Effects of Altering Heparan Sulfate Proteoglycan Binding and Capsid Hydrophilicity on Retinal Transduction by Adeno-associated Virus

Abstract

ABSTRACT Adeno-associated viruses (AAVs) have recently emerged as the leading vector for retinal gene therapy. However, AAV vectors that are capable of achieving clinically relevant levels of transgene expression and widespread retinal transduction are still an unmet need. Using rationally designed AAV2-based capsid variants, we investigate the role of capsid hydrophilicity and hydrophobicity as it relates to retinal transduction. We show that hydrophilic, single-amino-acid mutations (V387R, W502H, E530K, L583R) in AAV2 negatively impact retinal transduction when heparan sulfate proteoglycan (HSPG) binding remains intact. Conversely, addition of hydrophobic point mutations to an HSPG binding-deficient capsid (AAV2ΔHS) leads to increased retinal transduction in both mouse and macaque. Our top performing vector, AAV2(4pMut)ΔHS, achieved robust rod and cone photoreceptor (PR) transduction in macaque, especially in the fovea, and demonstrates the ability to spread laterally beyond the borders of the subretinal injection (SRI) bleb. This study both evaluates biophysical properties of AAV capsids that influence retinal transduction and assesses the transduction and tropism of a novel capsid variant in a clinically relevant animal model. IMPORTANCE Rationally guided engineering of AAV capsids aims to create new generations of vectors with enhanced potential for human gene therapy. By applying rational design principles to AAV2-based capsids, we evaluated the influence of hydrophilic and hydrophobic amino acid mutations on retinal transduction as it relates to vector administration route. Through this approach, we identified a largely deleterious relationship between hydrophilic amino acid mutations and canonical HSPG binding by AAV2-based capsids. Conversely, the inclusion of hydrophobic amino acid substitutions on an HSPG binding-deficient capsid (AAV2ΔHS) generated a vector capable of robust rod and cone photoreceptor (PR) transduction. This vector AAV2(4pMut)ΔHS also demonstrates a remarkable ability to spread laterally beyond the initial subretinal injection (SRI) bleb, making it an ideal candidate for the treatment of retinal diseases that require a large area of transduction.