Impact of Heparan Sulfate Binding on Transduction of Retina by Recombinant Adeno-Associated Virus Vectors

Abstract

Adeno-associated viruses (AAVs) currently are being developed to efficiently transduce the retina following noninvasive, intravitreal (Ivt) injection. However, a major barrier encountered by intravitreally delivered AAVs is the inner limiting membrane (ILM), a basement membrane rich in heparan sulfate (HS) proteoglycan. The goal of this study was to determine the impact of HS binding on retinal transduction by Ivt-delivered AAVs. The heparin affinities of AAV2-based tyrosine-to-phenylalanine (Y-F) and threonine-to-valine (T-V) capsid mutants, designed to avoid proteasomal degradation during cellular trafficking, were established. In addition, the impact of grafting HS binding residues onto AAV1, AAV5, and AAV8(Y733F) as well as ablation of HS binding by AAV2-based vectors on retinal transduction was investigated. Finally, the potential relationship between thermal stability of AAV2-based capsids and Ivt-mediated transduction was explored. The results show that the Y-F and T-V AAV2 capsid mutants bind heparin but with slightly reduced affinity relative to that of AAV2. The grafting of HS binding increased Ivt transduction by AAV1 but not by AAV5 or AAV8(Y733F). The substitution of any canonical HS binding residues ablated Ivt-mediated transduction by AAV2-based vectors. However, these same HS variant vectors displayed efficient retinal transduction when delivered subretinally. Notably, a variant devoid of canonical HS binding residues, AAV2(4pMut)ΔHS, was remarkably efficient at transducing photoreceptors. The disparate AAV phenotypes indicate that HS binding, while critical for AAV2-based vectors, is not the sole determinant for transduction via the Ivt route. Finally, Y-F and T-V mutations alter capsid stability, with a potential relationship existing between stability and improvements in retinal transduction by Ivt injection.

Importance: AAV has emerged as the vector of choice for gene delivery to the retina, with attention focused on developing vectors that can mediate transduction following noninvasive, intravitreal injection. HS binding has been postulated to play a role in intravitreally mediated transduction of retina. Our evaluation of the HS binding of AAV2-based variants and other AAV serotype vectors and the correlation of this property with transduction points to HS affinity as a factor controlling retinal transduction following Ivt delivery. However, HS binding is not the only requirement for improved Ivt-mediated transduction. We show that AAV2-based vectors lacking heparin binding transduce retina by subretinal injection and display a remarkable ability to transduce photoreceptors, indicating that other receptors are involved in this phenotype.

FIG 1

Schematic of the eye detailing the position of neural retina in the posterior segment, along with a higher-magnification view illustrating basic retinal anatomy. The inner limiting membrane (ILM) is a typical basement membrane at the vitreoretinal junction and is created by the end feet of Müller glia (pink). Müller glia span the entire width of the retina, forming barriers at each end, including the ILM in the inner retina and outer limiting membrane (OLM) in the outer retina. Between the ILM and OLM exist multiple nuclear and plexiform layers. The relative ability of previously described, AAV2-based vectors to transduce retinal neurons within these various layers following intravitreal injection is shown on the right. Strong transduction is depicted by large font/boldface letters, whereas weak transduction is depicted by smaller font/gray letters. While AAV2 transduces primarily ganglion cells within the inner retina, AAV2(quadY-F+T-V) promotes efficient transduction of middle retina, i.e., Müller cells (pink), bipolar cells (navy blue), horizontal cells (tan), and outer retina, i.e., photoreceptors (black/red/blue/green) and RPE (gray), following intravitreal delivery in mouse. GCL, ganglion cell layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segments of photoreceptors; OS, outer segments of photoreceptors.

FIG 2

Heparin binding assay and electron micrographs of various AAV2-based recombinant vectors containing tyrosine-to-phenylalanine and/or threonine-to-valine substitutions. (A) The heparin binding elution profiles of AAV2 (positive control), AAV2 mutants (tripleY-F, quadY-F, and quadY-F+T-V), and AAV5 (negative control) at different salt concentrations. (B) Quantification of HS binding data from panel A. (C) Negative-stain electron micrographs of the corresponding vectors from panel A observed at ×46,000 magnification. White scale bars are 50 nm in length and are located at the bottom left of each micrograph. L, load; FT, flowthrough; W, wash.

FIG 3

Capsid sequences of rAAV6, rAAV1, and rAAV1(E531K) and their transduction profiles following intravitreal or subretinal injection of rAAV in mouse retina. (A) rAAV1(E531K) is grafted with the HS binding determinant of AAV6 via a glutamic acid-to-lysine (E-K) substitution at position 531. (B) rAAV6-, rAAV1-, and rAAV1(E531K)-mediated mCherry expression (red) following Ivt or subretinal injection of between 1 × 10¹⁰ and 2 × 10¹⁰ vector genomes in C57BL/6J mice. IS/OS, inner segments/outer segments of photoreceptors; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 34 μm.

FIG 4

Capsid sequences, heparin binding, and electron micrographs of AAV5 and AAV8(Y733F) variants designed to bind HS. (A) Residues of AAV2 that were inserted into AAV5 and AAV8(Y733F) are highlighted in gray, with additional residues inserted by Opie et al. highlighted in black (29). (B) The heparin binding elution profiles of AAV2 (positive control), AAV5, and AAV8(Y733F) variants compared to those of their parent vectors AAV5 and AAV8(Y733F) (negative controls) at different salt concentrations. (C) Negative-stain electron micrographs of the corresponding vectors from panels A and B are visualized at ×46,000 magnification. Scale bar in panel C, 50 nm. L, load; FT, flowthrough; W, wash.

FIG 5

(A) rAAV5-, rAAV5+HS-, rAAV8(Y733F)-, and rAAV8(Y733F)+HS-mediated mCherry expression (red) following subretinal or intravitreal injection of between 2 × 10⁹ and 6 × 10⁹ vector genomes in C57BL/6J mice. The image of retina receiving AAV5 by subretinal injection was extended vertically to allow for visualization of transduction of RPE that was detached during the processing of tissue. IS/OS, inner segments/outer segments of photoreceptors; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 34 μm. (B) Transduction efficiency of unmodified AAV5 and AAV5+HS in 661W cone photoreceptor cells. Vectors were used at multiplicities of infection (MOI) of 10,000 and 50,000. mCherry expression was calculated with FACS by multiplying the percentage of positive cells by the mean fluorescence intensity in each sample. The red line indicates the value under which all signal is noise (no transduction is observed relative to that of blank cells).

FIG 6

Heparin binding of rAAV2 and rAAV2(4pMut) capsids. Heparin binding elution profiles of AAV2(4pMut) and AAV2 (for comparison) at different salt concentrations.

FIG 7

(A) Heparin binding elution profiles of the different AAV2 HS variants along with AAV2 (positive control) and AAV5 (negative control) at different salt concentrations. (B) mCherry expression (red) following Ivt or subretinal injection of between 2 × 10⁹ and 4 × 10¹⁰ vector genomes in C57BL/6J mice of rAAV2(quadY-F+T-V), rAAV2, rAAV2(4pMut), rAAV2(4pMut)+R-S, rAAV2(4pMut)+R-T, rAAV2(4pMut)+R-S+R-T, and rAAV2(4pMut)+R-S+R-T+R-G [also called AAV2(4pMut)ΔHS]. (C) Transduction efficiency of AAV2 variants in HEK293 cells. All vectors were used at a multiplicity of infection (MOI) of 5,000. mCherry expression is shown in arbitrary units on the y axis, calculated by multiplying the percentage of positive cells by the mean fluorescence intensity in each sample. The red line indicates the value under which all signal is noise (no transduction is observed relative to that of blank cells). (D) Negative-stain electron micrographs of the corresponding vectors from panel A observed at ×46,000 magnification. L, load; FT, flowthrough; W, wash; IS/OS, inner segments/outer segments of photoreceptors; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar in panel A, 40 μm. Scale bar in panel D, 50 nm.

FIG 8

In vivo imaging of luciferase gene expression at 4 weeks postintravascular delivery of AAV2, AAV2(4pMut)ΔHS, or AAV2(quadY-F+T-V). All vectors were delivered by injection to the retro-orbital venous sinus at a dose of 2 × 10¹¹ vector genomes. (A) Comparison of live images of bioluminescence from two representative mice (ventral position) per cohort. (B) Relative signal intensities. The values are means from the log base 10 value of flux relative to results for the non-luciferase-expressing control; the error bars represent standard deviations. p/s, photons per second.

FIG 9

Transduction profiles of rAAV2-based vectors with various HS affinities following subretinal or intravitreal injection in Nrl-GFP mice. rAAV2, rAAV2(quadY-F+T-V), rAAV2(4pMut), and rAAV2(4pMut)ΔHS vectors were delivered at a dose of 5 × 10⁹ vector genomes. Both in-life fundus images and representative retinal sections are shown. IS/OS, inner segments/outer segments of photoreceptors; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar in panel A, 50 μm. (B) AAV2(4pMut)ΔHS-mediated mCherry expression in non-rod neural retinal cells. Costaining with antibodies against cone arrestin (left), purkinje cell protein (PCP2) (middle), or calbindin (right) reveal the transduction of cone photoreceptors and, to a much lesser extent, bipolar cells (white arrows), horizontal cells (white arrows), and Muller cells (yellow arrows in the middle panel). IS/OS, inner/outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; GC, ganglion cell layer. Scale bar in panel B, 12 μm.

FIG 10

Relative transduction efficiencies of unmodified rAAV2, rAAV2(quadY-F+T-V), rAAV2(4pMut), rAAV2(4pMut)ΔHS, and rAAV5 in rod photoreceptors and non-rod neural retina and glia following intravitreal or subretinal injection in Nrl-GFP mice. (A, B, and D) FACS analysis was used to quantify the percentage of cells that were GFP positive (rods), mCherry positive (neural retinal cells/glia transduced by rAAV), and both GFP and mCherry positive (rods transduced by rAAV). Plots from pooled treated and untreated retinas are shown in panels A, B, and D. (C) Quantification of the percent transduction by rAAV2-based vectors, as determined by mCherry expression, of non-rod neural retina and rods in intravitreally (left) and subretinally (right) injected Nrl-GFP mice. Differences between cohorts were significant in all cases except those labeled NS (not significant). (D) Quantification of the percent transduction by rAAV5, as determined by mCherry expression, of non-rod neural retina and rods in subretinally injected Nrl-GFP mice. Error bars represent ± one standard deviation.

FIG 11

Transduction of rAAV2(4pMut) and rAAV2(quadY-F+T-V) vectors containing a photoreceptor-specific hGRK1 promoter and mCherry transgene following either subretinal or intravitreal injection at two doses in C57BL/6J mice. mCherry expression in representative fundus images (A) and retinal sections (B) reveals relatively higher transduction of photoreceptors (PRs) by AAV2(quadY-F+T-V) following either subretinal or intravitreal injection at both doses. (C) Transduction was quantified using relative mCherry-mediated pixel intensity in fundus images such as those seen in panel A (three per cohort). LD, low dose; HD, high dose; IS/OS, inner/outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; AU, arbitrary units. Scale bar in panel B, 20 μm.

FIG 12

Thermal profile of wtAAV2, AAV2-based capsid mutants, and AAV5 vectors which were determined by DSF. The peak temperature or melting temperature of each vector is recorded in the table and the standard deviation calculated from the experiments, all of which were done in triplicates.