The authors thank the Vector Core at the University of North Carolina for providing the scAAV8-GFP vectors used in this study, the CGIBD Histology Core, and the laboratory of Dr. Brian C. Gilger for their assistance with the clinical assessment aspects of this study. This study was supported by the Pfizer-NC Biotech Distinguished Postdoctoral Fellowship and a Career Development Award from the American Society of Gene &#38; Cell Therapy and the Cystic Fibrosis Foundation.&#160;The content is solely the responsibility of the authors and does not necessarily represent the official views of the American Society of Gene &#38; Cell Therapy or the Cystic Fibrosis Foundation.

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...

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P., Carter, R., Sabliov, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+polymeric+nanoparticles+in+the+eye:+implications+in+ocular+disease+therapy.">Distribution of polymeric nanoparticles in the eye: implications in ocular disease therapy.</a> Journal of Nanobiotechnology. 19 (1), 10 (2021).</li><li>Petit, L., Khanna, H., Punzo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+gene+therapy+for+diseases+of+the+eye.">Advances in gene therapy for diseases of the eye.</a> Humam Gene Therapy. 27 (8), 563-579 (2016).</li><li>Russell, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+safety+of+voretigene+neparvovec+(AAV2-hRPE65v2)+in+patients+with+RPE65-mediated+inherited+retinal+dystrophy:+a+randomised,+controlled,+open-label,+phase+3+trial.">Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial.</a> Lancet. 390 (10097), 849-860 (2017).</li><li>Atchison, R. W., Casto, B. C., Hammon, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-associated+defective+virus+particles.">Adenovirus-associated defective virus particles.</a> Science. 149 (3685), 754-756 (1965).</li><li>Atchison, R. W., Casto, B. C., Hammon, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopy+of+adenovirus-associated+virus+(AAV)+in+cell+cultures.">Electron microscopy of adenovirus-associated virus (AAV) in cell cultures.</a> Virology. 29 (2), 353-357 (1966).</li><li>Peng, Y., Tang, L., Zhou, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subretinal+injection:+a+review+on+the+novel+route+of+therapeutic+delivery+for+vitreoretinal+diseases.">Subretinal injection: a review on the novel route of therapeutic delivery for vitreoretinal diseases.</a> Ophthalmic Research. 58 (4), 217-226 (2017).</li><li>Gaudana, R., Jwala, J., Boddu, S. H., Mitra, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+perspectives+in+ocular+drug+delivery.">Recent perspectives in ocular drug delivery.</a> Pharmacological Research. 26 (5), 1197-1216 (2009).</li><li>Amado, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+efficacy+of+subretinal+readministration+of+a+viral+vector+in+large+animals+to+treat+congenital+blindness.">Safety and efficacy of subretinal readministration of a viral vector in large animals to treat congenital blindness.</a> Science Translational Medicine. 2 (21), (2010).</li><li>Li, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraocular+route+of+AAV2+vector+administration+defines+humoral+immune+response+and+therapeutic+potential.">Intraocular route of AAV2 vector administration defines humoral immune response and therapeutic potential.</a> Molecular Vision. 14, 1760-1769 (2008).</li><li>Ausayakhun, S., Yuvaves, P., Ngamtiphakom, S., Prasitsilp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+cytomegalovirus+retinitis+in+AIDS+patients+with+intravitreal+ganciclovir.">Treatment of cytomegalovirus retinitis in AIDS patients with intravitreal ganciclovir.</a> Journal of Medical Association of Thailand. 88, 15-20 (2005).</li><li>Miyadera, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrastromal+gene+therapy+prevents+and+reverses+advanced+corneal+clouding+in+a+canine+model+of+mucopolysaccharidosis+I.">Intrastromal gene therapy prevents and reverses advanced corneal clouding in a canine model of mucopolysaccharidosis I.</a> Molecular Therapy. 28 (6), 1455-1463 (2020).</li><li>Song, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotype+survey+of+AAV+gene+delivery+via+subconjunctival+injection+in+mice.">Serotype survey of AAV gene delivery via subconjunctival injection in mice.</a> Gene Therapy. 25 (6), 402-414 (2018).</li><li>Cheng, H. C., Yeh, S. I., Tsao, Y. P., Kuo, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subconjunctival+injection+of+recombinant+AAV-angiostatin+ameliorates+alkali+burn+induced+corneal+angiogenesis.">Subconjunctival injection of recombinant AAV-angiostatin ameliorates alkali burn induced corneal angiogenesis.</a> Molecular Vision. 13, 2344-2352 (2007).</li><li>Veneziale, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCH+412499:+biodistribution+and+safety+of+an+adenovirus+containing+P21(WAF-1/CIP-1)+following+subconjunctival+injection+in+Cynomolgus+monkeys.">SCH 412499: biodistribution and safety of an adenovirus containing P21(WAF-1/CIP-1) following subconjunctival injection in Cynomolgus monkeys.</a> Cutaneous and Ocular Toxicology. 26 (2), 83-105 (2007).</li><li>Liu, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+by+subconjunctival+injection+of+adenovirus+in+rats:+a+study+of+local+distribution,+transgene+duration+and+safety.">Gene delivery by subconjunctival injection of adenovirus in rats: a study of local distribution, transgene duration and safety.</a> PLoS One. 10 (12), 0143956 (2015).</li><li>Igarashi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+comparison+of+administration+routes+for+AAV8-mediated+ocular+gene+therapy.">Direct comparison of administration routes for AAV8-mediated ocular gene therapy.</a> Current Eye Research. 38 (5), 569-577 (2013).</li><li>Gaudana, R., Ananthula, H. K., Parenky, A., Mitra, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+drug+delivery.">Ocular drug delivery.</a> AAPS Journal. 12 (3), 348-360 (2010).</li><li>Short, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+evaluation+of+ocular+drug+delivery+formulations:+techniques+and+practical+considerations.">Safety evaluation of ocular drug delivery formulations: techniques and practical considerations.</a> Toxicologic Pathology. 36 (1), 49-62 (2008).</li><li>Stevens, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administering+a+subconjunctival+injection.">Administering a subconjunctival injection.</a> Community Eye Health. 22 (69), 15 (2009).</li><li>Song, L., Bower, J. J., Hirsch, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+administration+of+adeno-associated+virus+vectors+for+corneal+gene+delivery.">Preparation and administration of adeno-associated virus vectors for corneal gene delivery.</a> Methods in Molecular Biology. 2145, 77-102 (2020).</li><li>de Vries, V. A., Bassil, F. L., Ramdas, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+intravitreal+injections+on+intraocular+pressure+and+retinal+nerve+fiber+layer:+a+systematic+review+and+meta-analysis.">The effects of intravitreal injections on intraocular pressure and retinal nerve fiber layer: a systematic review and meta-analysis.</a> Scientific Reports. 10 (1), 13248 (2020).</li><li>Hartman, R. R., Kompella, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravitreal,+subretinal,+and+suprachoroidal+injections:+evolution+of+microneedles+for+drug+delivery.">Intravitreal, subretinal, and suprachoroidal injections: evolution of microneedles for drug delivery.</a> Journal of Ocular Pharmacology and Theraputics. 34 (1-2), 141-153 (2018).</li><li>Nuzzi, R., Scalabrin, S., Becco, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+intraocular+pressure+spikes+due+to+intravitreal+bevacizumab+injections+by+scleral+indentation+with+cotton+swab+or+digital+ocular+massage:+innovative+techniques+compared.">Reduction of intraocular pressure spikes due to intravitreal bevacizumab injections by scleral indentation with cotton swab or digital ocular massage: innovative techniques compared.</a> Clinical Ophthalmology. 14, 2533-2541 (2020).</li><li>Crabtree, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV-mediated+expression+of+HLA-G1/5+reduces+severity+of+experimental+autoimmune+uveitis.">AAV-mediated expression of HLA-G1/5 reduces severity of experimental autoimmune uveitis.</a> Scientific Reports. 9 (1), 19864 (2019).</li><li>Song, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+to+human+limbal+stem+cells+using+viral+vectors.">Gene delivery to human limbal stem cells using viral vectors.</a> Human Gene Therapy. 30 (11), 1336-1348 (2019).</li><li>Reichel, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+model+of+conjunctival+scarring+in+the+mouse+eye.">New model of conjunctival scarring in the mouse eye.</a> British Journal of Ophthalmology. 82 (9), 1072-1077 (1998).</li><li>Barnard, A. R., Rudenko, A. N., MacLaren, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vector+shedding+and+immunogenicity+sampling+for+retinal+gene+therapy.">Vector shedding and immunogenicity sampling for retinal gene therapy.</a> Methods in Molecular Biology. 1715, 359-371 (2018).</li><li>Gilger, B. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fixed-depth+microneedle+enhances+reproducibility+and+safety+for+corneal+gene+therapy.">A fixed-depth microneedle enhances reproducibility and safety for corneal gene therapy.</a> Cornea. 39 (3), 362-369 (2020).</li><li>Cheruvu, N. P., Kompella, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bovine+and+porcine+transscleral+solute+transport:+influence+of+lipophilicity+and+the+Choroid-Bruch's+layer.">Bovine and porcine transscleral solute transport: influence of lipophilicity and the Choroid-Bruch's layer.</a> Investigative Ophthalmology and Visual Science. 47 (10), 4513-4522 (2006).</li></ol>

The authors have no conflicts of interest to disclose.

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...

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 disease3,4.
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&#39;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 pain7,8. 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 destruction9,10,11,12. Thus, efforts to explore less invasive gene delivery routes such as SCJ injection have become increasingly important13,14,15,16,17.
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 uveitis18,19. 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&#160;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 specificity13,16,21. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>36 G NanoFil Needles</td>
			<td>World Precision Instruments</td>
			<td>NF36BV-2</td>
			<td></td>
		</tr>
		<tr>
			<td>AAV vector</td>
			<td>&#160; University of North Carolina at Chapel Hill</td>
			<td>&#160; /</td>
			<td></td>
		</tr>
		<tr>
			<td>Acepromazine</td>
			<td>Henry Schein</td>
			<td>NDC 11695-0079-8</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GFP antibody</td>
			<td>AVES labs Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>Cannon</td>
			<td>Cannon EOS T5i</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA/RNA extraction kit</td>
			<td>Qiagen</td>
			<td>80204</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Forceps</td>
			<td>Fine Science Tools</td>
			<td>F6521</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton</td>
			<td>7654-01</td>
			<td></td>
		</tr>
		<tr>
			<td>India ink</td>
			<td>StatLab</td>
			<td>NC9903975</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride injection solution</td>
			<td>Henry Schein</td>
			<td>NDC 0409-2051-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Moisture-resistant film</td>
			<td>Parafilm</td>
			<td>807-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>Becton Dickinson and Company</td>
			<td>427401</td>
			<td></td>
		</tr>
		<tr>
			<td>Proparacaine 0.1%</td>
			<td>Bausch Health US</td>
			<td>NDC 24208-730-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Rebound tonometer</td>
			<td>Tonovet</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium fluorescein solution</td>
			<td>Sigma-Aldich</td>
			<td>46960</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Infuse/Withdraw Pump 11 Pico Plus Elite Programmable Syringe Pump</td>
			<td>Harvard Bioscience</td>
			<td>70-4504</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscopye</td>
			<td>Leica</td>
			<td>Mz6</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracaine Hydrochloride Ophthalmic Solution 0.5%</td>
			<td>Bausch and Lomb</td>
			<td>Rx only</td>
			<td></td>
		</tr>
		<tr>
			<td>Topical ointment</td>
			<td>GenTeal</td>
			<td>NDC 0078-0429-47</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Akorn</td>
			<td>NDC 59399-110-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Zone-Quick Phenol Red Thread Box 100 Threads</td>
			<td>ZONE-QUICK</td>
			<td>PO6448</td>
			<td></td>
		</tr>
	</tbody>
</table>

subconjunctival administration of adeno-associated virus vectors in small animal models

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.

Solution injected into the subconjunctival space presents as a bleb depending on the injection volume. 
In this experiment, 7 &#181;L of AAV (7 &#215; 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 &#181;L/s. A bleb can appear upon injection (arrow). A microscopic view of AAV vector administration to the mu...

Watch this Scientific Journal Video about Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models at JoVE.com

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

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.

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ...

This protocol offers a method of gene therapy delivery to multiple ocular tissues in a safe, easy, and effective manner. It is also adaptable for intrastromal and subretinal injections in small animals. Use of the subconjunctival route of injection is minimal invasive.It poses fewer risks and holds great promise for AV delivery to treat multiple ocular diseases. With over 1 billion people worldwide suffering from visual impairment, using a safe and easy method for drug delivery offers broad applicability for the treatment of many different causes of blindness. The gene delivery methods described in this protocol are of particular interest to those who are developing potential intervention strategies and practicing ophthalmologists, who will eventually deliver these drugs to patients.The biggest challenge for someone who is performing this protocol for the first time is likely the insertion of the needle into the concept conjunctival space. To begin, assemble the injection system by placing a stereo microscope and a syringe pump in a bio-safety cabinet. Next, cut the polyethylene tubing to a length of approximately 50 centimeters.Insert the end of a 36-gauge needle into one end of the tubing. Then fill a disposable 3-milliliter syringe with sterile water and insert it into the side of the tube opposite the needle. Flush the tubing three times, alternating between sterile water and 70%alcohol to disinfect the tubing and ensure that there are no leaks, clogs, or damage throughout the tubing.Using the disposable 3-milliliter syringe, fill the tubing with sterile water and leave the tubing attached to the disposable syringe. Then place a piece of parafoam on the surface of the bio-safety cabinet, and add a pool of sterile water to it. Submerge the portion of the tubing connected to the needle into the pool of sterile water.Next, pull the disposable syringe out from the tube opening to prevent any air from entering the tubing system upon removal of the syringe. Then fill a 10-microliter Hamilton syringe with sterile water. Next, connect the Hamilton syringe needle to the open end of the tubing by submerging the needle tip of the Hamilton syringe into the pool of sterile water on the para film.Press and hold the fast reverse button on the pump screen to move the pusher blocks to the approximate length of the syringe. Next, unscrew the bracket clamping knobs to loosen the the retaining brackets on the pusher and the syringe holder blocks. Subsequently, load the Hamilton syringe onto the syringe holder block and secure the syringe following the manufacturer's instructions.To adjust the parameters in the pump setting screen, press the Force"button and set the force level at 30%Then accept the changes to go back to the setting screen. Press the Quick Start"button, select Method, and then select the Infuse Only"option. For the syringe option, select Hamilton 1700, then click on Glass"and select 10 microliters.Later, select the infusion rate and the injection volume. Eject the water from the Hamilton syringe while leaving the tubing and injection needle full of water by pressing the Infuse"button. Next, withdraw the virus by placing the injection needle into an aliquot of the virus stock.Then slightly pull back on the Hamilton syringe by pressing the reverse button to introduce a small air bubble in the tubing. After anesthetizing the animal, grab the conjunctiva with forceps. Then insert the needle into the conjunctiva until the bevel is entirely covered by the conjunctival membrane, and lay the needle against the globe.Simultaneously, start the injection by pressing the Start"button using the foot switch. To examine the distribution of the AAV vector, the ocular and surrounding tissues were stained with hematoxylin and eosin. The representative sagittal sections demonstrated that the dispersion of India ink occurred mainly adjacent to the extraocular muscles in the outer surface of the sclera in the periocular loose connective tissues.To confirm transduction of self-complimentary AAV 8 at eight weeks post-injection, green fluorescence protein abundance in the cross-sections was examined by immunofluorescence staining. The results revealed that AAV 8 vectors efficiently transduced the periocular muscles posterior to the eye and the cornea. To analyze the vector bio-distribution, vector genome copy numbers and distinct eye compartments in other organs, including liver and heart, were examined by quantitative polymerase chain reaction using primers and probes specific to the transgene.Expression of the transgene was confirmed by quantitative reverse transcription polymerase chain reaction, which suggested that the subconjunctival injection of AAV 8 results in transgene expression in the eyelid, conjunctiva, cornea, and optic nerve. The air bubble will serve as a barrier between the water in the tube and the therapeutic drug. This technique allows other researchers to explore the use of the subconjunctival route of administration for both ophthalmic solutions and ocular gene therapies for multiple diseases.

subconjunctival-administration-adeno-associated-virus-vectors-small

Research

JoVE Journal

Medicine

2.6K Views.  University of North Carolina at Chapel Hill. Questo protocollo offre un metodo di somministrazione della terapia genica a più tessuti oculari in modo sicuro, facile ed efficace. È anche adattabile per iniezioni intrastromali e sottoretiniche in piccoli animali. L'uso della via di iniezione subcongiuntivale è minimamente invasivo.Pone meno rischi e rappresenta una grande promessa per la somministrazione AV per il trattamento di più malattie oculari. Con oltre 1 miliardo di persone in tutto il mondo che soffrono di disabilità ...