Eyes Section丨Application of AAV in Gene Therapy: Serotype, Promoter Selection, and Injection Strategies
Release time：2024-12-24 11:43:40
rAAV (recombinant adeno-associated virus) is highly suitable for ocular gene therapy. The immune-privileged environment of the eye, combined with the low immunogenicity of rAAV, minimizes strong innate immune responses, thereby reducing the risk of adverse immune reactions. Additionally, rAAV can target a broad range of retinal cells, including photoreceptors, retinal pigment epithelium (RPE), and ganglion cells. Various AAV capsids have been developed to optimize transduction of specific cell types.
Serotypes such as AAV1, AAV2, AAV5, and AAV8 can effectively transduce photoreceptors and RPE cells. Among these, AAV2 is one of the most extensively studied and applied serotypes in ocular gene therapy and has been successfully used as a vector in numerous clinical trials, including those targeting Leber congenital amaurosis (LCA) and choroideremia. Other commonly used serotypes include AAV5 and AAV8, each offering unique advantages.
For example, subretinal injection of AAV5-hRKp.RPGR has been shown to be safe and well-tolerated, improving retinal sensitivity and functional vision in male patients with X-linked retinitis pigmentosa (XLRP-RPGR). AAV8, when administered via subretinal injection, can transduce photoreceptors more efficiently than AAV2.
Injection Methods for AAV Delivery to the Retina in Ophthalmic Therapy
In ocular treatment, rAAV can be delivered to the retina through three common administration routes: subretinal injection, intravitreal injection, suprachoroidal injections.
Figure 1: Schematic Diagram of AAV Injection for Ocular Gene Therapy
Advantages and Disadvantages of AAV Injection Methods for Retinal Delivery
Injection Method
Advantages
Disadvantages
1. Subretinal Injection
Directly targets photoreceptors and retinal pigment epithelial (RPE) cells.
Allows treatment of specific areas (e.g., macula).
Avoids adaptive immune response.
Complex operation.
Reflux into the vitreous cavity.
2. Intravitreal Injection
Transduces more retinal cells.
Suitable for inner retinal infections.
Dilution issues.
Humoral immune response.
3. Suprachoroidal Injection
Minimally invasive.
Reduces the risk of increased intraocular pressure.
Requires crossing multiple tissue layers to reach the outer retina.
Immune response.
Application Example
Example 1: AAV-Delivered Photoreceptor-Specific CRISPR/Cas9 System
Serotype: AAV8
Promoter: RK (human rhodopsin kinase promoter)
Experimental Animal: Nrl-L-EGFP mice, 2 weeks old
Injection Protocol: Subretinal injection with an expression duration of 10 weeks
Experimental Results:
The knockdown efficiency of the AAV-CRISPR/Cas9 system was evaluated using the enhanced green fluorescent protein (EGFP) gene as the target. A mixture of AAV8 (AAV-sgRNA-EGFP) and AAV-Cas9 was subretinally injected into 2-week-old Nrl-L-EGFP mice at a dosage of 2.5 × 10^9 vg/eye for both AAV-Cas9 and AAV-sgRNA-EGFP. Approximately 43% of rod cells transduced by sgRNA successfully knocked out the EGFP gene. The remaining 57% of cells failed to knock out EGFP due to potential factors such as lack of Cas9 expression, in-frame insertions/deletions that did not disrupt EGFP expression, and/or the presence of multiple EGFP gene copies in the mice, which exceeded the gene disruption capability of the CRISPR system.
Figure 2: AAV-CRISPR/Cas9 Suppresses EGFP in Mouse Retina
Example 2: Reactivation of CaMKII can protect RGCs from excitotoxicity or axonal injury
Serotype: AAV2
Promoter: mSncg (mouse g-synuclein promoter), CAG
Experimental Animals: C57BL/6 mice, 8 weeks old
Injection Scheme: Intravitreal injection, expression for 2 weeks
Experimental Results: To study whether enhancing the activity of CaMKII is sufficient to protect RGCs from excitotoxicity or axonal injury, AAV2 (AAV-CaMKIIa T286D, titer 2×10^13 vg/ml) was injected intravitreally 2 weeks before the injury to mediate the expression of the CaMKII mutant gene in RGCs. AAV2 effectively transduced more than 95% of retinal ganglion cells (RGCs), and the results showed that the expression level of the CaMKIIa T286D mutant in RGCs was 60% of the endogenous CaMKII. The CaMKIIa T286D mutant strongly protected RGCs. In mouse injury and disease models, reactivation of CaMKII activity via AAV gene therapy can protect RGCs and preserve visual function.
Figure 3 AAV-mediated gene transduction of RGCs
Example 3 TASK-3-mediated non-image-forming behavior and image-forming behavior
Serotype: AAV2
Promoter: NEFL (the RGC-specific promoter Ple345)
Experimental Animals: TASK-3 KO mice
Injection Scheme: Intravitreal injection, 2×10^10 vg/eye, expression for 3 weeks
Experimental Results: To test whether the TASK-3 channel in RGCs is sufficient to maintain PLR sensitivity, TASK-3 channels were overexpressed in RGCs of TASK-3 KO mice using a viral vector and RGC-specific promoter. AAV2-Ple345 (NEFL)-kcnk9-HA (AAV2-TASK-3) and AAV2-Ple345 (NEFL)-EGFP (AAV2-control) were packaged and injected intravitreally into both eyes of TASK-3 KO mice. The expression specificity was assessed 3 weeks later. The results showed that TASK-3 was specifically expressed in RGCs, and the sensitivity of PLR was enhanced in TASK-3 KO mice that received TASK-3 overexpression compared to those that received control virus injections.
Figure 4 AAV-mediated overexpression of TASK-3 in RGCs
Example 4: Efficient Cross-Species Ocular Gene Delivery Using AAV Virus
Serotype: AAVv128
Promoter: CMV
Experimental Animals: Cynomolgus monkeys (Macaca fascicularis)
Injection Scheme: Subchoroidal injection, 2×10^10 vg/eye, expression for 3 weeks
Experimental Results: The retinal transduction efficiency of AAV8 and AAVv128-eGFP vectors was evaluated in non-human primates (NHPs) via subchoroidal injection. eGFP expression was detectable on day 14 after injection. In vivo retinal fluorescence imaging of the animals was performed using a scanning laser ophthalmoscope (SLO) to detect eGFP expression, followed by immunofluorescence analysis. Subchoroidal injection of AAV in cynomolgus monkeys at a dose of 3.5×10^12 vg/eye showed significantly increased eGFP fluorescence in the peripheral retinal fundus when using the AAVv128 vector.
Figure 5 Evaluation of AAV8 and AAVv128 transduction efficiency after intraocular injection in NHPs
Reference
1、Wang JH, Zhan W, Gallagher TL, Gao G. Recombinant adeno-associated virus as a delivery platform for ocular gene therapy: A comprehensive review. Mol Ther. Published online October 28, 2024.
2、Yu W, Mookherjee S, Chaitankar V, et al. Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice. Nat Commun. 2017;8:14716.
3、Guo X, Zhou J, Starr C, et al. Preservation of vision after CaMKII-mediated protection of retinal ganglion cells. Cell. 2021;184(16):4299-4314.e12.
4、Wen X, Liao P, Luo Y, et al. Tandem pore domain acid-sensitive K channel 3 (TASK-3) regulates visual sensitivity in healthy and aging retina. Sci Adv. 2022;8(36):eabn8785.
5、Luo S, Jiang H, Li Q, et al. An adeno-associated virus variant enabling efficient ocular-directed gene delivery across species. Nat Commun. 2024;15(1):3780.
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