully using adeno-associated virus (AAV) vectors. Owing to the fragile nature of XLRS retina, translating this therapy to the clinic may require an alternative to invasive subretinal vector administration. Here we show that all layers of the retinoschisin knockout (Rs1-KO) mouse retina can be transduced efficiently with AAV vectors administered by simple vitreous injection. Retinoschisin expression was restricted to the neuroretina using a new vector that uses a 3.5-kb human retinoschisin promoter and an AAV type 8 capsid. Intravitreal administration to Rs1-KO mice resulted in robust retinoschisin expression with a retinal distribution similar to that observed in wild-type retina, including the expression by the photoreceptors lying deep in the retina. No off-target expression was observed. Rs1-KO mice treated with this vector showed a decrease in the schisis cavities and had improved retinal signaling evaluated by recording the electroretinogram 11-15 weeks after the application.

754. Intravitreal Administration of an AAV8 Retinoschisin Vector to the Rs1-KO Mouse Results in Cell Type-Specific Gene Expression and Rescue of the Disease Phenotype

754. Intravitreal Administration of an AAV8 Retinoschisin Vector to the Rs1-KO Mouse Results in Cell Type-Specific Gene Expression and Rescue of the Disease Phenotype

The Effects of Transient Retinal Detachment on Cavity Size and Glial and Neural Remodeling in a Mouse Model of X-Linked Retinoschisis

To determine the cellular consequences of retinal detachment in retinoschisin knockout (Rs1-KO) mice, a model for retinoschisin in humans. Experimental retinal detachments (RDs) were induced in the right eyes of both Rs1-KO and wild-type (wt) control mice. Immunocytochemistry was performed on retinal tissue at 1, 7, or 28 days after RD with antibodies to anti-GFAP, -neurofilament, and -rod opsin to examine cellular changes after detachment. Images of the immunostained tissue were captured by laser scanning confocal microscopy. Quantitative analysis was performed to measure the number of Hoechst-stained photoreceptor nuclei and their density, number, and size of inner retinal cavities, as well as the number of subretinal glial scars. Since detachments were created with balanced salt solution, by examination, all retinas had spontaneously reattached by 1 day. Cellular responses common to many photoreceptor degenerations occurred in the nondetached retinas of Rs1-KO mice, and, of importance, RD did not appear to significantly accentuate these responses. The number of schisis cavities was not changed after detachment, but their size was reduced. These data indicate that large short-term RD in Rs1-KO mice, followed by a period of reattachment may cause a slight increase in photoreceptor cell death, but detachments do not accentuate the gliosis and neurite sprouting already present and may in fact reduce the size of existing retinal cavities. This finding suggests that performing subretinal injections to deliver therapeutic agents may be a viable option in the treatment of patients with retinoschisis without causing significant cellular damage to the retina.

Synaptic Pathology in Retinoschisis Knockout (Rs1−/y) Mouse Retina and Modification by rAAV-Rs1Gene Delivery

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. The protein expression and morphology of the OPL in Rs1-KO mice was investigated at different ages, to explore the role of the synaptic layer in these ERG changes. Retinas of wild-type (Wt) and Rs1-KO mice from postnatal day (P)7 to 12 months were evaluated by light and electron microscopy (EM) and biochemistry. PSD95 (postsynaptic density protein), mGluR6 (metabotropic glutamate receptor subtype 6), retinoschisin (Rs1), the Müller cell proteins glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS), the bipolar cell marker protein kinase C alpha (PKCalpha), and the horizontal cell marker calbindin were localized by immunofluorescence and immuno-EM. Levels of PSD95 and mGluR6 were determined by quantitative Western blot. Rs1-KO mice treated by intravitreous injection of rAAV(2/2)-CMV-Rs1 in one eye at P14 were evaluated at 8 months by full-field scotopic ERG responses and retinal immunohistochemistry. Rs1 was associated with the outer surface of synaptic membranes in wild-type (Wt) retinas. PSD95 and mGluR6 were juxtaposed in the OPL of the Rs1-KO retinas by P14, implying that synaptic structures are formed. Light microscopic retinal morphology was similar in Wt and Rs1-KO at P14, but by P21, the OPL was disrupted in Rs1-KO, and some PSD95 and mGluR6 was mislocalized in the outer nuclear layer (ONL). GFAP expression spanned all retinal layers. EM showed synaptic structures adjacent to photoreceptor nuclei. PSD95 and mGluR6 levels were normal at 1 month on Western blot but declined to 59% (P < 0.001) and 55% (P < 0.05) of Wt, respectively, by 4 months. Levels thereafter showed no further reduction out to 12 months. Eyes injected with AAV-Rs1 were studied at 8 months by immunohistochemistry and had higher expression of PSD95 and mGluR6 and less GFAP expression compared with fellow untreated eyes. In the Rs1-KO mouse, retinal layer formation and synaptic protein expression in the OPL is normal up to P14, implying normal development of synaptic connections. Aberrant localization of synaptic proteins by P21 indicates that displacement of developing and/or mature synapses contributes to the b-wave reduction at young ages, when photoreceptor numbers and synaptic protein levels are normal. The subsequent decline in PSD95 and mGluR6 between 1 and 12 months in Rs1-KO retina mirrors the course of b-wave change and provides evidence of causal relationship between the ERG and OPL changes. These findings and the improved structural integrity of the OPL and b-wave amplitude after Rs1 gene transfer therapy provide a cellular and molecular basis for interpreting the changes in retinal signaling in this model.