Outer Plexiform Layer Structures Are Not Altered Following AAV-Mediated Gene Transfer in Healthy Rat Retina

Area of Science:

• Ophthalmology
• Molecular Biology
• Neuroscience

Background:

• Ocular gene therapy, particularly using adeno-associated virus (AAV) vectors, shows promise for treating retinal diseases.
• Current delivery methods involve subretinal injections, which cause a temporary retinal detachment.
• The impact of this transient detachment on retinal structure and function is not fully understood.

Purpose of the Study:

• To investigate the effects of subretinal injection and AAV-mediated gene transfer on retinal morphology and apoptotic status in healthy rat retinas.
• To assess whether the temporary retinal detachment and viral vector presence alter the outer plexiform layer (OPL) and neuronal circuitry.

Main Methods:

• Subretinal injection of AAV2/5.CMV.d2GFP or sham injection in rat retinas.
• Immunohistochemistry, confocal microscopy, and electron microscopy to analyze OPL morphology and synaptic structures.
• Quantification of synaptic contacts and assessment of apoptotic and inflammatory markers using TUNEL assay.

Main Results:

• No significant differences in the size or shape of pre- and postsynaptic structures in the OPL between injected and non-injected areas.
• Absolute numbers of synaptic ribbons remained unchanged, with no signs of gliosis.
• TUNEL assay revealed no variation in retinal cell apoptosis, indicating no adverse effects from the procedure or transgene expression.

Conclusions:

• Subretinal injection for ocular gene therapy, including AAV vector delivery and transgene expression, does not induce significant structural or apoptotic changes in healthy rat retinas.
• The temporary retinal detachment associated with this delivery method appears safe for retinal circuitry.
• Further studies in disease models, like RPE65 deficiency in dogs, are needed to evaluate transgene expression in diseased retinas.