Heparan sulfate affinity dictates transduction of photoreceptors from the vitreous by capsid mutated AAV2 variants.

Abstract

 

Purpose

 

Developing AAV vectors capable of efficiently transducing retina from the vitreous would be a major step forward in translating gene therapy to the clinic. It is known that mutagenesis of surface exposed tyrosine residues prevents proteosomal degradation and increases nuclear transport of AAV thereby increasing its transduction efficiency. A variant with four Y-F mutations, AAV(quadY-F), transduces distal layers of the retina, including photoreceptors when delivered to the vitreous. The mechanism for this enhanced “penetration”is not fully understood. The primary cellular receptor for AAV2 is heparan sulfate (HS) and heparan sulfate proteoglycan (HSPG) is a primary component of the inner limiting membrane. We hypothesized that the relative ‘penetrating’ ability of capsid mutants relies on their respective HS affinities.

 

Methods

 

AAV2 based vectors containing combinations of Y-F and/or T-V mutations were analyzed by chromatography on heparin agarose columns to determine respective affinities. A new set of capsid variants was created using atriple Y-F/single T-V variant (AAV2 MAX) as template. Structure informed mutations of the known HS binding residues of AAV2 were performed with the goal of generating vectors with a range of HS binding or no binding whatsoever (AAV2 MAX delta HS). New variants are currently being evaluated for transduction efficiencies in vitro and in vivo via subretinal and intravitreal injections in mice.

 

Results

 

AAV2 variants that primarily transduce inner retina from the vitreous (AAV2wt and AAV2tripleY-F) have strong affinity for HS, while those that transduce photoreceptors have moderate affinity (AAV2quadY-F and AAV2quadY-F+T-V). AAV2 MAX delta HS, the variant with ablated HS binding does not transduce cells in vitro but is highly efficient at transducing photoreceptors when delivered subretinally.

 

Conclusions

 

HS affinity of AAV2 based vectors is a key factor in their ability to transduce photoreceptors from the vitreous. Photoreceptor transduction by AAV2 is not dependent on HS binding, suggesting another ligand-receptor pair is responsible for attachment of virus to the surface of photoreceptors. Therefore, changes in HS affinity are unlikely to negatively impact the recognition of the target cell type (photoreceptors), suggesting that further improvements using this strategy are achievable.

 

 

Figure 1. HS binding of AAV2-based capsid mutants

 

Keywords: 538 gene transfer/gene therapy • 688 retina • 541 glycoconjugates/glycoproteins