Evaluation of the dystrophin carboxy-terminal domain for micro-dystrophin gene therapy in cardiac and skeletal muscles in the DMDmdx rat model.

Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the gene encoding dystrophin. Gene therapy using micro-dystrophin (MD) transgenes and recombinant adeno-associated virus (rAAV) vectors hold great promise. To overcome the limited packaging capacity of rAAV vectors, most MD do not include dystrophin carboxy-terminal (CT) domain. Yet, the CT domain is known to recruit α1- and ß1-syntrophins and α-dystrobrevin, a part of the dystrophin-associated protein complex (DAPC), which is a signaling and structural mediator of muscle cells. In this study, we explored the impact of inclusion of the dystrophin CT domain on ΔR4-23/ΔCT MD (MD1), in DMDmdx rats, which allows for relevant evaluations at muscular and cardiac levels. We showed by LC-MS/MS that MD1 expression is sufficient to restore the interactions at a physiological level of most DAPC partners in skeletal and cardiac muscles, and that inclusion of the CT domain increases the recruitment of some DAPC partners at supra-physiological levels. In parallel, we demonstrated that inclusion of the CT domain does not improve MD1 therapeutic efficacy on DMD muscle and cardiac pathologies. Our work highlights new evidences of the therapeutic potential of MD1 and strengthens the relevance of this candidate for gene therapy of DMD.

Safety and Long-Term Efficacy of AAV4 Gene Therapy in Patients with RPE65 Leber Congenital Amaurosis.

The aim of this study was the evaluation of the safety and efficacy of unilateral subretinal injection of the adeno-associated vector (AAV) serotypes 2 and 4 (AAV2/4) RPE65-RPE65 vector in patients with Leber congenital amaurosis (LCA) associated with RPE65 gene deficiency. We evaluated ocular and general tolerance and visual function up to 1 year after vector administration in the most severely affected eye in nine patients with retinal degeneration associated with mutations in the RPE65 gene. Patients received either low (1.22 × 1010 to 2 × 1010 vector genomes [vg]) or high (between 3.27 × 1010 and 4.8 × 1010 vg) vector doses. An ancillary study, in which six of the original nine patients participated, extended the follow-up period to 2-3.5 years. All patients showed good ophthalmological and general tolerance to the rAAV2/4-RPE65-RPE65 vector. We observed a trend toward improved visual acuity in patients with nystagmus, stabilization and improvement of the visual field, and cortical activation along visual pathways during fMRI analysis. OCT analysis after vector administration revealed no retinal thinning, except in cases of macular detachment. Our findings show that the rAAV2/4.RPE65.RPE65 vector was well tolerated in nine patients with RPE65-associated LCA. Efficacy parameters varied between patients during follow-up.

AAV-ID: A Rapid and Robust Assay for Batch-to-Batch Consistency Evaluation of AAV Preparations.

Adeno-associated virus (AAV) vectors are promising clinical candidates for therapeutic gene transfer, and a number of AAV-based drugs may emerge on the market over the coming years. To insure the consistency in efficacy and safety of any drug vial that reaches the patient, regulatory agencies require extensive characterization of the final product. Identity is a key characteristic of a therapeutic product, as it ensures its proper labeling and batch-to-batch consistency. Currently, there is no facile, fast, and robust characterization assay enabling to probe the identity of AAV products at the protein level. Here, we investigated whether the thermostability of AAV particles could inform us on the composition of vector preparations. AAV-ID, an assay based on differential scanning fluorimetry (DSF), was evaluated in two AAV research laboratories for specificity, sensitivity, and reproducibility, for six different serotypes (AAV1, 2, 5, 6.2, 8, and 9), using 67 randomly selected AAV preparations. In addition to enabling discrimination of AAV serotypes based on their melting temperatures, the obtained fluorescent fingerprints also provided information on sample homogeneity, particle concentration, and buffer composition. Our data support the use of AAV-ID as a reproducible, fast, and low-cost method to ensure batch-to-batch consistency in manufacturing facilities and academic laboratories.

AAV-mediated Gene Therapy Halts Retinal Degeneration in PDE6ß-deficient Dogs.

We previously reported that subretinal injection of AAV2/5 RK.cpde6ß allowed long-term preservation of photoreceptor function and vision in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6ß deficiency. The present study builds on these earlier findings to provide a detailed assessment of the long-term effects of gene therapy on the spatiotemporal pattern of retinal degeneration in rcd1 dogs treated at 20 days of age. We analyzed the density distribution of the retinal layers and of particular photoreceptor cells in 3.5-year-old treated and untreated rcd1 dogs. Whereas no rods were observed outside the bleb or in untreated eyes, gene transfer halted rod degeneration in all vector-exposed regions. Moreover, while gene therapy resulted in the preservation of cones, glial cells and both the inner nuclear and ganglion cell layers, no cells remained in vector-unexposed retinas, except in the visual streak. Finally, the retinal structure of treated 3.5-year-old rcd1 dogs was identical to that of unaffected 4-month-old rcd1 dogs, indicating near complete preservation. Our findings indicate that gene therapy arrests the degenerative process even if intervention is initiated after the onset of photoreceptor degeneration, and point to significant potential of this therapeutic approach in future clinical trials.

Successful gene therapy in the RPGRIP1-deficient dog: a large model of cone-rod dystrophy.

For the development of new therapies, proof-of-concept studies in large animal models that share clinical features with their human counterparts represent a pivotal step. For inherited retinal dystrophies primarily involving photoreceptor cells, the efficacy of gene therapy has been demonstrated in canine models of stationary cone dystrophies and progressive rod-cone dystrophies but not in large models of progressive cone-rod dystrophies, another important cause of blindness. To address the last issue, we evaluated gene therapy in the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1)-deficient dog, a model exhibiting a severe cone-rod dystrophy similar to that seen in humans. Subretinal injection of AAV5 (n = 5) or AAV8 (n = 2) encoding the canine Rpgrip1 improved photoreceptor survival in transduced areas of treated retinas. Cone function was significantly and stably rescued in all treated eyes (18-72% of those recorded in normal eyes) up to 24 months postinjection. Rod function was also preserved (22-29% of baseline function) in four of the five treated dogs up to 24 months postinjection. No detectable rod function remained in untreated contralateral eyes. More importantly, treatment preserved bright- and dim-light vision. Efficacy of gene therapy in this large animal model of cone-rod dystrophy provides great promise for human treatment.

Liver-specific transcriptional modules identified by genome-wide in silico analysis enable efficient gene therapy in mice and non-human primates.

The robustness and safety of liver-directed gene therapy can be substantially improved by enhancing expression of the therapeutic transgene in the liver. To achieve this, we developed a new approach of rational in silico vector design. This approach relies on a genome-wide bio-informatics strategy to identify cis-acting regulatory modules (CRMs) containing evolutionary conserved clusters of transcription factor binding site motifs that determine high tissue-specific gene expression. Incorporation of these CRMs into adeno-associated viral (AAV) and non-viral vectors enhanced gene expression in mice liver 10 to 100-fold, depending on the promoter used. Furthermore, these CRMs resulted in robust and sustained liver-specific expression of coagulation factor IX (FIX), validating their immediate therapeutic and translational relevance. Subsequent translational studies indicated that therapeutic FIX expression levels could be attained reaching 20-35% of normal levels after AAV-based liver-directed gene therapy in cynomolgus macaques. This study underscores the potential of rational vector design using computational approaches to improve their robustness and therefore allows for the use of lower and thus safer vector doses for gene therapy, while maximizing therapeutic efficacy.

Forelimb treatment in a large cohort of dystrophic dogs supports delivery of a recombinant AAV for exon skipping in Duchenne patients.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by mutations in the dystrophin gene, without curative treatment yet available. Our study provides, for the first time, the overall safety profile and therapeutic dose of a recombinant adeno-associated virus vector, serotype 8 (rAAV8) carrying a modified U7snRNA sequence promoting exon skipping to restore a functional in-frame dystrophin transcript, and injected by locoregional transvenous perfusion of the forelimb. Eighteen Golden Retriever Muscular Dystrophy (GRMD) dogs were exposed to increasing doses of GMP-manufactured vector. Treatment was well tolerated in all, and no acute nor delayed adverse effect, including systemic and immune toxicity was detected. There was a dose relationship for the amount of exon skipping with up to 80% of myofibers expressing dystrophin at the highest dose. Similarly, histological, nuclear magnetic resonance pathological indices and strength improvement responded in a dose-dependent manner. The systematic comparison of effects using different independent methods, allowed to define a minimum threshold of dystrophin expressing fibers (>33% for structural measures and >40% for strength) under which there was no clear-cut therapeutic effect. Altogether, these results support the concept of a phase 1/2 trial of locoregional delivery into upper limbs of nonambulatory DMD patients.

Humoral and cellular capsid-specific immune responses to adeno-associated virus type 1 in randomized healthy donors.

A major impediment to the use of adeno-associated virus (AAV)-mediated gene delivery to muscle in clinical applications is the pre-existing immune responses against the vector. Pre-existing humoral response to different AAV serotypes is now well documented. In contrast, cellular responses to AAV capsid have not been analyzed in a systematic manner, despite the risk of T cell reactivation upon gene transfer. AAV1 has been widely used in humans to target muscle. In this study, we analyzed PBMCs and sera of healthy donors for the presence of AAV1 capsid-specific T cell responses and AAV1 neutralizing factors. Approximately 30% of donors presented AAV1 capsid-specific T cells, mainly effector memory CD8(+) cells. IFN-γ-producing cells were also observed among effector memory CD4(+) cells for two of these donors. Moreover, to our knowledge, this study shows for the first time on a large cohort that there was no correlation between AAV1-specific T cell and humoral responses. Indeed, most donors presenting specific Ig and neutralizing factors were negative for cellular response (and vice versa). These new data raise the question of prescreening patients not only for the humoral response, but also for the cellular response. Clearly, a better understanding of the natural immunology of AAV serotypes will allow us to improve AAV gene therapy and make it an efficient treatment for genetic disease.

Mannose-coupled AAV2: A second-generation AAV vector for increased retinal gene therapy efficiency.

Inherited retinal diseases are a leading and untreatable cause of blindness and are therefore candidate diseases for gene therapy. Recombinant vectors derived from adeno-associated virus (rAAV) are currently the most promising vehicles for in vivo therapeutic gene delivery to the retina. However, there is a need for novel AAV-based vectors with greater efficacy for ophthalmic applications, as underscored by recent reports of dose-related inflammatory responses in clinical trials of rAAV-based ocular gene therapies. Improved therapeutic efficacy of vectors would allow for decreases in the dose delivered, with consequent reductions in inflammatory reactions. Here, we describe the development of new rAAV vectors using bioconjugation chemistry to modify the rAAV capsid, thereby improving the therapeutic index. Covalent coupling of a mannose ligand, via the formation of a thiourea bond, to the amino groups of the rAAV capsid significantly increases vector transduction efficiency of both rat and nonhuman primate retinas. These optimized rAAV vectors have important implications for the treatment of a wide range of retinal diseases.

Human galectin 3 binding protein interacts with recombinant adeno-associated virus type 6.

Recombinant adeno-associated viruses (rAAVs) hold enormous potential for human gene therapy. Despite the well-established safety and efficacy of rAAVs for in vivo gene transfer, there is still little information concerning the fate of vectors in blood following systemic delivery. We screened for serum proteins interacting with different AAV serotypes in humans, macaques, dogs, and mice. We report that serotypes rAAV-1, -5, and -6 but not serotypes rAAV-2, -7, -8, -9, and -10 interact in human sera with galectin 3 binding protein (hu-G3BP), a soluble scavenger receptor. Among the three serotypes, rAAV-6 has the most important capacities for binding to G3BP. rAAV-6 also bound G3BP in dog sera but not in macaque and mouse sera. In mice, rAAV-6 interacted with another protein of the innate immune system, C-reactive protein (CRP). Furthermore, interaction of hu-G3BP with rAAV-6 led to the formation of aggregates and hampered transduction when the two were codelivered into the mouse. Based on these data, we propose that species-specific interactions of AAVs with blood proteins may differentially impact vector distribution and efficacy in different animal models.

PCR-based detection of gene transfer vectors: application to gene doping surveillance.

Athletes who illicitly use drugs to enhance their athletic performance are at risk of being banned from sports competitions. Consequently, some athletes may seek new doping methods that they expect to be capable of circumventing detection. With advances in gene transfer vector design and therapeutic gene transfer, and demonstrations of safety and therapeutic benefit in humans, there is an increased probability of the pursuit of gene doping by athletes. In anticipation of the potential for gene doping, assays have been established to directly detect complementary DNA of genes that are top candidates for use in doping, as well as vector control elements. The development of molecular assays that are capable of exposing gene doping in sports can serve as a deterrent and may also identify athletes who have illicitly used gene transfer for performance enhancement. PCR-based methods to detect foreign DNA with high reliability, sensitivity, and specificity include TaqMan real-time PCR, nested PCR, and internal threshold control PCR.

Integration frequency and intermolecular recombination of rAAV vectors in non-human primate skeletal muscle and liver.

The comprehensive characterization of recombinant adeno-associated viral (rAAV) integration frequency and persistence for assessing rAAV vector biosafety in gene therapy is severely limited due to the predominance of episomal rAAV vector genomes maintained in vivo. Introducing rAAV insertional standards (rAIS), we show that linear amplification-mediated (LAM)-PCR and deep sequencing can be used for validated measurement of rAAV integration frequencies. Integration of rAAV2/1 or rAAV2/8, following intramuscular (IM) or regional intravenous (RI) administration of therapeutically relevant vector doses in nine adult non-human primates (NHP), occurs at low frequency between 10(-4) and 10(-5) both in NHP liver and muscle, but with no preference for specific genomic loci. High resolution mapping of inverted terminal repeat (ITR) breakpoints in concatemeric and integrated vector genomes reveals distinct vector recombination hotspots, including large deletions of up to 3 kb. Moreover, retrieval of integrated rAAV genomes indicated approximately threefold increase in liver compared to muscle. This molecular analysis of rAAV persistence in NHP provides a promising basis for a reliable genotoxic risk assessment of rAAV in clinical trials.

Restoration of vision in the pde6ß-deficient dog, a large animal model of rod-cone dystrophy.

Defects in the ß subunit of rod cGMP phosphodiesterase 6 (PDE6ß) are associated with autosomal recessive retinitis pigmentosa (RP), a childhood blinding disease with early retinal degeneration and vision loss. To date, there is no treatment for this pathology. The aim of this preclinical study was to test recombinant adeno-associated virus (AAV)-mediated gene addition therapy in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6ß deficiency that strongly resembles the human pathology. A total of eight rcd1 dogs were injected subretinally with AAV2/5RK.cpde6ß (n = 4) or AAV2/8RK.cpde6ß (n = 4). In vivo and post-mortem morphological analysis showed a significant preservation of the retinal structure in transduced areas of both AAV2/5RK.cpde6ß- and AAV2/8RK.cpde6ß-treated retinas. Moreover, substantial rod-derived electroretinography (ERG) signals were recorded as soon as 1 month postinjection (35% of normal eyes) and remained stable for at least 18 months (the duration of the study) in treated eyes. Rod-responses were undetectable in untreated contralateral eyes. Most importantly, dim-light vision was restored in all treated rcd1 dogs. These results demonstrate for the first time that gene therapy effectively restores long-term retinal function and vision in a large animal model of autosomal recessive rod-cone dystrophy, and provide great promise for human treatment.

Evaluation of an AAV9-mini-dystrophin gene therapy candidate in a rat model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by loss-of-function mutations in the dystrophin gene and is characterized by muscle wasting and early mortality. Adeno-associated virus-mediated gene therapy is being investigated as a treatment for DMD. In the nonclinical study documented here, we determined the effective dose of fordadistrogene movaparvovec, a clinical candidate adeno-associated virus serotype 9 vector carrying a human mini-dystrophin transgene, after single intravenous injection in a dystrophin-deficient (DMDmdx) rat model of DMD. Overall, we found that transduction efficiency, number of muscle fibers expressing the human mini-dystrophin polypeptide, improvement of the skeletal and cardiac muscle tissue architecture, correction of muscle strength and fatigability, and improvement of diastolic and systolic cardiac function were directly correlated with the amount of vector administered. The effective dose was then tested in older DMDmdx rats with a more dystrophic phenotype similar to the pathology observed in older patients with DMD. Except for a less complete rescue of muscle function in the oldest cohort, fordadistrogene movaparvovec was also found to be therapeutically effective in older DMDmdx rats, suggesting that this product may be appropriate for evaluation in patients with DMD at all stages of disease.

Efficient intracerebral delivery of AAV5 vector encoding human ARSA in non-human primate.

Metachromatic leukodystrophy (MLD) is a lethal neurodegenerative disease caused by a deficiency in the lysosomal arylsulfatase A (ARSA) enzyme leading to the accumulation of sulfatides in glial and neuronal cells. We previously demonstrated in ARSA-deficient mice that intracerebral injection of a serotype 5 adeno-associated vector (AAV) encoding human ARSA corrects the biochemical, neuropathological and behavioral abnormalities. However, before considering a potential clinical application, scaling-up issues should be addressed in large animals. Therefore, we performed intracerebral injection of the same AAV vector (total dose of 3.8 x 10(11) or 1.9 x 10(12) vector genome, three sites of injection in the right hemisphere, two deposits per site of injection) into three selected areas of the centrum semiovale white matter, or in the deep gray matter nuclei (caudate nucleus, putamen, thalamus) of six non-human primates to evaluate vector distribution, as well as expression and activity of human ARSA. The procedure was perfectly tolerated, without any adverse effect or change in neurobehavioral examination. AAV vector was detected in a brain volume of 12-15 cm(3) that corresponded to 37-46% of the injected hemisphere. ARSA enzyme was expressed in multiple interconnected brain areas over a distance of 22-33 mm. ARSA activity was increased by 12-38% in a brain volume that corresponded to 50-65% of injected hemisphere. These data provide substantial evidence for potential benefits of brain gene therapy in patients with MLD.

A 10 patient case report on the impact of plasmapheresis upon neutralizing factors against adeno-associated virus (AAV) types 1, 2, 6, and 8.

Adeno-associated viruses (AAV) are small, nonenveloped single-stranded DNA viruses which require helper viruses to facilitate efficient replication. These recombinant viruses are some of the most promising candidates for therapeutic gene transfer to treat many genetic and acquired diseases. Nevertheless, the presence of humoral responses to the wild-type AAV common among humans is one of the limitations of in vivo transduction efficacy in humans using cognate recombinant vector. In this study, based on the serum samples that we were able to collect from various clinical situations, we studied the impact of one to five plasmapheresis (PP), at 1-5 day intervals on neutralizing factor (NAF) titers specific for AAV types 1, 2, 6, and 8 in seropositive patients with diverse pathologies and immunosuppressor treatments. We show that frequent sessions of PP result in drastic reduction of NAF specific for AAV1, 2, 6, and 8 to undetectable levels or titers <1:5, mainly when initial titers, i.e., before the first PP were ≤1:20. Altogether, these results show that the use of PP and its possible association with pharmacological immunosuppressive treatments may help to design optimal management of seropositive patients for AAV gene therapy treatments.

Safe, efficient, and reproducible gene therapy of the brain in the dog models of Sanfilippo and Hurler syndromes.

Recent trials in patients with neurodegenerative diseases documented the safety of gene therapy based on adeno-associated virus (AAV) vectors deposited into the brain. Inborn errors of the metabolism are the most frequent causes of neurodegeneration in pre-adulthood. In Sanfilippo syndrome, a lysosomal storage disease in which heparan sulfate oligosaccharides accumulate, the onset of clinical manifestation is before 5 years. Studies in the mouse model showed that gene therapy providing the missing enzyme α-N-acetyl-glucosaminidase to brain cells prevents neurodegeneration and improves behavior. We now document safety and efficacy in affected dogs. Animals received eight deposits of a serotype 5 AAV vector, including vector prepared in insect Sf9 cells. As shown previously in dogs with the closely related Hurler syndrome, immunosuppression was necessary to prevent neuroinflammation and elimination of transduced cells. In immunosuppressed dogs, vector was efficiently delivered throughout the brain, induced α-N-acetyl-glucosaminidase production, cleared stored compounds and storage lesions. The suitability of the procedure for clinical application was further assessed in Hurler dogs, providing information on reproducibility, tolerance, appropriate vector type and dosage, and optimal age for treatment in a total number of 25 treated dogs. Results strongly support projects of human trials aimed at assessing this treatment in Sanfilippo syndrome.

Cellular and Structural Characterization of VP1 and VP2 Knockout Mutants of AAV3B Serotype and Implications for AAV Manufacturing.

AAV virion biology is still lacking a complete understanding of the role that the various structural subunits (VP1, 2, and 3) play in virus assembly, infectivity, and therapeutic delivery for clinical indications. In this study, we focus on the less studied adeno-associated virus AAV3B and generate a collection of AAV plasmid substrates that assemble virion particles deficient specifically in VP1, VP2, or VP1 and 2 structural subunits. Using a collection of biological and structural assays, we observed that virions devoid of VP1, VP2, or VP1 and 2 efficiently assembled virion particles, indistinguishable by cryoelectron microscopy (cryo-EM) from that of wild type (WT), but unique in virion transduction (WT > VP2 > VP1 > VP1 and 2 mutants). We also observed that the missing structural subunit was mostly compensated by additional VP3 protomers in the formed virion particle. Using cryo-EM analysis, virions fell into three classes, namely full, empty, and partially filled, based on comparison of density values within the capsid. Further, we characterize virions described as "broken" or "disassembled" particles, and provide structural information that supports the particle dissolution occurring through the two-fold symmetry sites. Finally, we highlight the unique value of employing cryo-EM as an essential tool for release criteria with respect to AAV manufacturing.

Neonatal systemic delivery of scAAV9 in rodents and large animals results in gene transfer to RPE cells in the retina.

Systemic delivery of recombinant adeno-associated virus (rAAV) vectors has recently been shown to cross the blood brain barrier in rodents and large animals and to efficiently target cells of the central nervous system. Such approach could be particularly interesting to treat lysosomal storage diseases or neurodegenerative disorders characterized by multiple organs injuries especially neuronal and retinal dysfunctions. However, the ability of rAAV vector to cross the blood retina barrier and to transduce retinal cells after systemic injection has not been precisely determined. In this study, gene transfer was investigated in the retina of neonatal and adult rats after intravenous injection of self-complementary (sc) rAAV serotype 1, 5, 6, 8, and 9 carrying a CMV-driven green fluorescent protein (GFP), by fluorescence fundus photography and histological examination. Neonatal rats injected with scAAV2/9 vector displayed the strongest GFP expression in the retina, within the retinal pigment epithelium (RPE) cells. Retinal tropism of scAAV2/9 vector was further assessed after systemic delivery in large animal models, i.e., dogs and cats. Interestingly, efficient gene transfer was observed in the RPE cells of these two large animal models following neonatal intravenous injection of the vector. The ability of scAAV2/9 to transduce simultaneously neurons in the central nervous system, and RPE cells in the retina, after neonatal systemic delivery, makes this approach potentially interesting for the treatment of infantile neurodegenerative diseases characterized by both neuronal and retinal damages.

Regulation of retinal function but nonrescue of vision in RPE65-deficient dogs treated with doxycycline-regulatable AAV vectors.

In previous studies, we demonstrated that recombinant adeno-associated virus (rAAV)-mediated gene transfer of the doxycycline (Dox)-regulatable system allows for the regulation of erythropoietin (EPO) expression in the retina of nonhuman primates after intravenous or oral administration of Dox. In addition, it was shown that administrating different amounts of Dox resulted in a dose-response dynamic of transgene expression. Adeno-associated viral gene therapy has raised hope for the treatment of patients with Leber congenital amaurosis, caused by mutations in the retinal pigment epithelium (RPE)-specific gene RPE65. The preliminary results of three clinical trials suggest some improvement in visual function. However, further improvements might be necessary to optimize vision recovery and this means developing vectors able to generate transgene expression at physiological levels. The purpose of this study was to investigate the ability of the Dox-regulatable system to regulate retinal function in RPE65(-/-) Briard dogs. rAAV vectors expressing RPE65 under the control of either the TetOff and TetOn Dox-regulated promoters or the cytomegalovirus (CMV) constitutive promoter were generated and administered subretinally to seven RPE65-deficient dogs. We demonstrate that the induction and deinduction of retinal function, as assessed by electroretinography (ERG), can be achieved using a Dox-regulatable system, but do not lead to any recovery of vision.