Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

In this video article, we described the limbal-approach subretinal injection technique in detail with representative results of RPE/choroid/scleral flat-mount. This is an easy and convenient technique for subretinal injection of viral vectors into RPE. Direct visualization of bleb formation during the injection is an important step for accurate delivery for the beginners. There are some subretinal injection techniques introduced in Journal of Visualized Experiments^8-10. Subretinal space is the potential space between retina and RPE, therefore there are two possible routes to approach the subretinal space. One is an inside approach to the subretinal space via vitreous and retina and the other is an outside approach via RPE and sclera. This limbal-approach subretinal injection technique is the former one via intravitreal space and retina. This technique has an advantage to provide more posterior bleb formation compared to the latter because the latter approach makes a bleb from the peripheral side via scleral tunneling or incision, which should be made over the peripheral retina. In addition, simple limbal puncture is easier than scleral tunneling without retinal penetration. For the neonatal mice, however, it would be more appropriate to use the outside approach⁹. Because neonatal mouse’s eyeball is very small and full of the lens, limbal-approach technique can cause unwanted cataract formation or even phthisis bulbi.

There are some critical steps in this protocol. First of all, initial puncture should be place slightly posterior to the limbus because too anterior puncture at the cornea makes iris incarceration, or too posterior puncture makes direct hole at the peripheral retina. Secondly, it is recommended to be careful to avoid hitting the lens with needle while making the initial puncture. It is better not to insert the entire bevel of the needle to avoid lens puncture. Thirdly, the best approach angle of the needle is about 45 degrees against iris plane, and the blunt needle should be pushed posteriorly toward peripapillary area. Finally, the needle should be placed in the subretinal space during the injection, resulting in uniform bleb formations, otherwise the displaced needle causes intravitreal injection.

The eye is a small organ with unique features that make it an attractive target for gene therapy¹¹. Specifically, it is a small-enclosed compartment with immune-privilege that enables gene therapy with relatively low toxicity and immune reactions in the eye and adverse systemic effects. Easy visualization and ready accessibility to surgical approaches are other advantages of the eye for gene therapy. The retina has a highly organized laminar structure with 10 distinct layers. Thus, accurate spatial gene delivery can only be achieved by the appropriate methods of intraocular injection. The two most common methods for intraocular delivery of viral vectors are intravitreal injection and subretinal injection⁶. In general, intravitreal injection is the preferred route for inner retina targeting retinal ganglion cells and Müller cells. Subretinal injection is the preferred route for outer retina targeting photoreceptor cells and RPE cells. For an efficient transduction in RPE, it is necessary to deliver viral vectors subretinally despite the potential for damage to the retina.

In this study, we used LV vectors with GFP and RFP for the visualization. Because viral vectors have tropism for specific cell types of the retina, viral vectors should be chosen according to tropism of AAV, LV, and Ad to target cells⁵. For example, AAV2/2, AAV2/6 and AAV2/8 are the highest transduction efficiency to RGC and Müller cells. AAV2/5, 2/7, 2/8 and 2/9 efficiently transduce photoreceptor cells and RPE with AAV2/8 being the most efficient serotype in mice^5,12. AAV2/1, AAV2/2, AAV2/4, AAV2/6, and AAV2/8 efficiently transduce RPE⁵. For LV vectors, there is a high-level of expression in RPE cells after subretinal injections, but little or no expression after intravitreal injections¹³.

For effective gene delivery to RPE, viral promoters should be also considered. Viral promoters, such as cytomegalovirus (CMV) promoter and chimeric chicken beta-actin/CMV enhancer promoter are strong and ubiquitous promoters, thus, they are used in majority of gene therapy studies. In RPE, the CMV promoter is estimated to express ten-fold as much protein compared with the RPE65 promoter¹⁴. Of importance, the optimal combination of the promoter and viral vectors including serotype should be achieved for better results as demonstrated by superior expression of RPE65 when the human RPE65 promoter are packaged in serotype rAAV2/4 than rAAV2/2¹⁵. Focusing on gene delivery to RPE cells, RPE-specific promoters such as the RPE65 or the VMD2 promoter reduces the off-target safety issues¹⁶.

AMD is a complex disease that has two phases with a degenerative phase referred to as dry AMD and a phase characterized by choroidal neovascularization referred to as wet AMD. Also, AMD is a disease in which numerous genetic susceptibilities including complement factor H (CFH), complement C3, complement factor I (CFI), Age-related maculopathy susceptibility 2 (ARMS2) and ApoE are combined with environmental risk factors¹⁷. Due to the complex nature of genetic risk factors in AMD, there is no single candidate gene for its gene therapy. Thus, the current gene therapy in AMD is focused on gene transfer to express anti-angiogenic proteins targeting choroidal neovascularization in wet AMD¹⁸. There is no candidate gene yet for treating dry AMD with a gene therapy. Recently, we suggested that intracellular amyloid beta accumulation could be related to dry AMD features in transgenic mice¹⁹. In addition, subretinally injected amyloid beta was taken up by RPE in mice (data not published). We can cleave intracellular amyloid beta with viral protease²⁰. Thus, we have further studied the gene therapy in dry AMD with potential targets.

In conclusion, gene therapy in ocular disease is more promising than in any other organ. In addition, subretinal injection is a fundamental technique for gene therapy in retinal diseases, especially in relation to photoreceptor cells and RPE. Thus, we hope that this technique becomes more widely used and can contribute to the advancement of gene therapy not only in monogenic inherited retinopathy but also in age-related macular degeneration.