Tropism of the AAV6.2 Vector in the Murine Retina

Abstract

Retinitis pigmentosa (RP) is a progressive inherited retinal dystrophy (IRD) that primarily affects rod photoreceptor cells, leading to the degeneration of photoreceptors and the gradual loss of vision. While RP is one of the most studied IRDs, other neurodegenerative diseases affecting the retina and optic nerve, such as glaucoma, also involve common mechanisms of cellular stress and degeneration. Current therapeutic approaches under investigation include gene therapy, retina prosthesis, and neuroprotection. Among these approaches, gene therapy has shown promise, though challenges related to viral vector tropism and transduction efficiency persist. The adeno-associated virus (AAV) vector is commonly employed for gene delivery, but novel serotypes and engineered variants are being explored to improve specificity and efficacy. This study evaluates the gene transfer efficiency of the AAV6.2 vector following intravitreal injection into the murine retina. Male C57BL/6 mice (9 weeks old) were intravitreally injected with 1 µL of AAV2-CMV-EGFP, AAV6-CMV-EGFP, or AAV6.2-CMV-EGFP at a titer of 3.2 × 10¹² vg/mL per eye. Retinal transduction was assessed using in vivo fluorescence imaging, flat-mount imaging, and immunohistochemistry. EGFP expression in retinal ganglion cells, Müller cells, amacrine cells, and bipolar cells was quantitatively analyzed. All three AAV serotypes effectively transduced retinal ganglion cells, but AAV6.2 exhibited enhanced transduction in Müller cells and other neuronal retinal cells, including bipolar and amacrine cells. AAV6.2 demonstrated more localized expression around retinal blood vessels compared to the diffuse expression observed with AAV2. Immunohistochemical analysis revealed that AAV6.2 had significantly higher transduction efficiency in Müller cells (p < 0.001) compared to AAV2 and AAV6. AAV6.2 shows superior transduction efficiency in Müller cells, positioning it as a promising vector for gene therapies targeting retinal degenerative diseases such as RP. Its ability to effectively transduce Müller cells suggests potential applications in neuroprotection and gene replacement therapies.

Keywords: AAV; AAV6.2; IRD; Müller cells; RP; gene therapy.

Figure A1

Immunohistochemistry on transverse retinal cryosections from murine retinas with intravitreal injection of AAV2-CMV-EGFP for (A–C) retinal ganglion cells labeled with RBPMS, (D–F) Müller cells labeled with GS, (G–I) bipolar cells labeled with PKCα, and (J–L) amacrine cells labeled with Syntaxin. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars, 20 µm in (A–L).

Figure A2

Immunohistochemistry on transverse retinal cryosections from murine retinas with intravitreal injection of AAV6-CMV-EGFP for (A–C) retinal ganglion cells labeled with RBPMS, (D–F) Müller cells labeled with GS, (G–I) bipolar cells labeled with PKCα, and (J–L) amacrine cells labeled with Syntaxin. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars, 20 µm in (A–L).

Figure A3

Immunohistochemistry on transverse retinal cryosections from murine retinas with intravitreal injection of AAV6.2-CMV-EGFP for (A–C) retinal ganglion cells labeled with RBPMS, (D–F) Müller cells labeled with GS, (G–I) bipolar cells labeled with PKCα, and (J–L) amacrine cells labeled with Syntaxin. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars, 20 µm in (A–L).

Figure 1

Time course of EGFP expression in the retina following AAV intravitreal injection. (a) Representative fundus photographs illustrating EGFP expression in the retinas of live mice at 1, 2, 3, 4, 5, and 6 weeks post-injection. The mice were injected intravitreally with AAV2-CMV-EGFP, AAV6-CMV-EGFP, or AAV6.2-CMV-EGFP. Asterisks indicate optic discs. (b) Quantification of EGFP fluorescence intensity from fundus fluorescent photographs. The mean fluorescence values were measured over the circular imaging range. Error bars represent the standard error of the mean. a.u.: arbitrary unit.

Figure 2

Distribution of EGFP expression. (a–c) Representative confocal images of retinal flat-mounts at 6 weeks post-injection, showing EGFP expression following intravitreal injection of AAV2-CMV-EGFP (a), AAV6-CMV-EGFP (b), and AAV6.2-CMV-EGFP (c). Asterisks indicate retinal vessels. Blood vessels were identified based on their characteristic appearance under bright-field microscopy.

Figure 3

Immunostaining of each retinal cell type of AAV vectors intravitreally injected into the mouse retina and evaluation of EGFP expression. (a) Immunohistochemistry on transverse retinal cryosections from murine retinas with intravitreal injection of AAV2-CMV-EGFP, AAV6-CMV-EGFP, AAV6.2-CMV-EGFP for (A–C) retinal ganglion cells labeled with RBPMS, (E–G) Müller cells labeled with GS, (I–K) bipolar cells labeled with PKCα, and (M–O) amacrine cells labeled with Syntaxin. The individual channels and DAPI staining can be found in Appendix A. (b)The number of fluorescent cells was quantified within one field of view on each retinal cross-section and then averaged. (D) Quantification of the EGFP-positive rate in RBPMS-positive cells. (H) Quantification of the EGFP-positive rate in GS-positive cells. (L) Quantification of the EGFP-positive rate in PKCα-positive cells. (P) Quantification of the EGFP-positive rate in Syntaxin-positive cells. The criterion for statistical significance was p < 0.05, and an unpaired t-test and one-way ANOVA with Bonferroni correction was performed. n = 12 images; n = 3 retinas. Of note, 4 images were taken per retina. Error bars represent the standard error of the mean. ns, not significant. * p < 0.05. ** p < 0.01. **** p < 0.0001. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars, 20 µm in (A–C,E–G,I–K,M–O).