Abstract
Purpose
Subretinal injections (SRis) are commonly used in retinal gene therapy procedures to deliver adeno-associated virus (AAV) to photoreceptors and retinal pigment epithelial cells. We present an optimized surgical protocol to minimize off-target application of AAV in the vitreous, which in turn reduces the risk of extensive biodistribution and inflammation, ultimately leading to enhanced safety of the therapy.Design
Experimental animal research study.Participants
Eight cynomolgus monkeys (Macaca fascicularis).Methods
Subretinal injections with an AAV2/8 vector were performed. The animals were allocated to 2 different vector dose groups (1×10ˆ 11 and 5×10ˆ 11 viral genomes [vg]). Samples of intravitreal fluid were taken at the end of the SRi procedure and again after a 3-minute lavage (wash-out) with balanced salt solution (BSS).Main outcome measures
Intravitreal vector genome copies were analyzed with quantitative polymerase chain reaction and compared between groups.Results
Even uneventful SRi leads to dissemination of millions of AAV particles (0.1-0.7% of viral vector loading dose) into the vitreous cavity. Three minutes of lavage led to a substantial decrease (on average 96%) of intravitreal vector load.Conclusions
Multiple studies have shown that the intravitreal space is not as immune privileged as the subretinal space. Intravitreal AAV particles disseminate into the bloodstream, lead to increased biodistribution into lymphatic tissue, and help to stage an immune response with implications for both safety and efficacy. Therefore, minimizing off-target vector application after reflux of vector from the subretinal space is of significant interest. We show that a simple lavage of intravitreal fluid efficiently decreases the intravitreal vector load. Such a step should be considered when performing subretinal gene therapy.