Targeting photoreceptors via intravitreal delivery using novel, capsid-mutated AAV vectors

Abstract

Development of viral vectors capable of transducing photoreceptors by less invasive methods than subretinal injection would provide a major advancement in retinal gene therapy. We sought to develop novel AAV vectors optimized for photoreceptor transduction following intravitreal delivery and to develop methodology for quantifying this transduction in vivo. Surface exposed tyrosine (Y) and threonine (T) residues on the capsids of AAV2, AAV5 and AAV8 were changed to phenylalanine (F) and valine (V), respectively. Transduction efficiencies of self-complimentary, capsid-mutant and unmodified AAV vectors containing the smCBA promoter and mCherry cDNA were initially scored in vitro using a cone photoreceptor cell line. Capsid mutants exhibiting the highest transduction efficiencies relative to unmodified vectors were then injected intravitreally into transgenic mice constitutively expressing a Rhodopsin-GFP fusion protein in rod photoreceptors (Rho-GFP mice). Photoreceptor transduction was quantified by fluorescent activated cell sorting (FACS) by counting cells positive for both GFP and mCherry. To explore the utility of the capsid mutants, standard, (non-self-complementary) AAV vectors containing the human rhodopsin kinase promoter (hGRK1) were made. Vectors were intravitreally injected in wildtype mice to assess whether efficient expression exclusive to photoreceptors was achievable. To restrict off-target expression in cells of the inner and middle retina, subsequent vectors incorporated multiple target sequences for miR181, an miRNA endogenously expressed in the inner and middle retina. Results showed that AAV2 containing four Y to F mutations combined with a single T to V mutation (quadY-F+T-V) transduced photoreceptors most efficiently. Robust photoreceptor expression was mediated by AAV2(quadY-F+T-V) -hGRK1-GFP. Observed off-target expression was reduced by incorporating target sequence for a miRNA highly expressed in inner/middle retina, miR181c. Thus we have identified a novel AAV vector capable of transducing photoreceptors following intravitreal delivery to mouse. Furthermore, we describe a robust methodology for quantifying photoreceptor transduction from intravitreally delivered AAV vectors.

Figure 1. Transduction efficiency of unmodified and capsid mutated vectors in vitro .

661W cells were infected with scAAV2, scAAV2(quadY-F), scAAV2(quadY-F +T-V), scAAV5, scAAV5(singleY-F), scAAV5(doubleY-F), and scAAV8, scAAV8(doubleY-F), and scAAV8(doubleY-F+T-V) at a multiplicity of infection (MOI) of 10,000. 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.

Figure 2. Qualitative comparison of unmodified and capsid mutated AAV vectors in vivo .

Fundoscopy (red channel only) of Rho-GFP mice 4 weeks post-injection with unmodified and capsid-mutated scAAV-smCBA-mCherry vectors (1.5×10⁹ vg delivered). Exposure and gain settings were the same across all images.

Figure 3. Quantitative comparison of unmodified and capsid mutated AAV vectors in vivo .

Transduction efficiency of unmodified and capsid-mutated scAAV2, scAAV5 and scAAV8 vectors in Rho-GFP mice. FACS analysis was used to quantify the percentage of cells that were GFP positive (PRs), mCherry positive (any retinal cells transduced with AAV) and both GFP and mCherry positive (PRs transduced by AAV). Representative plots for a negative control (uninjected retina) and 2 pooled retinas injected with scAAV2(quadY-F+T-V) are shown in Panels A and B, respectively. Cells that were both GFP and mCherry positive are shown in the top right of Panels A and B and represent the percent of transduced PRs. The bottom right of Panels A and B show cells that were mCherry positive but GFP negative, representing off-target transduction. The percentage of mCherry positive PRs (a measure of in vivo PR transduction efficiency for each vector) in retinas injected with unmodified or capsid-mutated scAAV vectors is shown in Panel C.

Figure 4. In vivo analysis of AAV2-based vectors containing the hGRK1 promoter.

Fundus images paired with immunohistochemistry of frozen retinal cross-sections from C57BL/6 mice taken 4 weeks post injection with AAV2, AAV2(quad Y-F), and AAV2(quad Y-F +T-V) vectors containing hGRK1-GFP (7.5×10⁹ vg delivered). Identical gain and exposures were used for fundoscopy. All tissue sections were imaged at 20X, with identical gain and exposure settings. GFP expression is shown in green. Nuclei were counterstained with DAPI (blue). RPE- retinal pigment epithelium, IS/OS- inner segments/outer segments, ONL- outer nuclear layer, INL- inner nuclear layer, GCL- ganglion cell layer.

Figure 5. In vivo analysis of AAV5-based vectors containing the hGRK1 promoter.

Fundus images paired with IHC of frozen retinal cross-sections from C57BL/6 mice taken 4 weeks post injection with capsid mutated AAV5 vectors containing hGRK1-GFP. For analysis of AAV5(singleY-F) and AAV5(doubleY-F) vectors 8.5×10¹⁰ vg and 5.3×10⁹ vg were delivered, respectively. Retinal tissue sections containing optic nerve head (Panels B and E) and peripheral retinal cross sections (Panels C and F) are shown. White arrows demarcate the optic nerve head. Identical gain and exposures were used for fundoscopy. All cross sections were imaged at 20X, with identical gain and exposure settings. GFP expression is shown in green. Nuclei were counterstained with DAPI (blue). RPE- retinal pigment epithelium, IS/OS- inner segments/outer segments, ONL- outer nuclear layer, INL- inner nuclear layer, GCL- ganglion cell layer.

Figure 6. In vivo analysis of AAV8-based vectors containing the hGRK1 promoter.

Fundus images paired with immunohistochemistry of frozen retinal cross-sections from C57BL/6 mice taken 4 weeks post injection with capsid mutated AAV8 vectors containing hGRK1-GFP. For analysis of AAV8(doubleY-F) and AAV8(doubleY-F+T-V) vectors, 1.3×10¹¹ and 6.0×10¹⁰ vg were delivered, respectively. Retinal cross sections containing optic nerve head (Panels B and E) and peripheral retinal cross sections (Panels C and F) are shown. White arrows demarcate the optic nerve head. Identical gain and exposures were used for fundoscopy. All cross sections were imaged at 20X, with identical gain and exposure settings. GFP expression is shown in green. Nuclei were counterstained with DAPI (blue). RPE- retinal pigment epithelium, IS/OS- inner segments/outer segments, ONL- outer nuclear layer, INL- inner nuclear layer, GCL- ganglion cell layer.

Figure 7. MicroRNA-mediated regulation of transgene expression.

Both hGRK1-GFP and hGRK1-GFP-miR181c were packaged in AAV2(quadY-F+T-V) and delivered intravitreally to C57BL/6 mice (1.5×10¹⁰ vg). Fundoscopy at 4 weeks post injection is shown adjacent to immunohistochemistry of frozen retinal cross-sections. Identical gain and exposures were used for fundoscopy. All cross sections were imaged at 20X, with identical gain and exposure settings. GFP expression is shown in green. Nuclei were counterstained with DAPI (blue). RPE- retinal pigment epithelium, IS/OS- inner segments/outer segments, ONL- outer nuclear layer, INL- inner nuclear layer, GCL- ganglion cell layer.