Inherited retinal degenerations are a leading and untreatbale cause of blindness, and as such they are targets for gene therapy. Numerous gene therapy treatments have progressed from laboratory research to clinical trails, and a pioneering gene therapy received the first ever FDA approval for treating patients. However, currently retinal gene therapy mostly involves subretinal injection of the therapeutic agent, which treats a limited area, entails retinal detachment and other potential complications, and requires general anesthesia with consequent risks, costs and prolonged recovery. Therefore there is great impetus to develop safer, less invasive and cheapter methods of gene delivery. A promising method is intravitreal injection, that does not cause retinal detachment, can lead to pan-retinal transduction and can be performed under local anesthesia in out-patient clinics. Intravitreally-injected vectors face several obstacles. First, the vector is diluted by the vitreous and has to overcome a long diffusion distance to the target cells. Second, the vector is exposed to the host’s immune response, risking neutralization by pre-existing antibodies and triggering a stronger immune response to the injection. Third, the vector has to cross the inner limiting membrane which is both a physical and a biological barrier as it contains binding sites that could cause the vector’s sequestration. Finally, in the target cell the vector is prone to proteasome degradation before delivering the transgene to the nucleus. Strategies to overcome these obstacles include modifications of the viral capsid, through rational design or directed evolution, which allow resistance to the immune system, enhancement of penetration through the inner limiting membrane or reduced degradation by intracellular proteasomes. Furthermore, physical and chemical manipulations of the inner limiting membrane and vitreous aim to improve vector penetration. Finally, compact non-viral vectors that can overcome the immunological, physical and anatomical and barriers have been developed. This paper reviews ongoing efforts to develop novel, safe and efficacious methods for intravitreal delivery of therapeutic genes for inherited retinal degenerations. To date, the most promising results are achieved in rodents with robust, pan-retinal transduction following intravitreal delivery. Trials in larger animal models demonstrate transduction mostly of inner retinal layers. Despite ongoing efforts, currently no intravitreally-injected vector has demonstrated outer retinal transduction efficacy comparable to that of subretinal delivery. Further work is warranted to test promising new viral and non-viral vectors on large animal models of inherited retinal degenerations. Positive results will pave the way to development of the next generation of treatments for inherited retinal degeneration.

{These authors contributed equally to this work.Applied Genetic Technologies Corporation (AGTC) is developing a recombinant adeno-associated virus (rAAV) vector expressing the human CNGA3 gene designated AGTC-402 (rAAV2tYF-PR1.7-hCNGA3) for the treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. The results are herein reported of a study evaluating safety and efficacy of AGTC-402 in CNGA3-deficient sheep. Thirteen day-blind sheep divided into three groups of four or five animals each received a subretinal injection of an AAV vector expressing a CNGA3 gene in a volume of 500 lL in the right eye. Two groups (n = 9) received either a lower or higher dose of the AGTC-402 vector, and one efficacy control group (n = 4) received a vector similar in design to one previously shown to rescue cone photoreceptor responses in the day-blind sheep model (rAAV5-PR2.1-hCNGA3). The left eye of each animal received a subretinal injection of 500 lL of vehicle (n = 4) or was untreated (n = 9). Subretinal injections were generally well tolerated and not associated with systemic toxicity. Most animals had mild to moderate conjunctival hyperemia, chemosis, and subconjunctival hemorrhage immediately after surgery that generally resolved by postoperative day 7. Two animals treated with the higher dose of AGTC-402 and three of the efficacy control group animals had microscopic findings of outer retinal atrophy with or without inflammatory cells in the retina and choroid that were procedural and/or test-article related. All vector-treated eyes showed improved cone-mediated electroretinography responses with no change in rod-mediated electroretinography responses. Behavioral maze testing under photopic conditions showed significantly improved navigation times and reduced numbers of obstacle collisions in all vector-treated eyes compared to their contralateral control eyes or predose results in the treated eyes. These results support the use of AGTC-402 in clinical studies in patients with achromatopsia caused by CNGA3 mutations, with careful evaluation for possible inflammatory and/ or toxic effects.

Achromatopsia causes severely reduced visual acuity, photoaversion, and inability to discern colors due to cone photoreceptor dysfunction. In 2010, we reported on day-blindness in sheep caused by a stop-codon mutation of the ovine CNGA3 gene and began gene augmentation therapy trials in this naturally occurring large animal model of CNGA3 achromatopsia. The purpose of this study was to evaluate long-term efficacy and safety results of treatment, findings that hold great relevance for clinical trials that started recently in CNGA3 achromatopsia patients. Nine day-blind sheep were available for long-term follow up. The right eye of each sheep was treated with a single subretinal injection of an Adeno-Associated Virus Type 5 (AAV5) vector carrying either a mouse (n = 4) or a human (n = 5) CNGA3 transgene under control of the 2.1-Kb red/green opsin promoter. The efficacy of treatment was assessed periodically with photopic maze tests and electroretinographic (ERG) recordings for as long as 74 months postoperatively. Safety was assessed by repeated ophthalmic examinations and scotopic ERG recordings. The retinas of three animals that died of unrelated causes >5 years post-treatment were studied histologically and immunohistochemically using anti-hCNGA3 and anti-red/green cone opsin antibodies. Passage time and number of collisions of treated sheep in the photopic maze test were significantly lower at all follow-up examinations as compared with pretreatment values (p = 0.0025 and p < 0.001, respectively). ERG Critical Flicker Fusion Frequency and flicker amplitudes at 30 and 40 Hz showed significant improvement following treatment (p < 0.0001) throughout the study. Ophthalmic examinations and rod ERG recordings showed no abnormalities in the treated eyes. Immunohistochemistry revealed the presence of CNGA3 protein in red/green opsin-positive cells (cones) of the treated eyes. Our results show significant, long-term improvement in cone function, demonstrating a robust rescue effect up to six years following a single treatment with a viral vector that provides episomal delivery of the transgene. This unique follow-up duration confirms the safe and stable nature of AAV5 gene therapy in the ovine achromatopsia model.

Achromatopsia is an inherited retinal disease characterized by loss of cone photoreceptor function. Day blind CNGA3 mutant Improved Awassi sheep provide a large animal model for achromatopsia. This study measured refractive error and axial length parameters of the eye in this model and evaluated chromatic pupillary light reflex (cPLR) testing as a potential screening test for loss of cone function. Twenty-one CNGA3 mutant, Improved Awassi, 12 control Afec-Assaf and 12 control breed-matched wild-type (WT) Awassi sheep were examined using streak retinoscopy and B-mode ocular ultrasonography. Four CNGA3 mutant and four Afec-Assaf control sheep underwent cPLR testing. Statistical tests showed that day-blind sheep are significantly more myopic than both Afec-Assaf and WT Awassi controls. Day-blind sheep had significantly longer vitreous axial length compared to WT Awassi (1.43 ± 0.13 and 1.23 ± 0.06 cm, respectively, p < 0.0002) and no response to bright red light compared to both controls. Lack of response to bright red light is consistent with cone dysfunction, demonstrating that cPLR can be used to diagnose day blindness in sheep. Day-blind sheep were found to exhibit myopia and increased vitreous chamber depth, providing a naturally occurring large animal model of myopia.

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