RESUMO
Blue cone monochromacy (BCM) is a rare X-linked retinal disease characterized by the absence of L- and M-opsin in cone photoreceptors, considered a potential gene therapy candidate. However, most experimental ocular gene therapies utilize subretinal vector injection which would pose a risk to the fragile central retinal structure of BCM patients. Here we describe the use of ADVM-062, a vector optimized for cone-specific expression of human L-opsin and administered using a single intravitreal (IVT) injection. Pharmacological activity of ADVM-062 was established in gerbils, whose cone-rich retina naturally lacks L-opsin. A single IVT administration dose of ADVM-062 effectively transduced gerbil cone photoreceptors and produced a de novo response to long-wavelength stimuli. To identify potential first-in-human doses we evaluated ADVM-062 in non-human primates. Cone-specific expression of ADVM-062 in primates was confirmed using ADVM-062.myc, a vector engineered with the same regulatory elements as ADVM-062. Enumeration of human OPN1LW.myc-positive cones demonstrated that doses ≥3 × 1010 vg/eye resulted in transduction of 18%-85% of foveal cones. A Good Laboratory Practice (GLP) toxicology study established that IVT administration of ADVM-062 was well tolerated at doses that could potentially achieve clinically meaningful effect, thus supporting the potential of ADVM-062 as a one-time IVT gene therapy for BCM.
Assuntos
Opsinas , Células Fotorreceptoras Retinianas Cones , Animais , Humanos , Células Fotorreceptoras Retinianas Cones/metabolismo , Opsinas/genética , Primatas/genética , Primatas/metabolismo , Opsinas de Bastonetes/genética , Opsinas de Bastonetes/metabolismo , Terapia Genética/métodosRESUMO
PURPOSE: To determine whether short-latency changes in multifocal electroretinography (mfERG) observed in experimental glaucoma (EG) are secondary solely to retinal ganglion cell (RGC) loss or whether there is a separate contribution from elevated intraocular pressure (IOP). METHODS: Prior to operative procedures, a series of baseline mfERGs were recorded from six rhesus macaques using a 241-element unstretched stimulus. Animals then underwent hemiretinal endodiathermy axotomy (HEA) by placing burns along the inferior 180° of the optic nerve margin in the right eye (OD). mfERG recordings were obtained in each animal at regular intervals following for 3-4 months to allow stabilization of the HEA effects. Laser trabecular meshwork destruction (LTD) to elevate IOP was then performed; first-order kernel (K1) waveform root-mean-square (RMS) amplitudes for the short-latency segment of the mfERG wave (9-35 ms) were computed for two 7-hexagon groupings-the first located within the superior (non-axotomized) macula and the second within the inferior (axotomized) macula. Immunohistochemistry for glial fibrillary acidic protein (GFAP) was done. RESULTS: By 3 months post HEA, there was marked thinning of the inferior nerve fiber layer as measured by optical coherence tomography. Compared with baseline, no statistically significant changes in 9-35 ms K1 RMS amplitudes were evident in either the axotomized or non-axotomized portions of the macula. Following LTD, mean IOP in HEA eyes rose to 46 ± 9 compared with 20 ± 2 mmHg (SD) in the fellow control eyes. In the HEA + EG eyes, statistically significant increases in K1 RMS amplitude were present in both the axotomized inferior and non-axotomized superior portions of the OD retinas. No changes in K1 RMS amplitude were found in the fellow control eyes from baseline to HEA epoch, but there was a smaller increase from baseline to HEA + EG. Upregulation of GFAP in the Müller cells was evident in both non-axotomized and axotomized retina in eyes with elevated IOP. CONCLUSIONS: The RMS amplitudes of the short-latency mfERG K1 waveforms are not altered following axotomy but undergo marked increases following elevated IOP. This suggests that the increase in mfERG amplitude was not solely a result of RGC loss and may reflect photoreceptor and bipolar cell dysfunction and/or changes in Müller cells.
Assuntos
Glaucoma , Células Ganglionares da Retina , Animais , Células Ganglionares da Retina/fisiologia , Eletrorretinografia/métodos , Axotomia , Macaca mulatta/fisiologia , Glaucoma/diagnóstico , Retina , Pressão IntraocularRESUMO
Multiple sclerosis (MS) is a common cause of neurologic disease in young adults that is primarily treated with disease-modifying therapies which target the immune and inflammatory responses. Promotion of remyelination has opened a new therapeutic avenue, but how best to determine efficacy of remyelinating drugs remains unresolved. Although prolongation and then shortening of visual evoked potential (VEP) latencies in optic neuritis in MS may identify demyelination and remyelination, this has not been directly confirmed. We recorded VEPs in a model in which there is complete demyelination of the optic nerve, with subsequent remyelination. We examined the optic nerves microscopically during active disease and recovery, and quantitated both demyelination and remyelination along the length of the nerves. Latencies of the main positive component of the control VEP demonstrated around 2-fold prolongation during active disease. VEP waveforms were nonrecordable in a few subjects or exhibited a broadened profile which precluded peak identification. As animals recovered neurologically, the VEP latencies decreased in association with complete remyelination of the optic nerve but remained prolonged relative to controls. Thus, it has been directly confirmed that VEP latencies reflect the myelin status of the optic nerve and will provide a surrogate marker in future remyelination clinical trials.
RESUMO
We report a focal disturbance in myelination of the optic nerve in the osteopetrotic (op/op) mouse, which results from a spontaneous compression of the nerve resulting from stenosis of the optic canal. The growth of the op/op optic nerve was significantly affected, being maximally suppressed at postnatal day 30 (P30; 33% of age matched control). Myelination of the nerve in the optic canal was significantly delayed at P15, and myelin was almost completely absent at P30. The size of nerves and myelination were conserved both in the intracranial and intraorbital segments at P30, suggesting that the axons in the compressed site are spared in all animals at P30. Interestingly, we observed recovery both in the nerve size and the density of myelinated axons at 7 months in almost half of the optic nerves examined, although some nerves lost axons and became atrophic. In vivo and ex vivo electrophysiological examinations of P30 op/op mice showed that nerve conduction was significantly delayed but not blocked with partial recovery in some mice by 7 months. Transcardial perfusion of FITC-labeled albumin suggested that local ischemia was at least in part the cause of this myelination failure. These results suggest that the primary abnormality is dysmyelination of the optic nerve in early development. This noninvasive model system will be a valuable tool to study the effects of nerve compression on the function and survival of oligodendrocyte progenitor cells/oligodendrocytes and axons and to explore the mechanism of redistribution of oligodendrocyte progenitor cells with compensatory myelination.
Assuntos
Doenças Desmielinizantes/genética , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Síndromes de Compressão Nervosa/patologia , Doenças do Nervo Óptico/genética , Doenças do Nervo Óptico/patologia , Nervo Óptico/patologia , Animais , Camundongos , Camundongos Mutantes Neurológicos , Síndromes de Compressão Nervosa/genética , Fibras Nervosas Mielinizadas/patologia , Fibras Nervosas Mielinizadas/fisiologia , Condução Nervosa/genética , Oligodendroglia/patologia , Oligodendroglia/fisiologia , Nervo Óptico/fisiologia , Osteopetrose/genética , Células-Tronco/patologia , Células-Tronco/fisiologiaRESUMO
We compared the suitability of pentobarbital sodium (PB) and propofol (PF) anesthetics for multifocal electroretinograms (mfERGs) in rhesus macaques. mfERGs were collected from 4 ocularly normal rhesus macaques. All animals were pre-anesthetized with intramuscular ketamine (10-15 mg/kg). Intravenous PB induction/maintenance levels were 15 mg/kg/2-10 mg/kg and for PF, 2-5 mg/kg/6-24 mg/kg/h. There were 3 testing sessions with PB anesthesia and 5-7 testing sessions with PF anesthesia. All PB sessions were carried out before PF. First-order (K1) and second-order (first slice) kernels (K2.1) response density amplitude (RDA), implicit time (IT), and root mean square signal-to-noise ratios (RMS SNR) of the low-frequency (LFC) and high-frequency (HFC) components were evaluated. The use of PF or PB anesthesia resulted in robust, replicable mfERGs in rhesus macaques; however, RMS SNR of K1 LFC in ring and quadrant analyses was significantly larger for PF than for PB. Additionally, K1 RDA under PF was significantly larger than under PB for N1, P1, and P2 components (ring and quadrant) and for N2 (quadrant). PF IT was significantly prolonged (<1 ms) relative to PB IT for N1, P1 (ring), and N1 (quadrant), while PB IT was significantly prolonged (0.8-4.2 ms) relative to PF IT for N2 and P2 (ring and quadrant). K1 HFC and K2.1 LFC did not differ significantly between PB and PF in the ring or quadrant analyses. The response differences found with PB and PF anesthesia likely arise from variable relative effects of the anesthetics on retinal γ-aminobutyric acid (GABA(A)) receptors, and in part, on glycine and on glutamate receptors. Given the advantages of a stable anesthetic plane with continuous intravenous infusion and a smoother, more rapid recovery, PF is an appealing alternative for mfERG testing in rhesus macaques.
Assuntos
Anestesia/métodos , Eletrorretinografia/efeitos dos fármacos , Pentobarbital/farmacologia , Propofol/farmacologia , Retina/efeitos dos fármacos , Anestésicos Intravenosos/farmacologia , Animais , Potenciais Evocados Visuais/efeitos dos fármacos , Feminino , Hipnóticos e Sedativos/farmacologia , Macaca mulatta , Retina/fisiologia , Retinoscopia , Razão Sinal-RuídoRESUMO
PURPOSE: To evaluate sweep VEP (sVEP) in preverbal children with optic nerve hypoplasia (ONH) and to assess associations between sVEP results, patient clinical characteristics and future recognition visual acuity. METHODS: The medical records of children with ONH who had sVEP testing and documented recognition visual acuity at the University of Wisconsin from 2005 to 2013 were reviewed retrospectively. Optic nerve size, amblyopia treatment, and neurologic diagnoses were collected. RESULTS: A total of 57 patients were included: 41 (71%) with bilateral ONH and 27 (47%) with neurologic abnormality. Mean age at initial sVEP was 13.3 months (range, 1-32). Mean duration between initial sVEP and final recognition acuity was 5.5 years (range, 3.5-7). Sweep VEP was associated with ONH severity (P < 0.05). Sweep VEP, and the combination of ONH severity and neurologic status, were significant predictors (P < 0.05) of logMAR optotype acuity, together accounting for 54%-61% of the variance in final recognition acuity. CONCLUSIONS: Sweep VEP in preverbal children with ONH depends on ONH severity and correlates with final recognition visual acuity. Children with milder degrees of ONH without neurologic abnormalities had better final vision, and patients with severe ONH and neurologic diagnoses had worse vision outcomes.
Assuntos
Ambliopia , Hipoplasia do Nervo Óptico , Ambliopia/diagnóstico , Criança , Potenciais Evocados Visuais , Humanos , Estudos Retrospectivos , Acuidade VisualRESUMO
The purpose of this study was to evaluate the relationship between elevations of intraocular pressure (IOP) and the multifocal electroretinogram (mfERG) in non-human primates. Experimental glaucoma was induced in 4 rhesus and 4 cynomolgus monkeys by laser trabecular meshwork destruction (LTD) in one eye. To evaluate the contribution of ganglion cells to mfERG changes, one monkey of each species had previously underwent unilateral optic nerve transection (ONT). After >or=44 weeks of elevation, the IOP was reduced by trabeculectomy in 2 non-transected animals. In the intact (non-transected) animals, there was an increase in the amplitude of the early mfERG waveforms (N1 and P1) of the first-order kernel (K1) throughout the period of IOP elevation in all of the rhesus, but not all of the cynomolgus monkeys. A species difference was also present as a decrease of the second-order kernel, first slice (K2.1) in all of the cynomolgus monkeys but only in 1 of the rhesus monkeys (the 1 with the ONT). Similar IOP effects on the mfERG were seen in the ONT animals. Surgical lowering of IOP resulted in a return of the elevated K1 amplitudes to baseline levels. However, the depressed K2.1 RMS in the cynomolgus monkeys did not recover. These results demonstrate species-specific changes in cone-driven retinal function during periods of elevated IOP. These IOP-related effects can occur in the absence of retinal ganglion cells and may be reversible.
Assuntos
Modelos Animais de Doenças , Glaucoma/fisiopatologia , Pressão Intraocular/fisiologia , Retina/fisiopatologia , Células Ganglionares da Retina/fisiologia , Animais , Eletrorretinografia , Feminino , Macaca fascicularis , Macaca mulatta , Masculino , Compressão Nervosa , Traumatismos do Nervo Óptico/fisiopatologia , TrabeculectomiaRESUMO
PURPOSE: We present 3 cases of Alström syndrome (ALMS) that highlight the importance of the ophthalmic exam, as well as the diagnostic challenges and management considerations of this ultra-rare disease. OBSERVATIONS: The first case is of a 2-year-old boy with history of spasmus nutans who presented with head bobbing and nystagmus. The second patient is a 5-year-old boy with history of infantile dilated cardiomyopathy status post heart transplant, Burkitt lymphoma status post chemotherapy, obesity, global developmental delay, and high hyperopia previously thought to have cortical visual impairment secondary to heart surgery/possible ischemic event. This patient presented with nystagmus, photophobia, and reduced vision. The third case involves a 8-year-old boy with history of obesity, bilateral optic nerve atrophy, hyperopic astigmatism, exotropia, and nystagmus. Upon presentation to the consulting pediatric ophthalmologist, none of the patients had yet been diagnosed with ALMS. All 3 cases were subsequently found to have an electroretinogram (ERG) that exhibited severe global depression and to carry ALMS1 pathogenic variants. CONCLUSIONS AND IMPORTANCE: ALMS is an autosomal recessive disease caused by ALMS1 variations, characterized by cone-rod dystrophy, obesity, progressive sensorineural hearing loss, cardiomyopathy, insulin resistance, and multiorgan dysfunction. Retinal dystrophy diagnosis is critical given clinical criteria and detection rates of genetic testing. Early diagnosis is extremely important because progression to flat ERG leads to the inability to differentiate between rod-cone or cone-rod involvement, either of which have their own differential diagnoses. In our series, the ophthalmic exam and abnormal ERG prompted further genetic testing and the subsequent diagnosis of ALMS. Multidisciplinary care ensures the best possible outcome with the ophthalmologist playing a key role.
RESUMO
Glaucoma, a multifactorial neurodegenerative disease characterized by progressive loss of retinal ganglion cells and their axons in the optic nerve, is a leading cause of irreversible vision loss. Intraocular pressure (IOP) is a risk factor for axonal damage, which initially occurs at the optic nerve head (ONH). Complex cellular and molecular mechanisms involved in the pathogenesis of glaucomatous optic neuropathy remain unclear. Here we define early molecular events in the ONH in an inherited large animal glaucoma model in which ONH structure resembles that of humans. Gene expression profiling of ONH tissues from rigorously phenotyped feline subjects with early-stage glaucoma and precisely age-matched controls was performed by RNA-sequencing (RNA-seq) analysis and complementary bioinformatic approaches applied to identify molecular processes and pathways of interest. Immunolabeling supported RNA-seq findings while providing cell-, region-, and disease stage-specific context in the ONH in situ. Transcriptomic evidence for cell proliferation and immune/inflammatory responses is identifiable in early glaucoma, soon after IOP elevation and prior to morphologically detectable axon loss, in this large animal model. In particular, proliferation of microglia and oligodendrocyte precursor cells is a prominent feature of early-stage, but not chronic, glaucoma. ONH microgliosis is a consistent hallmark in both early and chronic stages of glaucoma. Molecular pathways and cell type-specific responses strongly implicate toll-like receptor and NF-κB signaling in early glaucoma pathophysiology. The current study provides critical insights into molecular pathways, highly dependent on cell type and sub-region in the ONH even prior to irreversible axon degeneration in glaucoma.
Assuntos
Glaucoma/metabolismo , Microglia/metabolismo , Disco Óptico/metabolismo , Nervo Óptico/metabolismo , Animais , Gatos , Proliferação de Células/fisiologia , Modelos Animais de Doenças , Potenciais Evocados Visuais/fisiologia , Perfilação da Expressão Gênica , Glaucoma/patologia , Inflamação/metabolismo , Inflamação/patologia , Microglia/patologia , Disco Óptico/patologia , Nervo Óptico/patologia , Transdução de Sinais/fisiologia , TranscriptomaRESUMO
Purpose: Vigabatrin (VGB) is an effective antiepileptic that increases concentrations of inhibitory γ-aminobutyric acid (GABA) by inhibiting GABA transaminase. Reports of VGB-associated visual field loss limit its clinical usefulness, and retinal toxicity studies in laboratory animals have yielded conflicting results. Methods: We examined the functional and morphologic effects of VGB in C57BL/6J mice that received either VGB or saline IP from 10 to 18 weeks of age. Retinal structure and function were assessed in vivo by optical coherence tomography (OCT), ERG, and optomotor response. After euthanasia, retinas were processed for immunohistochemistry, and retinal GABA, and VGB quantified by mass spectrometry. Results: No significant differences in visual acuity or total retinal thickness were identified between groups by optomotor response or optical coherence tomography, respectively. After 4 weeks of VGB treatment, ERG b-wave amplitude was enhanced, and amplitudes of oscillatory potentials were reduced. Dramatic rod and cone bipolar and horizontal cell remodeling, with extension of dendrites into the outer nuclear layer, was observed in retinas of VGB-treated mice. VGB treatment resulted in a mean 3.3-fold increase in retinal GABA concentration relative to controls and retinal VGB concentrations that were 20-fold greater than brain. Conclusions: No evidence of significant retinal thinning or ERG a- or b-wave deficits were apparent, although we describe significant alterations in ERG b-wave and oscillatory potentials and in retinal cell morphology in VGB-treated C57BL/6J mice. The dramatic concentration of VGB in retina relative to the target tissue (brain), with a corresponding increase in retinal GABA, offers insight into the pathophysiology of VGB-associated visual field loss.
Assuntos
Anticonvulsivantes/farmacologia , GABAérgicos/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Retina/efeitos dos fármacos , Vigabatrina/farmacologia , Animais , Masculino , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/fisiologia , Músculos Oculomotores/efeitos dos fármacos , Distribuição Aleatória , Retina/fisiopatologia , Doenças Retinianas/tratamento farmacológico , Doenças Retinianas/fisiopatologia , Tomografia de Coerência Óptica , Campos Visuais/fisiologiaRESUMO
Laser-induced experimental glaucoma (ExGl) in non-human primates (NHPs) is a common animal model for ocular drug development. While many features of human hypertensive glaucoma are replicated in this model, structural and functional changes in the unlasered portions of trabecular meshwork (TM) of laser-treated primate eyes are understudied. We studied NHPs with ExGl of several years duration. As expected, ExGl eyes exhibited selective reductions of the retinal nerve fiber layer that correlate with electrophysiologic measures documenting a link between morphologic and elctrophysiologic endpoints. Softening of unlasered TM in ExGl eyes compared to untreated controls was observed. The degree of TM softening was consistent, regardless of pre-mortem clinical findings including severity of IOP elevation, retinal nerve fiber layer thinning, or electrodiagnostic findings. Importantly, this softening is contrary to TM stiffening reported in glaucomatous human eyes. Furthermore, microscopic analysis of unlasered TM from eyes with ExGl demonstrated TM thinning with collapse of Schlemm's canal; and proteomic analysis confirmed downregulation of metabolic and structural proteins. These data demonstrate unexpected and compensatory changes involving the TM in the NHP model of ExGl. The data suggest that compensatory mechanisms exist in normal animals and respond to elevated IOP through softening of the meshwork to increase outflow.
Assuntos
Olho/metabolismo , Glaucoma/metabolismo , Hipertensão/metabolismo , Modelos Animais , Malha Trabecular/fisiologia , Animais , Fenômenos Eletrofisiológicos , Olho/patologia , Glaucoma/etiologia , Humanos , Hipertensão/complicações , Pressão Intraocular , Lasers , Primatas , ProteomaRESUMO
OBJECTIVE: To develop a quantitative measure of regional variation in choroidal blood flow (ChBF). METHODS: Five million 15-microm fluorescent microspheres were injected into the left ventricles of 4 rabbits and 3 monkeys. The fixed globes were bleached, flat mounted, and photomicrographed. After image analysis to locate each microsphere, regional densities and blood flow were determined. RESULTS: Regional variation in ChBF was clearly evident. In the rabbit, a high density of spheres was seen in the visual streak. This was surrounded by a middle peripheral area of low sphere density and a far peripheral region of moderately high density. In the monkeys, sphere density was markedly greater in the macula compared with the periphery. Contour plots produced lines of constant flow that were oval and extended farther nasally than temporally in the monkeys. The ratio of central to peripheral ChBF was much greater in the monkeys than in the rabbits. CONCLUSION: Quantitative assessment of regional ChBF can be performed using a modification of the fluorescent microsphere impaction method. CLINICAL RELEVANCE: This method of determining regional ChBF will be useful for studying the vascular effects of pharmacologic agents and for characterizing animal models of human disease involving the outer retina.
Assuntos
Corioide/irrigação sanguínea , Corantes Fluorescentes , Hemorreologia/métodos , Microesferas , Animais , Velocidade do Fluxo Sanguíneo , Macaca fascicularis , Macaca mulatta , Microscopia de Fluorescência , Coelhos , Fluxo Sanguíneo Regional/fisiologiaRESUMO
PURPOSE: To evaluate monkey multifocal visual evoked cortical potentials (mfVEPs) recorded from central and peripheral fields for reliability and isolation from electroretinographic (ERG) activity. METHODS: The mfVEP stimulus consisted of a 7-element hexagonal array that subtended 80 degrees of the central visual field. Recordings were made under intravenous pentobarbital sodium (15 mg/kg) anesthesia. Two monkeys with absent optic nerve and ganglion cell function after combined unilateral optic nerve transection and experimental ocular hypertension (ONT/OHT) were followed longitudinally. In a second study, 16 ophthalmologically normal monkeys were tested once. RESULTS: Testing of the non-transected eye in two transected animals revealed robust first- and second-order kernel, first slice (K1 and K2.1) mfVEPs. Stimulation of the transected eye revealed no contamination of the mfVEP from the concurrently recorded multifocal ERGs. There was complete separation of the root-mean-square (RMS) mfVEP amplitudes from the transected and the fellow eyes tested repeatedly across a 4- to 17- month period. The largest amplitude mfVEP was generated by the central element; however, mfVEPs were recorded from outside the central 20 degrees element. The 16 normal animals showed waveforms similar to the normal eyes of the ONT/OHT animals both in shape and distribution throughout the visual field. A scalar-product measure showed both K1 and K2.1 mfVEPs from central and some peripheral elements were statistically distinct from noise. CONCLUSIONS: mfVEPs can be reliably recorded from non-human primates anesthetized with pentobarbital. Under the recording conditions described, mfVEPs are not contaminated by ERG activity. mfVEPs may be useful in animal models of diseases that differentially affect macular and peripheral visual field responsiveness.
Assuntos
Anestesia Intravenosa , Córtex Cerebral/fisiologia , Potenciais Evocados Visuais/fisiologia , Vias Visuais/fisiologia , Animais , Eletrorretinografia , Hipnóticos e Sedativos/administração & dosagem , Macaca fascicularis , Macaca mulatta , Nervo Óptico/cirurgia , Pentobarbital/administração & dosagem , Reprodutibilidade dos Testes , Retina/fisiologia , Células Ganglionares da Retina/fisiologiaRESUMO
Applied Genetic Technologies Corporation (AGTC) is developing rAAV2tYF-PR1.7-hCNGB3, a recombinant adeno-associated virus (rAAV) vector expressing the human CNGB3 gene, for treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. We report here results of a study evaluating safety and biodistribution of rAAV2tYF-PR1.7-hCNGB3 in CNGB3-deficient mice. Three groups of animals (n = 35 males and 35 females per group) received a subretinal injection in one eye of 1 µl containing either vehicle or rAAV2tYF-PR1.7-hCNGB3 at one of two dose concentrations (1 × 10(12) or 4.2 × 10(12) vg/ml) and were euthanized 4 or 13 weeks later. There were no test-article-related changes in clinical observations, body weights, food consumption, ocular examinations, clinical pathology parameters, organ weights, or macroscopic observations at necropsy. Cone-mediated electroretinography (ERG) responses were detected after vector administration in the treated eyes in 90% of animals in the higher dose group and 31% of animals in the lower dose group. Rod-mediated ERG responses were reduced in the treated eye for all groups, with the greatest reduction in males given the higher dose of vector, but returned to normal by the end of the study. Microscopic pathology results demonstrated minimal mononuclear cell infiltrates in the retina and vitreous of some animals at the interim euthanasia and in the vitreous of some animals at the terminal euthanasia. Serum anti-AAV antibodies developed in most vector-injected animals. No animals developed antibodies to hCNGB3. Biodistribution studies demonstrated high levels of vector DNA in vector-injected eyes but little or no vector DNA in nonocular tissue. These results support the use of rAAV2tYF-PR1.7-hCNGB3 in clinical studies in patients with achromatopsia caused by CNGB3 mutations.
Assuntos
Defeitos da Visão Cromática/terapia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , DNA Recombinante/efeitos adversos , Dependovirus/genética , Terapia Genética , Vetores Genéticos/efeitos adversos , Animais , Defeitos da Visão Cromática/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/deficiência , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , DNA Recombinante/administração & dosagem , Feminino , Vetores Genéticos/administração & dosagem , Humanos , Injeções Intraoculares , Masculino , Camundongos , Retina/metabolismoRESUMO
Applied Genetic Technologies Corporation (AGTC) is developing rAAV2tYF-PR1.7-hCNGB3, a recombinant adeno-associated viral (rAAV) vector expressing the human CNGB3 gene, for treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. We report here results of a study evaluating the safety and biodistribution of rAAV2tYF-PR1.7-hCNGB3 in cynomolgus macaques. Three groups of animals (n = 2 males and 2 females per group) received a subretinal injection in one eye of 300 µl containing either vehicle or rAAV2tYF-PR1.7-hCNGB3 at one of two concentrations (4 × 10(11) or 4 × 10(12) vector genomes/ml) and were evaluated over a 3-month period before being euthanized. Administration of rAAV2tYF-PR1.7-hCNGB3 was associated with a dose-related anterior and posterior segment inflammatory response that was greater than that observed in eyes injected with the vehicle control. Most manifestations of inflammation improved over time except that vitreous cells persisted in vector-treated eyes until the end of the study. One animal in the lower vector dose group was euthanized on study day 5, based on a clinical diagnosis of endophthalmitis. There were no test article-related effects on intraocular pressure, visual evoked potential responses, hematology or clinical chemistry parameters, or gross necropsy observations. Histopathological examination demonstrated minimal mononuclear infiltrates in all vector-injected eyes. Serum anti-AAV antibodies developed in all vector-injected animals. No animals developed antibodies to CNGB3. Biodistribution studies demonstrated high levels of vector DNA in the injected eye but minimal or no vector DNA in any other tissue. These results support the use of rAAV2tYF-PR1.7-hCNGB3 in clinical studies in patients with achromatopsia caused by CNGB3 mutations.
Assuntos
Defeitos da Visão Cromática/terapia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , DNA Recombinante/efeitos adversos , Dependovirus/genética , Terapia Genética , Vetores Genéticos/efeitos adversos , Animais , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , DNA Recombinante/administração & dosagem , Feminino , Vetores Genéticos/administração & dosagem , Humanos , Injeções Intraoculares , Macaca fascicularis , MasculinoRESUMO
Applied Genetic Technologies Corporation is developing rAAV2tYF-CB-hRS1, a recombinant adeno-associated virus (rAAV) vector for treatment of X-linked retinoschisis (XLRS), an inherited retinal disease characterized by splitting (schisis) of retinal layers causing poor vision. We report here results of a study evaluating the safety and biodistribution of rAAV2tYF-CB-hRS1 in normal cynomolgus macaques. Three groups of male animals (n = 6 per group) received an intravitreal injection in one eye of either vehicle, or rAAV2tYF-CB-hRS1 at one of two dose levels (4 × 10(10) or 4 × 10(11) vg/eye). Half the animals were sacrificed after 14 days and the others after 91 or 115 days. The intravitreal injection procedure was well tolerated in all groups. Serial ophthalmic examinations demonstrated a dose-related anterior and posterior segment inflammatory response that improved over time. There were no test article-related effects on intraocular pressure, electroretinography, visual evoked potential, hematology, coagulation, clinical chemistry, or gross necropsy observations. Histopathological examination demonstrated minimal or moderate mononuclear infiltrates in 6 of 12 vector-injected eyes. Immunohistochemical staining showed RS1 labeling of the ganglion cell layer at the foveal slope in vector-injected eyes at both dose levels. Serum anti-AAV antibodies were detected in 4 of 6 vector-injected animals at the day 15 sacrifice and all vector-injected animals at later time points. No animals developed antibodies to RS1. Biodistribution studies demonstrated high levels of vector DNA in the injected eye but minimal or no vector DNA in any other tissue. These results support the use of rAAV2tYF-CB-hRS1 in clinical studies in patients with XLRS.
Assuntos
Dependovirus/genética , Proteínas do Olho/genética , Expressão Gênica , Vetores Genéticos/genética , Vetores Genéticos/farmacocinética , Retinosquise/genética , Retinosquise/terapia , Transgenes , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linhagem Celular , Protocolos Clínicos , Modelos Animais de Doenças , Genes Reporter , Vetores Genéticos/administração & dosagem , Vetores Genéticos/efeitos adversos , Humanos , Imuno-Histoquímica , Injeções Intravítreas , Macaca fascicularis , Masculino , Retina/metabolismo , Retina/patologia , Retinosquise/fisiopatologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Tempo , Distribuição Tecidual , Eliminação de Partículas ViraisRESUMO
PURPOSE: Systemic neoplastic processes can affect the retina through autoimmune retinopathy. This process may present in a variety of patterns. A novel pattern of paraneoplastic autoimmune retinopathy has been described. METHODS: Two patients presented with paraneoplastic vision changes. Optical coherence tomography and multifocal electroretinography were performed, which showed a unique focal retinal change in both patients. Case 1 is a 20-year-old woman with history of cutaneous melanoma. Case 2 is 67-year-old woman with history of breast cancer. RESULTS: Both patients showed a localized area of foveal thinning that corresponded with areas of depression on multifocal electroretinography. Each tested positive for antiretinal antibodies. CONCLUSION: This is a novel manifestation of paraneoplastic autoimmune retinopathy. Previously described cases have all affected the retina diffusely. These cases highlight the need to consider distant neoplastic processes when evaluating patient with similar presentations to those depicted here.
Assuntos
Doenças Autoimunes/diagnóstico , Síndromes Paraneoplásicas Oculares/diagnóstico , Doenças Retinianas/diagnóstico , Idoso , Neoplasias da Mama/complicações , Feminino , Humanos , Melanoma/complicações , Neoplasias Cutâneas/complicações , Adulto JovemRESUMO
PURPOSE: Outer retinal injury has been well described in glaucoma. To better understand the source of this injury, we wanted to develop a reliable model of partial retinal ganglion cell (RGC) axotomy. METHODS: Endodiathermy spots were placed along the inferior 180° adjacent to the optic nerve margin in the right eyes of four cynomolgus monkeys. Fluorescein angiography, spectral domain optical coherence tomography (SD-OCT), and multifocal electroretinography (mfERG) were performed at various intervals. Two animals were sacrificed at 3 months. Two animals were sacrificed at 4 months, at which time they underwent an injection of fluorescent microspheres to measure regional choroidal blood flow. Retinal immunohistochemistry for glial fibrillary acidic protein (GFAP), rhodopsin, S-cone opsin, and M/L-cone opsin were performed, as were axon counts of the optic nerves. RESULTS: At 3 months, there was marked thinning of the inferior nerve fiber layer on SD-OCT. The mfERG waveforms were consistent with inner but not outer retinal injury. Greater than 95% reduction in axons was seen in the inferior optic nerves but no secondary degeneration superiorly. There was marked thinning of the nerve fiber and ganglion cell layers in the inferior retinas. However, the photoreceptor histology was similar in the axotomized and nonaxotomized areas. Regional choroidal blood flow was not affected by the axotomy. CONCLUSIONS: Unlike experimental glaucoma, hemiretinal endodiathermy axotomy (HEA) of the RGCs produces no apparent anatomic, functional, or blood flow effects on the outer retina and choroid.
Assuntos
Axotomia , Modelos Animais de Doenças , Fibras Nervosas/patologia , Retina/fisiologia , Degeneração Retiniana/fisiopatologia , Células Ganglionares da Retina/patologia , Animais , Corioide/irrigação sanguínea , Eletrocoagulação , Eletrorretinografia , Feminino , Angiofluoresceinografia , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Macaca fascicularis , Masculino , Opsinas/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Vasos Retinianos/fisiologia , Rodopsina/metabolismo , Tomografia de Coerência ÓpticaRESUMO
OBJECTIVE: To characterize functional and anatomic sequelae of a bleb induced by subretinal injection. METHODS: Subretinal injections (100 µL) of balanced salt solution were placed in the superotemporal macula of 1 eye in 3 cynomolgus macaques. Fellow eyes received intravitreal injections (100 µL) of balanced salt solution. Fundus photography, ocular coherence tomography, and multifocal electroretinography were performed before and immediately after injection and again at intervals up to 3 months postinjection. Histopathologic analyses included transmission electron microscopy and immunohistochemistry for glial fibrillary acidic protein, rhodopsin, M/L-cone opsin, and S-cone opsin. RESULTS: Retinas were reattached by 2 days postinjection (seen by ocular coherence tomography). Multifocal electroretinography waveforms were suppressed post-subretinal injection within the subretinal injection bleb and, surprisingly, also in regions far peripheral to this area. Multifocal electroretinography amplitudes were nearly completely recovered by 90 days. The spectral-domain ocular coherence tomography inner segment-outer segment line had decreased reflectivity at 92 days. Glial fibrillary acidic protein and S-cone opsin staining were unaffected. Rhodopsin and M/L-cone opsins were partially displaced into the inner segments. Transmission electron microscopy revealed disorganization of the outer segment rod (but not cone) discs. At all postinjection intervals, eyes with intravitreal injection were similar to baseline. CONCLUSIONS: Subretinal injection is a promising route for drug delivery to the eye. Three months post-subretinal injection, retinal function was nearly recovered, although reorganization of the outer segment rod disc remained disrupted. Understanding the functional and anatomic effects of subretinal injection is important for interpretation of the effects of compounds delivered to the subretinal space. CLINICAL RELEVANCE: Subretinal injection is a new potential route for drug delivery to the eye. Separating drug effects from the procedural effects is critical.
Assuntos
Acetatos/administração & dosagem , Minerais/administração & dosagem , Retina/efeitos dos fármacos , Retina/fisiologia , Cloreto de Sódio/administração & dosagem , Animais , Combinação de Medicamentos , Sistemas de Liberação de Medicamentos , Eletrorretinografia , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Injeções Intravítreas , Macaca fascicularis , Microscopia Eletrônica , Oftalmoscopia , Opsinas/metabolismo , Retina/metabolismo , Retina/ultraestrutura , Rodopsina/metabolismo , Tomografia de Coerência ÓpticaRESUMO
Purpose. The scanning laser polarimetry with variable corneal compensation (GDx VCC) methodology was established and verified in monkeys with experimental glaucoma (ExpG). Terminal GDx parameters were correlated with axon counts and electrophysiologic measures. The effects of memantine on these parameters were investigated. Methods. ExpG was induced in monkeys and intraocular pressure monitored weekly. Some monkeys received memantine in their diet before and after ExpG induction (1-10 months). GDx VCC scans, stereophotographs, and multifocal visual evoked potential (mfVEP) data were collected at baseline and every 6 to 8 weeks until euthanasia. Optic nerves were prepared for axon counting and other morphologic analysis. Results. There was no difference in IOP elevation exposure between memantine-treated and no-memantine-treated monkeys. The percentage of the optic nerve area composed of connective tissue septa was significantly greater in ExpG eyes than in Fellow eyes. There was a strong positive correlation between axon counts and terminal GDx parameter measures. Animals not receiving memantine exhibited significantly lower mfVEP amplitudes in ExpG eyes compared with the ipsilateral baseline or the final value in the Fellow eye. ExpG eyes from memantine-treated animals had higher overall mean amplitudes that were not significantly different relative to the ipsilateral baseline and final amplitudes in the Fellow eye. Conclusions. The authors' studies confirm that GDx VCC can be utilized in monkey ExpG studies to detect early retinal structural changes and that these changes are highly correlated with optic nerve axon counts. These structural changes may or may not lead to central functional changes as shown by the mfVEP in response to investigational therapies.