1.1 Retinal tissue structure
The retina is obviously stratified, and the access paths of the outer photoreceptor cells, RPE and inner ganglion cells are different.
1.2 AAV serotype selection
AAV2 is often used for inner layer/vitreous related design; AAV5, AAV8 and engineered capsids are often used for outer layer or subretinal delivery.
1.3 Retina-specific promoter
VMD2/RPE65 is selected for RPE, GRK1 is selected for photoreceptor cells, and SNCG can be assessed for ganglion cells.
1.4 Administration method and dosage reference
Subretinal (SR): the gold standard pathway targeting the RPE and photoreceptors. Mouse 1×10^8-3×10^9 vg/eye, 1-2 μL, 33 G Hamilton blunt needle, slowly inject 1-2 μL/min and retain the needle for 30 s; NHP 7×10^11-7×10^12 vg/eye (AAV8), 100-200 μL, 39 G injection cannula/36-42 G retinal needle, AAV and Healon Mix 1:1 to form bleb; dog 1.0-1.4×10^11 vg/eye, 200 μL; pig 1×10^10-1.25×10^11 vg/eye [1-4, 1-5, 1-8].
Intravitreal cavity (IVT): Targeting RGCs and inner retina. Mouse 1×10^9-1×10^10 vg/eye, 1-2 μL, 33 G blunt needle; rat 1-5×10^9 vg/eye, 5 μL; NHP 7×10^11 vg/eye, 100 μL, 30 G via pars plana. The internal limiting membrane (ILM) is the main barrier for IVT transduction of the outer retina, and engineering capsids such as AAV2.7m8 or ILM removal can improve penetration [1-7,1-8].
Coverage and limitations: SR only transduces RPE+ photoreceptors in the bleb area (the mouse bleb covers about 20-40% of the retina), and the off-target is extremely low; IVT is mainly RGC/inner layer, and large animals have limited penetration of the outer retina. The peak expression is 3-6 weeks on average, and SR can last for >1.5 years. The transduction efficiency and immunogenicity of SR in NHP are better than those in IVT [1-5,1-6].
1.5 Key research evidence
Acland et al. confirmed that AAV subretinal injection restored vision in the RPE65 mutant dog model, laying the foundation for RPE65 gene therapy [1-4]; Yiu et al. established the AAV2/8 photoreceptor transduction dose threshold in monkeys (7×10^12 vg/eye for extensive transduction, 7×10^11 vg/eye (no obvious expression) [1-5]; The RD-CURE Alliance compared SR and IVT in NHP, and the retinal gene transfer and biodistribution of SR were significantly better than that of IVT [1-6]; Dalkara et al. demonstrated that ILM is a physical barrier to intravitreal transduction [1-7]. The design should report cell layer, transduction area, functional endpoints, and intraocular inflammation simultaneously.
1.6 References for this chapter
[1-1] Russell S, et al. Voretigene neparvovec phase 3 trial. Lancet. 2017;390:849-860. DOI:10.1016/S0140-6736(17)31868-8. PMID:28712537. [H]
[1-2] Fischer MD, et al. Subretinal gene therapy targeting cone photoreceptors. JAMA Ophthalmol. 2020;138:643-651. [H]
[1-3] He X, et al. AAV for gene therapy in ocular diseases. Research. 2023;6:0291. [N]
[1-4] Acland GM, Aguirre GD, Ray J, et al. Gene therapy restores vision in a canine model of childhood blindness. Nat Genet. 2001;28:92-95. DOI:10.1038/88357. PMID:11385066. [H]
[1-5] Yiu G, et al. Dosage thresholds for AAV2 and AAV8 photoreceptor gene therapy in monkey. Sci Transl Med. 2011;3:88ra54. DOI:10.1126/scitranslmed.3002103. PMID:21849668. [H]
[1-6] RD-CURE Consortium. Superior retinal gene transfer and biodistribution profile of subretinal versus intravitreal delivery of AAV8 in nonhuman primates. Invest Ophthalmol Vis Sci. 2017;58:5792-5801. DOI:10.1167/iovs.17-22473. PMID:29186476. [H]
[1-7] Dalkara D, et al. Inner limiting membrane barriers to AAV-mediated retinal transduction from the vitreous. Mol Ther. 2009;17:2096-2102. DOI:10.1038/mt.2009.181. PMID:19773742. [H]
[1-8] Boye SE, et al. Suprachoroidal and subretinal injections of AAV using transscleral microneedles for retinal gene delivery in nonhuman primates. Hum Gene Ther. 2019;30:1123-1134. DOI:10.1089/hum.2019.052. [N]