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

Summary

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

Abstract

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Introduction

In ophthalmology, gene therapy has emerged as the treatment modality in monogenic inherited retinopathies. There are inherited retinopathies associated with genes in retinal pigment epithelium (RPE) including Leber congenital amurosis^1,2, retinitis pigmentosa³, and choroideremia⁴. The research field of gene therapy is expanding in both preclinical studies and clinical trials using viral vectors such as adeno-associated virus (AAV), lentivirus (LV) and adenovirus (Ad)⁵. Different viral vectors have different tropism in the retina. For a safe and effective gene therapy, viral vectors should be carefu....

Protocol

All experiments on animals were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and the guidelines and regulations set forth by the Seoul National University Institutional Animal Care and Use Committee and Seoul National University Hospital Biosafety Committee.

1. Preparing Injection Kit and Viral Vectors

1. Prepare the microliter syringe equipped with a 33 G blunt needle sterilized using ethylene oxide gas. Dilute the viral vectors in PBS for the adequate titer in the micro tube (i.e., 1 x 10⁶ TU/µl). Flush....

Results

To evaluate the efficacy of the subretinal injection on viral gene transduction by this protocol, we used commercially available LV vectors with CMV promoter expressing both GFP and RFP for the indicator. Eyes were enucleated after the appropriate time period according to the research purpose. For the representative results, eyes were enucleated 10 weeks and 20 weeks after subretinal injection. After complete removal of the retina using the method described above, the flat mount of RPE/choroid/sclera complex was evaluate.......

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
TWEEZERS DUMONT #5 11cm DUMOSTAR 0.1 x 0.06 mm TIPS	WPI	500233
VANNAS Scissors S/S, 105 mm	WPI	555583S
33 G Blunt needle	WPI	NF33BL-2
NanoFil Syringe, 10 microliter	WPI	NANOFIL
RPE-KIT	WPI	RPE-KIT	For easy one hand injection
30 G x 1/2 (0.3 mm x 13 mm) BD PrecisionGlideTM Needle	BD	305107	Initial puncture for subretinal injection
Microscope Cover Glasses (No. 1 3 mm diameter)	Warner Instruments	64-0720  (CS-3R)
Leica operating microscope	Leica	LM M80
Fluoresecein microscope	Nikon	Eclipse 80i
Lentivirus	Thermo scientific	TMO.LV-Ctr	Used to dilute vectors
PBS	Gibco	10010-015	Used to dilute vectors
Troperin (Phenylephrin 0.5% - Tropicamide 0.5%)	Hanmi		For dilation
Proparacaine Hydrochloride Ophthalmic Solution USP, 0.5% (Sterile)	Bausch&Lomb		For topical anesthesia
Healon GV OVD	Abbott Medical Optics Inc.
Zoletil 50 (tiletamine hypochloride and zolazepam hypochloride)	Virbac		For general anesthesia
Rompun injection (Xylazine HCl)	Bayer		For general anesthesia