Abstract
Purpose :
Gene therapy is becoming realistic for clinical against inherited retinal degenerative diseases (IRDs) in recent years. Adeno-associated virus (AAV) vectors are commonly used in gene therapy; however, each serotype has different infection efficiency and tissue specificity. Thus, it is important to evaluate the characteristics of AAV to select the appropriate vector for therapeutic applications. AAV6.2, developed by an F129L point mutation in AAV6 that increases its heparin affinity. In this study, we evaluated the tropism of AAV6.2 to the retina and examined its potential application.
Methods :
One microliter of AAV-2-CMV-EGFP, AAV-6-CMV-EGFP, or AAV-6.2-CMV-EGFP vector at a titer of 3.2 × 10e12 vg/ml was injected intravitreally in each eye of 9-week-old male C57BL/6 mice (n=6 for each vector). In vivo fluorescence imaging, flat-mount imaging, and immunohistological staining of sections were performed to analyze tropism of AAV-2, AAV-6, and AAV-6.2 vectors. The criterion for statistical significance was p<0.05, and One-way ANOVA with Bonferroni correction was performed.
Results :
Expression of each vector reached its peak at 30 days past-injection. In vivo fluorescence imaging, AAV6.2 exhibited a tendency for earlier expression compared to AAV2 and AAV6. AAV6 and AAV6.2 showed patchy expression in fundus and whole-mount images, unlike the uniform expression of AAV2. In flat-mount imaging, AAV6 and AAV6.2 showed patchy expression patterns, contrasting with the uniform expression of AAV2 and the expression of AAV6 and AAV6.2 often aligned with retinal vessels. Immunohistological staining of AAV6.2 retinal sections showed EGFP expression in retinal ganglion cells, bipolar cells, and Müller cells. Co-labeling with Glutamine Synthetase (GS) in Müller cells showed that AAV6.2 was significantly more efficient than AAV2 and AAV6 (24.1 ± 2.50 cells/212.13μm^2 for AAV6.2 vs. 10.9 ± 1.01 and 9.4 ± 1.08 cells/212.13μm^2 for AAV2 and AAV6, respectively; p<0.01).
Conclusions :
AAV6.2 successfully and efficiently introduced reporter genes into mouse Müller cells through intravitreal injection. AAV6.2 has potential applications in gene therapy targeting Müller cells.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.