Immune function in X-linked retinoschisis subjects in an AAV8-RS1 phase I/IIa gene therapy trial.

This study explored systemic immune changes in 11 subjects with X-linked retinoschisis (XLRS) in a phase I/IIa adeno-associated virus 8 (AAV8)-RS1 gene therapy trial (ClinicalTrials.gov: NCT02317887). Immune cell proportions and serum analytes were compared to 12 healthy male controls. At pre-dosing baseline the mean CD4/CD8 ratio of XLRS subjects was elevated. CD11c+ myeloid dendritic cells (DCs) and the serum epidermal growth factor (EGF) level were decreased, while CD123+ plasmacytoid DCs and serum interferon (IFN)-γ and tumor necrosis factor (TNF)-α were increased, indicating that the XLRS baseline immune status differs from that of controls. XLRS samples 14 days after AAV8-RS1 administration were compared with the XLRS baseline. Frequency of CD11b+CD11c+ DCc was decreased in 8 of 11 XLRS subjects across all vector doses (1e9-3e11 vector genomes [vg]/eye). CD8+human leukocyte antigen-DR isotype (HLA-DR)+ cytotoxic T cells and CD68+CD80+ macrophages were upregulated in 10 of 11 XLRS subjects, along with increased serum granzyme B in 8 of 11 XLRS subjects and elevated IFN-γ in 9 of 11 XLRS subjects. The six XLRS subjects with ocular inflammation after vector application gave a modestly positive correlation of inflammation score to their respective baseline CD4/CD8 ratios. This exploratory study indicates that XLRS subjects may exhibit a proinflammatory, baseline immune phenotype, and that intravitreal dosing with AAV8-RS1 leads to systemic immune activation with an increase of activated lymphocytes, macrophages, and proinflammatory cytokines.

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).

"Para-retinal" Vector Administration into the Deep Vitreous Enhances Retinal Transgene Expression.

Intravitreal administration for human adeno-associated vector (AAV) delivery is easier and less traumatic to ocular tissues than subretinal injection, but it gives limited retinal transduction. AAV vectors are large (about 4,000 kDa) compared with most intraocular drugs, such as ranibizumab (48 kDa), and the large size impedes diffusion to reach the retina from the usual injection site in the anterior/mid-vitreous. Intuitively, a preferred placement for the vector would be deep in the vitreous near the retina, which we term "para-retinal" delivery. We explored the consequences of para-retinal intravitreal delivery in the rabbit eye and in non-human primate (NHP) eye. 1 h after para-retinal administration in the rabbit eye, the vector concentration near the retina remained four times greater than in the anterior vitreous, indicating limited vector diffusion through the gelatinous vitreous matrix. In NHP, para-retinal placement showed greater transduction in the fovea than vector applied in the mid-vitreous. More efficient retinal delivery translates to using lower vector doses, with reduced risk of ocular inflammatory exposure. These results indicate that para-retinal delivery yields more effective vector concentration near the retina, thereby increasing the potential for better retinal transduction in human clinical application.

Repeat administration of proteins to the eye with a single intraocular injection of an adenovirus vector.

Delivery of therapeutic proteins, such as antiangiogenic proteins, to the eye is a demonstrated method for the control of age-related macular degeneration (AMD). However, one of the key limitations is the requirement for frequent and repeated intraocular injections. In this article, we demonstrate that repeated protein production in the eye can be stimulated from the cytomegalovirus (CMV) promoter without repeat intraocular injections using a small molecule, all-trans retinoic acid (ATRA). ATRA by systemic delivery can stimulate protein production multiple times in the eye. Administration of ATRA resulted in stimulation of gene expression to relevant levels that block abnormal blood vessel growth in an experimental animal model for AMD. These data support the principles of this technological discovery to therapeutic applications for chronic ocular diseases.

Persistent expression of PEDF in the eye using high-capacity adenovectors.

Ocular neovascularization, the growth of abnormal blood vessels in the eye, is a factor shared by the most common blinding diseases in developed countries. Pigment epithelium-derived factor (PEDF) is a potent antiangiogenic and neuroprotective protein that is normally produced in the eye. When delivered via an adenovector, PEDF can block the growth of new blood vessels and trigger the selective regression of abnormal vessels in animal models of ocular disease. Because of the absence of adenoviral genes, high-capacity (HC) adenovectors offer the potential for persistent transgene expression and enhanced tolerability. We have assessed the durability of PEDF expression and the induction of ocular inflammation following delivery of a PEDF-expressing HC adenovector compared to earlier generation vectors. The HC vector mediated prolonged PEDF expression in tissue-cultured pigmented epithelial cells and when delivered by intravitreal injection into the mouse eye. Delivery of first-generation adenovectors resulted in a dose-dependent increase in cytokine/chemokine gene expression, which correlated with the infiltration of inflammatory cells in the eye. In comparison, the levels of inflammatory gene expression and the intraocular infiltrate were substantially reduced following delivery of the HC vector. These results support the development of the HC adenovector gene delivery system for ocular disease.

Alternate serotype adenovector provides long-term therapeutic gene expression in the eye.

PURPOSE: To determine whether the duration of transgene expression from an alternate adenovector serotype, Ad35, can provide advantages over an Ad5 serotype vector following a single intravitreal (IVT) administration. METHODS: To assess the transgene expression profile, mice received one IVT injection of Ad5- or Ad35-based vectors expressing green fluorescent protein (GFP), luciferase or pigment epithelium-derived factor (PEDF). At specified time points following vector administration, eyes were monitored for GFP expression, or eyes were harvested and assayed for adenovector genomes, luciferase activity or PEDF levels. Ad35-based vector in vivo biologic activity was investigated using a mouse model of laser-induced choroidal neovascularization (CNV). On Day 0, mice received one IVT injection of Ad5.PEDF or Ad35.PEDF (HI-RGD) followed by laser-induced CNV on Day 28. Fourteen days later, animals were perfused with fluorescein-labeled dextran and CNV lesion size quantitated in choroidal flat mounts. RESULTS: These studies demonstrate that following a single IVT adenovector administration: 1) gene expression is prolonged following administration of an Ad35 compared to an Ad5-based vector; 2) the amount of vector genomes in the eye remain constant out to 60 days post injection of both Ad5 and Ad35-based vectors; and 3) an Ad35.PEDF (HI-RGD) vector inhibits CNV in a mouse model at 42 days post injection. CONCLUSIONS: These studies show that transgene and genome levels are prolonged in the eye following 1 IVT injection of an Ad35-based vector. Moreover, therapeutic gene levels from 1 IVT administration of Ad35.PEDF (HI-RGD) vector block abnormal blood vessel growth in a laser-induced CNV mouse model.

Trans-scleral delivery of antiangiogenic proteins.

PURPOSE: In this study, we investigated the penetration of various proteins into the mouse eye after a periocular injection of the protein or an adenoviral vector (Ad) expressing the protein. METHODS: At several time points after the injection, the retina, retinal pigmented epithelium/choroid, and sclera were dissected and enzyme-linked immunosorbent assays were performed. RESULTS: After a periocular injection of AdsFlt-1.10, AdTGFbeta.10, or AdPEDF.11, choroidal levels of pigment epithelium-derived factor (PEDF) and transforming growth factor-beta (TGF-beta) were not significantly different from scleral levels, and choroidal levels of sFlt-1 (soluble Flt-1 or soluble VEGF receptor 1) were only moderately reduced from scleral levels, indicating that each of these proteins penetrate the sclera well. In contrast, retinal levels of each of the three proteins were low compared to choroidal levels, suggesting poor penetration into the retina. Levels of PEDF in the choroid peaked 2 h after a periocular injection of PEDF protein and returned to baseline between 6 and 24 h, and peak levels in the retina were 8.6% of peak choroidal levels. Levels of green fluorescent protein, a protein unlikely to have any binding sites in mouse tissues, peaked in the choroid 2 h after the periocular injection and were undetectable by 4 h, while peak levels in the retina were 64.3% of peak choroidal levels. CONCLUSIONS: These data suggest that size and binding characteristics of proteins are likely to influence their ability to penetrate the eye from the periocular space, but in general, proteins as large as 50-75 kDa penetrate well into the choroid, but not into the retina. Periocular injections are feasible for the treatment of choroidal neovascularization with proteins or vectors that express them, but additional investigations are needed before they can be considered for treatment of retinal diseases.

Adenoviral vector-delivered pigment epithelium-derived factor for neovascular age-related macular degeneration: results of a phase I clinical trial.

Twenty-eight patients with advanced neovascular age-related macular degeneration (AMD) were given a single intravitreous injection of an E1-, partial E3-, E4-deleted adenoviral vector expressing human pigment epithelium- derived factor (AdPEDF.11). Doses ranging from 10(6) to 10(9.5) particle units (PU) were investigated. There were no serious adverse events related to AdPEDF.11 and no dose-limiting toxicities. Signs of mild, transient intraocular inflammation occurred in 25% of patients, but there was no severe inflammation. Six patients experienced increased intraocular pressure that was easily controlled by topical medication. All adenoviral cultures were negative. At 3 and 6 months after injection, 55 and 50%, respectively, of patients treated with 10(6)-10(7.5) PU and 94 and 71% of patients treated with 10(8)-10(9.5) PU had no change or improvement in lesion size from baseline. The median increase in lesion size at 6 and 12 months was 0.5 and 1.0 disk areas in the low-dose group compared with 0 and 0 disk areas in the high-dose group. These data suggest the possibility of antiangiogenic activity that may last for several months after a single intravitreous injection of doses greater than 10(8) PU of AdPEDF.11. This study provides evidence that adenoviral vector-mediated ocular gene transfer is a viable approach for the treatment of ocular disorders and that further studies investigating the efficacy of AdPEDF.11 in patients with neovascular AMD should be performed.

Repeated administration of adenovector in the eye results in efficient gene delivery.

PURPOSE: To determine whether repeat administration of an adenovector (Ad) into the eye results in efficient gene delivery and to test whether transgenes can be expressed from an adenovector expression system in the presence of preexisting, neutralizing anti-Ad antibodies. METHODS: To assess the efficiency of repeated gene delivery of an adenovector expression system, C57Bl/6 mice received one, two, or three injections (intravitreal [IVT] or periocular [PO]) of AdNull.11D (empty cassette) at 2-week intervals, followed by a single AdLuciferase (AdL.11D) IVT or PO injection. Mice were killed approximately 24 hours after AdL.11D injection and the eyes were enucleated and stored until assayed. Serum samples were also analyzed to determine whether repeated IVT or PO injections lead to induction of neutralizing antibodies directed against an adenovector delivery system. To determine whether preexisting neutralizing anti-Ad antibodies would block transgene expression, mice were preimmunized with one, two, or three intramuscular (IM) injection(s) of AdNull.11D (1 x 10(9) particle units [pu]). Fourteen days later, when systemic anti-Ad antibody titers were expected to exist, mice were given a single AdL.11D injection (IVT or PO) and killed, and the eyes and serum collected. RESULTS: These studies show that multiple injections at 2-week intervals with adenovectors (IM, IVT, or PO) did not prevent transgene expression in the eye. Moreover, measurement of neutralizing anti-Ad antibody titers revealed that measurable anti-Ad antibody titers in mice did not ablate transgene expression. CONCLUSIONS: These studies suggest that transgene expression after repeated adenovector administration into the eye is feasible and repeated injections, whether given IVT or PO, do not lead to an immediate increase in neutralizing anti-Ad antibody titers. Moreover, preimmunization of mice by systemic exposure to adenovector, does not block transgene expression in the eye. These studies indicate that repeat administration of adenovectors (IVT and PO) into the eye can be considered in designing future clinical trials and that the pre-existence of neutralizing anti-Ad antibodies probably does not mitigate activity.

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.

Laser photocoagulation and, to a lesser extent, photodynamic therapy target and enhance adenovirus vector-mediated gene transfer in the rat retina.

PURPOSE: To evaluate the transduction efficiency and localization of a reporter gene after intravitreous injection of adenovirus vector in laser photocoagulation (PC)- and photodynamic therapy (PDT)-treated eyes. METHODS: Adult Lewis rats received fundus PC, fundus PDT, or no treatment. Intravitreous injection of an adenovirus vector containing the construct expressing beta-galactosidase (AdlacZ.11D) was performed in each group. All eyes were then enucleated for histochemistry and processed for quantitative image analysis. RESULTS: In eyes with no treatment, there was moderate to intense staining for lacZ in the anterior segment, but little in the retina. In eyes treated with PC and PDT, there was significantly more LacZ staining in the retina. The increased staining corresponded closely with the sites treated with PC and PDT. Gene transduction in PC-treated eyes was enhanced and extended to at least 135 days after virus delivery, but not extended in PDT-treated eyes. Gene transfer and expression were targeted and enhanced at the site of laser burns, at all doses tested (3 x 10(5) to 3 x 10(9) particles per eye). CONCLUSIONS: Compared with untreated eyes, eyes treated with PC and to a lesser extent PDT, manifest increased transduction efficiency, in areas of the retina that are targeted by laser treatment. This finding suggests a new and promising strategy for the treatment of retinochoroidal neovascularization. Adenovirus gene therapy in combination with PC or PDT would have the advantage of increased transduction efficiency; increased duration of transgene expression; targeted delivery; and, potentially, a lower effective dose of virus.

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. It is now recognized that retinoschisis results from cyst formations within the retinal layers that interrupt normal visual neurosignaling and compromise structural integrity. Mutations in the human retinoschisin gene have been correlated with disease severity of the human XLRS phenotype. Introduction of a normal human retinoschisin cDNA into retinoschisin knockout mice restores retinal structure and improves neural function, providing proof-of-concept that gene replacement therapy is a plausible treatment 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.

Retinal neuroprotection against ischemic injury mediated by intraocular gene transfer of pigment epithelium-derived factor.

PURPOSE: To determine whether intraocular gene transfer of pigment epithelium-derived factor (PEDF) protects the retina from ischemia-reperfusion injury. METHODS: Four days before induction of pressure-induced ischemia, Lewis rats received intravitreous injection of 3 x 10(9) particles of an adenovirus vector expressing PEDF (AdPEDF.11) in one eye and 3 x 10(9) particles of an empty adenovirus vector (AdNull.11) in the contralateral eye. Seven days after reperfusion, eyes were enucleated and processed for morphometric analysis. Apoptotic cells stained by TdT-dUTP terminal nick-end labeling (TUNEL) in the retina were counted 12 hours after initiation of reperfusion. Retina levels of PEDF were measured by enzyme-linked immunosorbent assay. RESULTS: PEDF levels in retinal homogenates from eyes receiving AdPEDF.11 injection were well above the background levels in the untreated baseline and control eyes (P = 0.04). Retinal thickness was preserved in AdPEDF.11-treated eyes. Retinal cell density was significantly greater in the ganglion cell layer (GCL; P = 0.014), inner nuclear layer (INL; P = 0.008), and outer nuclear layer (ONL; P = 0.008) of AdPEDF.11-treated eyes compared with the corresponding layers in AdNull.11-treated eyes. AdNull.11-treated eyes also had significantly more TUNEL-positive cells in these layers than AdPEDF.11-treated eyes (P < 0.05). CONCLUSIONS: Adenoviral vector-mediated intraocular expression of PEDF significantly increases cell survival after ischemia-reperfusion injury of the retina. The protective effect may result from inhibition of ischemia-induced apoptosis. This study provides proof of concept for a gene transfer approach directed at interrupting programmed cell death induced by retinal ischemic insult.

Corneoscleral transplantation in congenital corneal staphyloma and Peters' anomaly.

An infant born with bilateral corneal clouding diagnosed clinically as congenital anterior staphyloma and Peters' anomaly was confirmed histopathologically. This case report demonstrates one clinical spectrum of Peters' anomaly. The clinical course and histopathologic findings are detailed as is a unique surgical approach of corneoscleral graft used to perserve the right globe.

Ocular localization and transduction by adenoviral vectors are serotype-dependent and can be modified by inclusion of RGD fiber modifications.

PURPOSE: To evaluate localization and transgene expression from adenoviral vector of serotypes 5, 35, and 28, ± an RGD motif in the fiber following intravitreal or subretinal administration. METHODS: Ocular transduction by adenoviral vector serotypes ± RGD was studied in the eyes of mice receiving an intravitreous or subretinal injection. Each serotype expressed a CMV-GFP expression cassette and histological sections of eyes were examined. Transgene expression levels were examined using luciferase (Luc) regulated by the CMV promoter. RESULTS: GFP localization studies revealed that serotypes 5 and 28 given intravitreously transduced corneal endothelial, trabecular, and iris cells. Intravitreous delivery of the unmodified Ad35 serotype transduced only trabecular meshwork cells, but, the modification of the RGD motif into the fiber of the Ad35 viral vector base expanded transduction to corneal endothelial and iris cells. Incorporation of the RGD motif into the fiber knob with deletion of RGD from the penton base did not affect the transduction ability of the Ad5 vector base. Subretinal studies showed that RGD in the Ad5 knob shifted transduction from RPE cells to photoreceptor cells. Using a CMV-Luc expression cassette, intravitreous delivery of all the tested vectors, such as Ad5-, Ad35- and Ad28- resulted in an initial rapid induction of luciferase activity that thereafter declined. Subretinal administration of vectors showed a marked difference in transgene activity. Ad35-Luc gene expression peaked at 7 days and remained elevated for 6 months. Ad28-Luc expression was high after 1 day and remained sustained for one month. CONCLUSIONS: Different adenoviral vector serotypes ± modifications transduce different cells within the eye. Transgene expression can be brief or extended and is serotype and delivery route dependent. Thus, adenoviral vectors provide a versatile platform for the delivery of therapeutic agents for ocular diseases.

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.

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.).

An empty E1, E3, E4 adenovirus vector protects photoreceptors from light-induced degeneration.

We have previously identified a neuroprotective effect associated with empty (E1(-), E3(-), E4(-)) adenovirus vector delivery in a model of light-induced, photoreceptor cell death. In this study, we further characterize this protective effect in light-injured retina and investigate its molecular basis. Dark-adapted BALB/c mice, aged 6-8 weeks, were exposed to standardized, intense fluorescent light for 96 or 144 h. Prior to dark adaptation, all mice received intravitreous injection of 1 x 10(9) particles of an empty (E1(-), E3(-), E4(-)) adenovirus vector in one eye and vehicle in the other. Following light challenge of 96 or 144 h, histopathological analysis and quantitative photoreceptor cell counts were conducted. Semiquantitative assessment of messenger ribonucleic acid (mRNA) for the apoptosis related genes: p50, p65, IkBa, caspase-1, caspase-3, Bad, c-Jun, Bax, Bak, Bcl-2, c-Fos, and p53 using quantitative reverse transcriptase polymerase chain reaction was performed on eyes following 12 h of light exposure. Following 96 h of light exposure, the photoreceptor cell density for E1(-), E3(-), E4(-) adenovirus vector and vehicle-injected eyes were 87.5 +/- 9.5 and 79.3 +/- 10.1, respectively, (p = 0.79). After 144 h of light exposure, the photoreceptor cell density was preserved in vector-injected eyes as compared to vehicle treated eyes, 68.9 +/- 10.0 and 49.2 +/- 4.6, respectively (p = 0.016). Relative mRNA levels of c-Fos and c-Jun at 12-h light exposure after injection differed significantly between vector- and vehicle-injected eyes (p = 0.036, 0.016, respectively). The expression of the other apoptosis-related genes evaluated was not significantly affected. This study investigates the molecular basis of photoreceptor neuroprotective pathway induction associated with E1(-), E3(-), E4(-) adenovirus vectors. The results indicate that empty adenovirus vectors protect photoreceptors from light-induced degeneration by the modulation of apoptotic pathways. Gene expression changes suggest that the suppression of c-Fos and c-Jun upregulation contributes significantly to the neuroprotective effect. Understanding the molecular basis of the neuroprotective pathway induction in photoreceptors is critical to the development of novel therapies for retinal degenerations.

Intraocular gene transfer of pigment epithelium-derived factor rescues photoreceptors from light-induced cell death.

In this study, we investigated whether intraocular gene transfer of pigment epithelium-derived factor (PEDF) ameliorates the extent of light-induced photoreceptor cell death. Lewis rats received intravitreous injection of 3 x 10(9) particles of adenoviral vector expressing PEDF (AdPEDF.11) in one eye and 3 x 10(9) particles of empty adenoviral vector (AdNull.11) in the contralateral eye. The rats were then dark-adapted for 3 days after which they were continuously exposed to fluorescent light (2,500 lux) for 0, 6, 24, 96, and 168 h. Both eyes were then enucleated and processed for morphometric analysis. Cell death in the retina was examined using TUNEL staining with a propidium iodide counterstain. The photoreceptor cell counts in each of the three groups were significantly different (P < 0.001). Eyes that received intravitreous injection of AdNull.11 or no injection showed a greater number of pyknotic photoreceptor cells and a reduced photoreceptor cell density as compared to eyes treated with intravitreous AdPEDF.11 injection. AdNull.11 treated eyes showed a lesser but still significant protection of photoreceptor cells when compared to untreated eyes. Fewer TUNEL-positive photoreceptor cells were present in AdPEDF.11 treated eyes than in AdNull.11 treated or untreated eyes (P = 0.004). The amplitudes of the ERG a-wave, b-wave, and oscillatory potentials (OPs) were increased significantly by treatment (P < 0.05). These data suggest that adenovirus vector-mediated intraocular expression of PEDF significantly increases photoreceptor cell survival following excessive light exposure. Neuroprotection may result from inhibition of light-induced apoptotic processes. This study provides proof of concept for a gene transfer approach to modulating retinal cell death resulting from photo-oxidative damage and supports the hypothesis that gene transfer of PEDF is broadly applicable to modulating apoptosis in the retina.