Gene therapy holds great promise for the treatment of eye diseases. Adeno-associated virus (AAV) serotypes, such as AAV2, AAV5, and AAV8, are widely used for retinal gene delivery. AAV2 is predominantly utilized for intravitreal injection (IVT), while AAV5 and AAV8 are often used for subretinal injection (SRI). Due to its minimally invasive nature and the ability to be performed in an outpatient setting, IVT represents a safe and promising approach for ocular gene therapy. IVT delivers AAV vectors into the vitreous space, primarily targeting the ganglion cell layer (GCL) and other inner retinal cells. However, the inner limiting membrane (ILM) poses a significant barrier, restricting the penetration of natural AAV serotypes into deeper retinal layers beyond the GCL.

To overcome these limitations, engineered AAV2 variants such as AAV2-YFs, AAV2-7M8, and AAV-4D-R100 have been developed through directed evolution, enabling improved delivery to the outer retina. However, significant challenges remain when translating these advancements to large-animal retinas, which differ substantially from rodent retinas. Large-animal retinas, such as those in dogs and non-human primates (NHPs), feature specialized high-acuity regions (e.g., the area centralis in dogs and the fovea in primates), a thicker vitreous, and a more robust ILM. Consequently, AAV2 may not be the optimal serotype for retina-targeting capsid engineering in NHPs using the IVT route.

To identify potential AAV capsids for further engineering, we systematically evaluated commonly used AAV serotypes (AAV2, AAV5, AAV6, AAV8, AAV9, and AAVrh.74) and a series of AAV2 variants (AAV2-Y444F-Y500F, AAV2-Y444F-A493V-Y500F, AAV2-GL, AAV2-NN, and AAV2-7M8) in the whole retina of Rhesus Macaque using barcode technology. The results showed that among the commonly used serotypes, AAV2 exhibited the highest DNA levels in the retina, followed by AAV5, AAV9, AAV8, and AAV6. AAV2-YFs slightly increased DNA levels. AAV2 variants selected from large-animal models (AAV2-7M8) demonstrated a ~10-fold increase in DNA levels compared with AAV2. However, AAV2 variants evolved using mouse models (AAV2-NN and AAV2-GL) showed either comparable or reduced DNA levels.

At the RNA level, AAV6 exhibited an ~8-fold increase in expression compared to AAV2. Notably, AAV2-7M8 demonstrated a several-hundred-fold increase in RNA expression relative to AAV2. Moreover, we identified a novel AAV capsid, designated AAV(Eye), which achieved a 3.4-fold increase in DNA levels and a 47-fold increase in RNA expression compared to AAV2. Additionally, AAV(Eye) demonstrated greater specificity in the retina compared to AAV2-7M8. Importantly, AAV(Eye) also exhibited a ~12-fold increase in RNA expression in Cynomolgus Monkeys compared to AAV2. Using single-cell molecular sequencing, we aim to identify the specific target cells for this capsid.

In summary, AAV(Eye) demonstrates strong potential as a template for future AAV capsid engineering efforts, providing an efficient platform for targeted retinal gene delivery especially in NHP models. Researchers are invited to collaborate on the ecodevelopment of AAV(Eye), with the goal of further enhancing its transduction efficiency across various non-human primate (NHP) models. By working together, we aim to optimize this AAV variant for improved gene delivery to ocular tissues, advancing its potential for treating eye-related disorders. This collaborative effort will leverage shared expertise and resources to accelerate the development of next-generation AAV vectors for translational research and therapeutic applications.