Preclinical Safety Evaluation of AAV2-sFLT01— A Gene Therapy for Age-related Macular Degeneration

Abstract

AAV2-sFLT01 is a vector that expresses a modified soluble Flt1 receptor designed to neutralize the proangiogenic activities of vascular endothelial growth factor (VEGF) for treatment of age-related macular degeneration (AMD) via an intravitreal injection. Owing to minimal data available for the intravitreal route of administration for adeno-associated virus (AAV), we initiated a 12-month safety study of AAV2-sFLT01 administered intravitreally at doses of 2.4 × 10(9) vector genomes (vg) and 2.4 × 10(10) vg to cynomolgus monkeys. Expression of sFlt01 protein peaked at ~1-month postadministration and remained relatively constant for the remainder of the study. Electroretinograms, fluorescein angiograms, and tonometry were assessed every 3 months, with no test article-related findings observed in any group. Indirect ophthalmoscopy and slit lamp exams performed monthly revealed a mild to moderate but self-resolving vitreal inflammation in the high-dose group only, which follow-up studies suggest was directed against the AAV2 capsid. Histological evaluation revealed no structural changes in any part of the eye and occasional inflammatory cells in the trabecular meshwork, vitreous and retina in the high-dose group. Biodistribution analysis in rats and monkeys found only trace amounts of vector outside the injected eye. In summary, these studies found AAV2-sFLT01 to be well-tolerated, localized, and capable of long-term expression.

Preclinical safety evaluation of subretinal AAV2.sFlt-1 in non-human primates

“…A recent study in non-human primates using a similar vector, AAV2.sFlt01, injected intravitreally, showed the vector to be safe overall, but a higher level of inflammation and immune response 50 than we report here was found. Whereas AAV2.sFlt-1 described here encodes the naturally occurring form of the soluble receptor driven by the CMV promoter, AAV2.sFLT01 encodes only one Flt-1 domain 2 linked by 9Gly to a human IgG1 heavy-chain Fc domain to generate a forced homodimer and is expressed by the chicken b-actin promoter.…”

“…Thus, the potential production of low amounts of sFlt-1 protein in the optic nerve is unlikely to represent any risk. Thus, our study on non-human primates demonstrated that, in contrast to intravitreally injected AAV2 constructs, 50 AAV2 constructs injected into the subretinal space remained localized in the eye. Density plots of B-cell populations (CD21 þ ) Ki67 expression are shown for two animals in the study, one AAV2.sFlt-1 treated and the other AAV2.GFP treated.…”

A New Promoter Allows Optogenetic Vision Restoration with Enhanced Sensitivity in Macaque Retina

“…25,43 For example, a recent study suggested that the engineered AAV variant AAV2-7m8 might be more immunogenic when used at high doses via intravitreal injections. 25 Therefore, we chose AAV2 for its known safety and efficacy in targeting RGCs in NHP studies [27][28][29] and the large body of work using this vector in human clinical trials. [20][21][22][23]44 High-level expression with a limited viral dose being a major parameter in obtaining functional expression, we designed a new RGC-specific promoter, driving strong transgene expression in RGCs.…”

“…This extended time point was chosen as previous studies established that after intravitreal delivery of AAV2, the transgene expression levels are stable after 6 months. 29 Based on this, we first wanted to examine whether the low dose (1 Â 10 11 vg) would lead to better functional results at 6 months. NHP5 was euthanized at 6 months post-injection and MEA was used to measure light responses as described previously.…”

“…AAV2 was chosen for gene delivery due to its efficacy in targeting RGCs and to the large body of knowledge using this serotype in non-human primate (NHP) studies 24,28,29 and in human clinical trials. [20][21][22][23]30 To obtain functional responses at lower light intensities, we optimized several parameters.…”