UC Berkeley

Adeno-associated virus (AAV) mediated gene therapies for inherited retinal degenerations require precise targeting of the outer retina, particularly in the retinal pigment epithelium (RPE), which plays an essential role in maintenance of the light-sensitive neuron, photoreceptors. Mutations in RPE-expressed genes, including RPE65, lead to blinding diseases such as Leber congenital amaurosis type 2 (LCA2). While subretinal injection achieves optimal RPE transduction, it is an invasive, inpatient surgical procedure associated with iatrogenic damage, including retinal detachment. Intravitreal (IVT) surgery is a far less invasive, outpatient procedure but has previously been shown to have limited penetration of the inner limiting membrane (ILM) of the retina and thus poor outer retina transduction. The goal of this study is to identify capsid variants capable of overcoming this barrier. Here, high-throughput screenings of libraries of over 1.28x109 capsids from different AAV serotypes were conducted, each with different outer surface structures to alter their binding properties, to be delivered with IVT injection in mice. Parallel screens were conducted in non-human primate (NHP) macaque and canine models for cross-species validation and better clinical translatability. A novel AAV serotype 2 based capsid, “Variant 3” (AAV2.Var3) was found to transduce approximately 10% of RPE cells in mouse and NHP eyes. For comparison, a leading, clinical-stage vector that is also AAV2-based, 7m8, effectively transduces the outer retina but has limited success targeting RPE through IVT delivery, where only 0-1% transduction was seen in this study. Previous studies have also determined a lack of RPE transduction with AAV2.7m8 IVT delivery in primates.To test the therapeutic efficacy of Variant 3, a therapeutic gene, human RPE65 (hRPE65), was packaged in AAV2.Var3, driven in expression by ubiquitous (CAG) and RPE-specific (BEST1) promoters. These constructs were injected intravitreally at postnatal day 14 (P14) in rd12 mice, a disease model for Leber congenital amaurosis type 2 (LCA2). AAV2.Var3-hRPE65 treated cohorts showed an increase in RPE65 protein expression confirmed by immunohistochemistry and Western blot. There was also a recovery of retinoid production evidenced by high-performance liquid chromatography (HPLC) assays to quantify 11-cis-retinal and all-trans-retinyl ester. Visual function assessed by both scotopic and photopic electroretinography (ERG) in response to light flashes demonstrated a slight improvement in rod-mediated visual function. With the translational feasibility of the AAV2.Var3 capsid, supported by demonstrated RPE transduction in the primate model following IVT delivery, these findings establish AAV2.Var3 as a promising vector for minimally invasive IVT gene delivery to the RPE, with potential clinical application in LCA2 and other RPE-associated inherited retinal diseases.