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The accumulation of N-retinylidene-N-retinylethanolamine (A2E, a toxic by-product of the visual pigment cycle) in the retinal pigment epithelium (RPE) is a major cause of visual impairment in the elderly. Photooxidation of A2E results in retinal pigment epithelium degeneration followed by that of associated photoreceptors. Present treatments rely on nutrient supplementation with antioxidants. 9’-cis-Norbixin (a natural diapocarotenoid, 97% purity) was prepared from Bixa orellana seeds. It was first evaluated in primary cultures of porcine retinal pigment epithelium cells challenged with A2E and illuminated with blue light, and it provided an improved photo-protection as compared with lutein or zeaxanthin. In Abca4-/- Rdh8-/- mice (a model of dry AMD), intravitreally-injected norbixin maintained the electroretinogram and protected photoreceptors against light damage. In a standard rat blue-light model of photodamage, norbixin was at least equally as active as phenyl-N-tert-butylnitrone, a free radical spin-trap. Chronic experiments performed with Abca4-/- Rdh8-/- mice treated orally for 3 months with norbixin showed a reduced A2E accumulation in the retina. Norbixin appears promising for developing an oral treatment of macular degeneration. A drug candidate (BIO201) with 9’-cis-norbixin as the active principle ingredient is under development, and its potential will be assessed in a forthcoming clinical trial. [ABSTRACT FROM AUTHOR]

The article discusses the challenge of testing retinal degeneration therapies due to anatomical differences between human and animal retinas. It presents three inducible cynomolgus macaque models of retinal degeneration such as optogenetics-based ablation, CRISPR-Cas9-induced rod-cone dystrophy, and an acute model using a polymer patch for photoreceptor separation.

Atrophic A\age-related macular degeneration (AMD) and Stargardt disease (STGD) are major blinding diseases affecting millions of patients worldwide, but no treatment is available. In dry AMD and STGD oxidative stress and subretinal accumulation of N -retinylidene- N -retinylethanolamine (A2E), a toxic by-product of the visual cycle, causes retinal pigment epithelium (RPE) and photoreceptor degeneration leading to visual impairment. Acute and chronic retinal degeneration following blue light damage (BLD) in BALB/c mice and aging of Abca4 -/- Rdh8 -/- mice, respectively, reproduce features of AMD and STGD. Efficacy of systemic administrations of 9'- cis -norbixin (norbixin), a natural di-apocarotenoid, prepared from Bixa orellana seeds with anti-oxidative properties, was evaluated during BLD in BALB/c mice, and in Abca4 -/- Rdh8 -/- mice of different ages, following three experimental designs: "preventive", "early curative" and "late curative" supplementations. Norbixin injected intraperitoneally in BALB/c mice, maintained scotopic and photopic electroretinogram amplitude and was neuroprotective. Norbixin chronic oral administration for 6 months in Abca4 -/- Rdh8 -/- mice following the "early curative" supplementation showed optimal neuroprotection and maintenance of photoreceptor function and reduced ocular A2E accumulation. Thus, norbixin appears promising as a systemic drug candidate for both AMD and STGD treatment.

Adeno-associated virus (AAV) has emerged as the vector of choice for delivering genes to the mammalian retina. From the first gene therapy to receive FDA approval for the inherited retinal disease (Luxturna™) to more recent clinical trials using microbial opsins to regain light sensitivity, therapeutic transgenes rely on AAV vectors for safe and efficient gene delivery to retinal cells. Such vectors are administered to the retina via subretinal (SR) injection or intravitreal (IVT) injection routes depending on the targeted retinal cell type. An attractive target for gene therapy is the fovea, bearing the highest concentration of cone cells responsible for our high acuity daylight vision. However, previous clinical trials and large animal studies reported that SR administration of vector under the cone-exclusive fovea disrupts its fine structure and might impair visual acuity. Due to its technical difficulty and potential risks, alternatives to vector injection under this delicate region have been investigated by using novel AAV capsid variants identified via rational design or directed evolution. We recently established new vector-promoter combinations to overcome the limitations associated with AAV-mediated cone transduction in the fovea. Our methods provide efficient foveal cone transduction without detaching this delicate region and rely on the use of engineered AAVs and optimal promoters compatible with optogenetic vision restoration. Here we describe in detail our AAV vectors, methods for intravitreal and subretinal injections as well as pre- and postoperative procedures as performed in cynomolgus macaques.

Intraocular injection of adeno-associated viral (AAV) vectors has been an evident route for delivering gene drugs into the retina. However, gaps in our understanding of AAV transduction patterns within the anatomically unique environments of the subretinal and intravitreal space of the primate eye impeded the establishment of noninvasive and efficient gene delivery to foveal cones in the clinic. Here, we establish new vector-promoter combinations to overcome the limitations associated with AAV-mediated cone transduction in the fovea with supporting studies in mouse models, human induced pluripotent stem cell-derived organoids, postmortem human retinal explants, and living macaques. We show that an AAV9 variant provides efficient foveal cone transduction when injected into the subretinal space several millimeters away from the fovea, without detaching this delicate region. An engineered AAV2 variant provides gene delivery to foveal cones with a well-tolerated dose administered intravitreally. Both delivery modalities rely on a cone-specific promoter and result in high-level transgene expression compatible with optogenetic vision restoration. The model systems described here provide insight into the behavior of AAV vectors across species to obtain safety and efficacy needed for gene therapy in neurodegenerative disorders.