Retinal Penetrating Adeno-Associated Virus

Abstract

Purpose: The most common method of delivery of genes to the outer retina uses recombinant adeno-associated virus (AAV) injected into the subretinal space using a surgical procedure. In contrast, most drugs are delivered to the retina using an intravitreal approach in an office setting. The objective of the current study was to develop AAV vectors that can reach the outer retina via intravitreal injection.

Methods: Recently, we described a molecular chaperone (Nuc1) that enhanced the penetration of small and large molecules, including AAV, into the retina. The Nuc1 amino acid sequence or a truncated version of Nuc1 (IKV) was genetically incorporated into an exposed loop of AAV2/9 VP1 protein. These novel recombinant AAV vectors expressing green fluorescent protein (GFP) or nuclear factor erythroid 2 p45-related factor 2 (Nrf2) were injected into the vitreous of C57Bl/6J or Nrf2 knockout mice, respectively. The amount of GFP expression or oxidative stress as measured by 8-Hydroxy-2'-deoxyguanosine staining in C57Bl/6J or Nrf2 knockout mice, respectively, was quantified.

Results: Incorporation of Nuc1 into AAV2/9 did not lead to significant expression of GFP in the murine retina. However, incorporation of IKV into AAV2/9 led to robust expression of GFP in photoreceptors and retinal pigment epithelium (RPE) via the intravitreal and subretinal routes of delivery. Furthermore, expression of Nrf2 using an IKV vector led to a reduction in oxidative stress in the retina of C57Bl/6J and Nrf2 knockout mice.

Conclusions: We have developed a novel AAV vector that enables delivery of transgenes to the outer retina of mice, including photoreceptors and RPE following intravitreal injection.

Figure 1.

Modeling of Nuc1 and IKV sequences into AAV9 VP1. (A) The Nuc1 or truncated Nuc1 (IKV) sequences flanked by glycine residues were inserted between amino acids 370 and 371 of AAV9 VP1 (PDB: 3UX1). (B) A fragment of VP1 (amino acids 263:383) containing either Nuc1 or IKV was modeled in AlphaFold 3 (Google Deepmind) and the results were superimposed on the previously published crystal structure of AAV9 VP1 using UCSF ChimeraX version 1.2.5.

Figure 2.

Incorporation of Nuc1 into the AAV capsid does not enhance infection of AAV2/9 via the subretinal route of injection. Subretinal injection of AAVNuc1CAGGFP did not lead to a significant infection of retinal cells relative to the parental virus AAV9CAGGFP. Co-staining of frozen sections with PKC (bipolar cells), Glutamine Synthase (Muller cells), cone, or rod opsin (photoreceptors) indicated that AAVNuc1CAGGFP infected primarily the rod and cone photoreceptors but in all cases, rather poorly relative to AAV9CAGGFP. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium.

Figure 3.

Incorporation of Nuc1 into the AAV capsid does not enhance infection of AAV2/9 via the intravitreal route of injection. (A) Intravitreal injection of AAVNuc1CAGGFP or AAV9CAGGFP did not lead to any significant expression of GFP in the retina. (B) In contrast, AAVNu1CAGGFP or AAV9CAGGFP could penetrate the retina when co injected with Nuc1. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium.

Figure 4.

AAVIKVGFP infection following subretinal injection. A single subretinal injection of AAVIKVGFP leads primarily to transduction of photoreceptors in addition to a variety of other retinal cells. Retinal cryostat sections were co-stained with rod opsin (A), cone opsin (B), glutamine synthase (C), and PKC (D). GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.

Figure 5.

AAVIKVGFP infection following intravitreal injection. A single intravitreal injection of AAVIKVGFP leads primarily to infection of photoreceptors (A) in addition to a variety of other retinal cells. Retinal cryostat sections were co-stained with cone opsin (B), rod opsin (C), PKC/ bipolar cells (D), tubulin (E), or glutamine synthase (F). GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar 100 µm. Where relevant, higher magnification images are also presented for some panels.

Figure 6.

Nuc1 mediated enhancement of retinal penetration by AAVIKV. Co-injection of AAVIKVGFP with Nuc1 peptide led to maximum transgene expression following intravitreal injection, occurring over the entire retinal surface (A). Higher magnification images indicated that photoreceptors were highly GFP-positive, including the retinal pigment epithelium (RPE) and choroid (B). Fundus imaging of live animals also revealed that transgene expression was across the entire retinal surface (C). Higher exposure of inner retina revealed GFP-positive inner plexiform layer (IPL) and ganglion cell layer (GCL), including some Muller cells (D). Longer exposure images of retinal sections revealed that cells in the inner nuclear layer (INL) were also positive, co-stained with PCK for bipolar cells (E). Scale bar 50 µm and 25 µm, respectively.

Figure 7.

Quantitation of GFP mRNA following intravitreal injection of AAV. Representative fundus images of GFP expression and quantitation of GFP mRNA following intravitreal injection of the various viruses described in this study. Relative to AAV9CAGGFP, co-injection of Nuc1 enhanced mRNA levels by over 4-fold. Nuc1 also enhanced expression of AAVIKVGFP by over eight-fold. Relative to AAV9GFP, IKVGFP + Nuc1 had approximately 300-fold greater levels of mRNA. Values are represented as mean ± SD (N = 4/eyes per group). *P < 0.05 versus controls (AAV9GFP and AAVIKVGFP, respectively) and #P < 0.05 versus AAV9GFP. Fundus image for AAVIKVGFP + Nuc1 is taken from Figure 6, presented here for easier comparison.

Figure 8.

Inhibition of oxidative stress in the outer retina via intravitreal AAV delivery. AAVIKV-Nrf2 injected C57/Bl6J eyes exhibited significantly less 8-OHdG staining relative to AAVIKV-GFP injected eyes (A). There was no detectable 8-OHdG staining in the ONL of PBS injected eyes. Similarly, AAVIKV-Nrf2 injected NRF2 knockout mice exhibited significantly less 8-OHdG staining relative to AAVIKV-GFP eyes (B). Quantitation of these retinas demonstrated a significant reduction in 8-OHdG staining in the ONL (C). GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Values are represented as mean ± SD (N = 5-6/retinas per group). *P < 0.05 versus AAVIKVGFP for C57BL/6J mice and #P < 0.05 versus Nrf2 knockout mice. Lower power images are included as insets for PBS, showing minimal 8-OHdG staining throughout the retina.