Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models

Summary

In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes.

Abstract

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SCJ) injections offer advantages over other intraocular administration routes as they are simple, safe, and usually performed in an outpatient setting. SCJ injections in small animals usually require the assistance of an operating microscope due to the size of the eye. Previous work has demonstrated that SCJ injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases.

Herein, a detailed protocol is presented for SCJ injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

Introduction

Ocular diseases encompass a broad range of both genetic and acquired disorders. In 2015, an estimated 36 million people were legally blind worldwide, and over 1 billion people suffer from at least some level of visual impairment, highlighting the need to scale up alleviation efforts at all levels¹. The main methods for delivering ocular medications include both topical and local administration, such as eye drops or subconjunctival (SCJ), intracameral, intravitreal, and subretinal injections. Although noninvasive topical therapy is the most common delivery method for ophthalmic drugs and is extensively used for many anterior segment disorders, the presence of corneal anatomical barriers presents a challenge for the bioavailability, biodistribution, and efficacy of topically administrated substances, suggesting that it may not be the best candidate treatment route for many diseases of the inner eye. Local injection into the specific ocular compartment affected by the disease is likely to be a more effective and targeted drug delivery approach². However, adverse effects resulting from repeated injections can complicate administration strategies. Ideally, a therapy should maintain long-term therapeutic efficacy following a single administration. Thus, gene therapy is a promising option for minimizing the number of required injections and providing sustained transgene expression for the treatment of ocular disease^3,4.

Numerous viral and nonviral vectors are available for gene therapy; however, AAV vectors are of high interest due to their excellent safety profile. AAV is a small, single-stranded, non-enveloped DNA virus that was initially discovered as a contaminant of an adenovirus preparation in 1965 by Atchison et al.^5,6 AAV was subsequently engineered as an efficient viral vector for gene delivery in the 1980s and has become the gene therapy vector of choice for many diseases, including ocular disorders, over the last few decades. The most notable of these is the first commercially available gene therapy drug, voretigene neparvovec, which was approved by the United States Food and Drug Administration to treat Leber's Congenital Amaurosis, a rare posterior eye disease. Although voretigene neparvovec has successfully overcome barriers to clinical development, challenges remain for the commercialization of additional ocular gene therapies. For example, voretigene neparvovec is administered to patients who retain viable retinal cells via subretinal injection. Thus, patients with more advanced forms of the disease who lack viable retinal cells are not eligible for treatment, as it would provide no clinical benefit. In addition, known complications associated with the subretinal injection procedure were observed, including eye inflammation, cataracts, retinal tearing, maculopathy, and pain^7,8. Other concerns related to this procedure include the possibility of hemorrhage, retinal detachment, endophthalmitis, and revocation of the ocular immune privileged status through eye tissue destruction^9,10,11,12. Thus, efforts to explore less invasive gene delivery routes such as SCJ injection have become increasingly important^{13,14,15,16,17}.

The conjunctiva is a thin membrane containing 3-5 layers of cells and connecting the anterior eye to the interior eyelid. SCJ injections are used clinically for ophthalmic drug delivery to both the anterior and/or posterior segments of the eye for the treatment of ocular diseases such as age-related macular degeneration, glaucoma, retinitis, and posterior uveitis^18,19. They are relatively simple to perform, employed routinely for ophthalmic drug delivery in an outpatient setting²⁰, somewhat painless, do not compromise ocular immune privilege, and allow administered drugs to spread through a large periorbital region that encompasses the optic nerve. Hence, SCJ injections are an attractive route of administration for AAV gene therapy applications. Natural AAV serotypes administered via SCJ injection in mice have previously been characterized for safety, transduction efficiency, serum immunogenicity, biodistribution, and tissue specificity^13,16,21. These data demonstrated that gene delivery to individual ocular tissues via SCJ administration is a formal possibility.

This paper describes a simple and adaptable protocol for SCJ injection to deliver AAV vectors in a mouse model. To ensure the reproducibility of this approach, an injection system consisting of a stereomicroscope, a programmable infusion/withdrawal syringe pump (which allows consistent and precise injection speed and pressure), and a gastight removable syringe coupled with microinjection needles is described. This system is adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. In addition, a fluorescein dye is often utilized to allow for visualization of the AAV injection site. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. Finally, common validation techniques for confirmation of AAV delivery to the desired tissues will be briefly discussed.

Protocol

All animal procedures were performed in accordance with the regulations of the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill. The use of AAV vectors is a Biosafety Level 1 biohazard risk. Wear proper personal protective equipment, including a lab coat, gloves, and goggles when handling AAV. For the experiment described herein, a recombinant AAV vector packaged with the serotype 8 capsid and encoding a generic ubiquitous cytomegalovirus (CMV) promoter controlling the expres…

Representative Results

Solution injected into the subconjunctival space presents as a bleb depending on the injection volume. In this experiment, 7 µL of AAV (7 × 109 viral genomes (vg)/eye) mixed with fluorescein at a final concentration of 0.1% was injected with a 36 G needle under a stereomicroscope, and the injection speed/pressure was held constant using a programmable syringe pump at 1 µL/s. A bleb can appear upon injection (arrow). A microscopic view of AAV vector administration to the mu…

Discussion

AAV-mediated gene therapy holds great potential for the treatment of ocular diseases. Current ocular gene therapy relies on two major local administration routes, intravitreal and subretinal injections. Unfortunately, both routes are invasive and can cause serious complications, including retinal detachment, cataract formation, and endophthalmitis. Thus, the investigation of relatively less invasive routes, such as SCJ injection, is of great interest.

Although this technique is relatively stra…

Materials

36 G NanoFil Needles	World Precision Instruments	NF36BV-2
AAV vector	University of North Carolina at Chapel Hill	/
Acepromazine	Henry Schein	NDC 11695-0079-8
anti-GFP antibody	AVES labs Inc.
Digital camera	Cannon	Cannon EOS T5i
DNA/RNA extraction kit	Qiagen	80204
Forceps	Fine Science Tools	F6521
Hamilton syringe	Hamilton	7654-01
India ink	StatLab	NC9903975
Ketamine hydrochloride injection solution	Henry Schein	NDC 0409-2051-05
Moisture-resistant film	Parafilm	807-6
Polyethylene tubing	Becton Dickinson and Company	427401
Proparacaine 0.1%	Bausch Health US	NDC 24208-730-06
Rebound tonometer	Tonovet	/
Sodium fluorescein solution	Sigma-Aldich	46960
Standard Infuse/Withdraw Pump 11 Pico Plus Elite Programmable Syringe Pump	Harvard Bioscience	70-4504
Stereo microscopye	Leica	Mz6
Tetracaine Hydrochloride Ophthalmic Solution 0.5%	Bausch and Lomb	Rx only
Topical ointment	GenTeal	NDC 0078-0429-47
Xylazine	Akorn	NDC 59399-110-20
Zone-Quick Phenol Red Thread Box 100 Threads	ZONE-QUICK	PO6448