An Optimized Treatment Protocol for Subretinal Injections Limits Intravitreal Vector Distribution

Area of Science:

• Ophthalmology
• Gene Therapy
• Molecular Biology

Background:

• Subretinal injections (SRis) are crucial for delivering adeno-associated virus (AAV) in retinal gene therapy.
• Off-target AAV delivery into the vitreous can increase biodistribution and inflammation, potentially compromising safety and efficacy.
• The intravitreal space is less immune-privileged than the subretinal space, making intravitreal AAV a concern.

Purpose of the Study:

• To optimize the surgical protocol for subretinal injections (SRis) to minimize off-target adeno-associated virus (AAV) delivery into the vitreous.
• To reduce the risk of extensive biodistribution and inflammation associated with intravitreal AAV.
• To enhance the overall safety of retinal gene therapy procedures.

Main Methods:

• An experimental animal study was conducted using eight cynomolgus monkeys (Macaca fascicularis).
• Subretinal injections of an AAV2/8 vector were performed at two different viral genome (vg) doses (1x10^11 and 5x10^11 vg).
• Intravitreal fluid samples were collected post-injection and after a 3-minute balanced salt solution (BSS) lavage to quantify vector genome copies using quantitative polymerase chain reaction.

Main Results:

• Even uneventful subretinal injections (SRis) resulted in the dissemination of millions of adeno-associated virus (AAV) particles into the vitreous cavity (0.1-0.7% of the total dose).
• A 3-minute lavage of the intravitreal space with balanced salt solution (BSS) significantly decreased the intravitreal vector load by an average of 96%.
• Quantitative polymerase chain reaction confirmed a substantial reduction in intravitreal vector genome copies after the lavage procedure.

Conclusions:

• Minimizing off-target adeno-associated virus (AAV) application after subretinal injection (SRi) reflux is critical for improving retinal gene therapy safety.
• A simple 3-minute lavage of the intravitreal space effectively reduces the intravitreal vector load.
• This lavage step should be considered an integral part of surgical protocols for subretinal gene therapy to mitigate risks associated with intravitreal vector dissemination.