Abstract
Purpose :
Adeno-associated virus (AAV) mediated gene therapies for retinal degeneration require targeting the outer retina where pathogenesis initiates. Subretinal injection achieves optimal outer retinal transfection but can cause iatrogenic damage as detrimental as the disease. We plan to identify novel AAV variants for RPE transfection via intravitreal (IVT) injection, which is much less invasive, by synthesizing and screening libraries of AAV variants.
Methods :
To find a dry AMD therapeutic that can be efficiently delivered to the RPE without causing retinal detachment in a diseased eye, we applied a concept pioneered by Nobel laureate, Dr. Frances Arnold, called “directed evolution.” It is unknown what AAV properties are necessary for transfection of the RPE from the vitreous. We created 1.48e+9 different 7-mer peptide insertions that adhere to the outside of the AAV2 capsid, changing its binding properties. Then, we screened these variants to find candidates that can transfect the RPE via intravitreal injection. Most of these AAV variants are nonfunctional; these are discarded. Others are reinjected at higher concentrations, narrowing it down to a small subset that can perform the task.
Results :
We screened the AAV2-7mer library in macaque, dog, and mouse, and quantified the top performing variants for RPE transfection using next-generation sequencing. Performance is measured by the number of counts of that variant in the posterior eyecup or RPE cells of the intravitreally injected eye. We then packaged the top 5 performing variants individually with the ubiquitous promoter chicken β-actin (CAG) and an RPE-specific promoter, VMD2. Each of the vectors included a GFP reporter. All 10 AAV constructs were intravitreally injected in C57Bl6 mice to visualize and quantify RPE transfection. At 6 weeks post-injection ,eyes were enucleated for analysis of RPE transduction. Additionally, we are synthesizing more AAV variant libraries to be screened using different serotypes and peptide insertion sequences.
Conclusions :
This data shows that our directed evolution screening of a variant library of AAVs is a promising strategy to identify AAV variants that will efficiently transfect RPE cells.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.