A modified AAV2/6 enhances retinal microglial transduction in a layer-specific manner

Abstract

Purpose : Microglia are a critical component to retinal homeostasis and disease progression, yet we still lack an effective strategy to manipulate them in vivo. Adeno-associated viruses (AAVs) are a widely used tool for transducing cells in the retina, and can also be translated to the clinic for targeted gene therapy approaches. So far, targeting microglia with AAV in the retina has not been explicitly addressed, since microglia transduction in other CNS regions has proven to be challenging.

Methods : We performed intravitreal and subretinal injections in adult C57Bl6/J mice using a self-complementary capsid-modified AAV2/6^TYF harboring the GFP transgene under the microglia-specific CD68 promoter (scAAV2/6^TYF-CD68-GFP). Then, we modified the viral capsid to reduce interaction with heparin-binding sites, and finally, we inserted a double-floxed inverted open reading frame (DIO) into the AAV2 transfer vector (scAAV2/6^d4-CD68-DIO-GFP) to increase specificity. Two weeks post-injection, we performed immunostaining for GFP and the ionized calcium binding adaptor molecule 1 (Iba1), which labels the entire microglial population. We quantified the ratio of GFP/Iba1 double-positive cells to total Iba1-positive cells within each plexiform layer to determine microglial transduction efficiency.

Results : Subretinal or intravitreal injection of scAAV2/6^TYF-CD68-GFP showed greater microglial transduction efficiency in the plexiform layer closest to AAV delivery location. However, the overall transduction efficiency was low regardless of injection method, and non-specific transgene expression was prominent, especially in photoreceptors and Müller glial cells. Subretinal injection of our modified scAAV2/6^d4-CD68-DIO-GFP resulted in microglia-specific transgene expression and moderately increased transduction efficiency in the outer plexiform layer (OPL). Strikingly, we observed up to a three-fold increase in microglial transduction efficiencies under conditions that stimulate microglia.

Conclusions : We show that AAV-mediated microglial transduction in the retina is feasible, yet remains a challenge. Our modified AAV2/6 ^d4 was microglia-specific and increased microglial transduction in the OPL, however our results suggest that additional factors contribute to substantial increases in transduction efficiency. Future work will need to identify how the retinal environment impacts AAV-mediated microglial transduction.

This is a 2021 ARVO Annual Meeting abstract.