Rational Design of AAV Vectors For Efficient Transduction of Müller Cells

Abstract

Purpose: Photoreceptor cell death is the cause of vision loss in many retinal degenerative diseases but their underlying genetic heterogeneity strongly supports development of a mutation-independent treatment strategy. One attractive approach is targeting Muller glia, cells which traverse the entire thickness of the retina, with vectors designed to produce secretable neurotrophins that slow or prevent photoreceptor cell death. Rational design of AAV has been successful in engineering variants with enhanced/novel transduction properties in the retina. Novel capsid variants that preferentially target Muller cells would potentially increase therapeutic benefit. Thus we investigated tropism and efficiency of AAV vectors rationally designed to target Müller cells.

Methods: AAV6 and AAV1 mutants were designed to 1) alter capsid affinity for heparan sulfate proteoglycan (HSPG) and 2) avoid proteosomal degradation via single or multiple tyrosine to phenylalanine (Y-F), serine to valine (S-V) and threonine to valine (T-V) mutations. Transduction efficiency was quantified by FACS following infection of HEK293, 661W and a biologically relevant Müller cell line (rMC-1). A subset of variants was evaluated by intravitreal or subretinal injection in mouse with fundoscopy and immunohistochemistry of retinal sections.

Results: AAV1 based vectors were highly efficient in HEK293 and 661W cells compared to AAV6 but the addition of HSPG binding via E531K mutation to AAV1 reduced efficiency in these cells. Interestingly, AAV6 transduction in rMC-1 cells was slightly improved over AAV1. The addition of HSPG binding to AAV1 and multiplexing of Y-F and T-V mutations to AAV6 greatly increased transduction efficiencies. In vivo studies revealed that intravitreally-delivered AAV6 and AAV1(E531K) transduced Müller cells more efficiently than AAV1. Analysis of AAV6 and AAV1E531K capsids containing Y-F, S-V and T-V mutations are underway.

Conclusions: By utilizing a biologically relevant cell line we have developed a high throughput strategy for identifying AAV variants with increased Müller cell transduction, thereby providing a means to more efficiently mediate glial-secretion of neurotrophins and prevent retinal degeneration.