Name: intravitreal injections in the ovine eye
Uploaded: 2022-07-05T14:00:00
Description: Watch this Scientific Journal Video about Intravitreal Injections in the Ovine Eye at JoVE.com

The authors would like to acknowledge Dr. Steve Heap (BVSc, CertVOphthal) for his assistance in establishing this protocol and performing the injections described by Murray et&#160;al.15. The authors also acknowledge funding from CureKids New Zealand, the Canterbury Medical Research Foundation, Neurogene Inc, and the Batten Disease Support and Research Association.

All experimental protocols were approved by the Lincoln University Animal Ethics committee and are in line with the US National Institutes of Health guidelines for the care and use of animals in research and the New Zealand Animal Welfare Act (1999). Borderdale sheep were diagnosed at birth14 and maintained at Lincoln University research farms. Three 3-month-old homozygous (CLN5-/-) ewes received a single IVT injection to the left eye, with the untreated right eye acting as an internal ...

<ol>
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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=Longitudinal+in+vivo+monitoring+of+the+CNS+demonstrates+the+efficacy+of+gene+therapy+in+a+sheep+model+of+CLN5+Batten+disease.">Longitudinal in vivo monitoring of the CNS demonstrates the efficacy of gene therapy in a sheep model of CLN5 Batten disease.</a> Molecular Therapy. 26 (10), 2366-2378 (2018).</li><li>Murray, S. J., Mitchell, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+history+of+retinal+degeneration+in+ovine+models+of+CLN5+and+CLN6+neuronal+ceroid+lipofuscinoses.">Natural history of retinal degeneration in ovine models of CLN5 and CLN6 neuronal ceroid lipofuscinoses.</a> Scientific Reports. 12 (1), 3670 (2022).</li><li>Russell, K. N., Mitchell, N. L., Wellby, M. P., Barrell, G. K., Palmer, D. 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J., 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=Intravitreal+gene+therapy+protects+against+retinal+dysfunction+and+degeneration+in+sheep+with+CLN5+Batten+disease.">Intravitreal gene therapy protects against retinal dysfunction and degeneration in sheep with CLN5 Batten disease.</a> Experimental Eye Research. 207, 108600 (2021).</li><li>Ross, M., 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=Outer+retinal+transduction+by+AAV2-7m8+following+intravitreal+injection+in+a+sheep+model+of+CNGA3+achromatopsia.">Outer retinal transduction by AAV2-7m8 following intravitreal injection in a sheep model of CNGA3 achromatopsia.</a> Gene Therapy. , (2021).</li><li>Boyd, R. 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A., 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=Antibody+neutralization+poses+a+barrier+to+intravitreal+adeno-associated+viral+vector+gene+delivery+to+non-human+primates.">Antibody neutralization poses a barrier to intravitreal adeno-associated viral vector gene delivery to non-human primates.</a> Gene Therapy. 22 (2), 116-126 (2015).</li><li>Whitehead, M., Osborne, A., Yu-Wai-Man, P., Martin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humoral+immune+responses+to+AAV+gene+therapy+in+the+ocular+compartment.">Humoral immune responses to AAV gene therapy in the ocular compartment.</a> Biological Reviews. 96 (4), 1616-1644 (2021).</li><li>Yun, C., Oh, J., Hwang, S. -. Y., Kim, S. -. 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L., Mueller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraocular+pressure+and+injection+forces+during+intravitreal+injection+into+enucleated+porcine+eyes.">Intraocular pressure and injection forces during intravitreal injection into enucleated porcine eyes.</a> European Journal of Pharmaceutics and Biopharmaceutics. 166, 87-93 (2021).</li><li>Ross, M., Ofri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+retinal+gene+therapy:+Evolving+from+subretinal+to+intravitreal+vector+delivery.">The future of retinal gene therapy: Evolving from subretinal to intravitreal vector delivery.</a> Neural Regeneration Research. 16 (9), 1751-1759 (2021).</li><li>Henein, 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=Hydrodynamics+of+intravitreal+injections+into+liquid+vitreous+substitutes.">Hydrodynamics of intravitreal injections into liquid vitreous substitutes.</a> Pharmaceutics. 11 (8), 371 (2019).</li><li>Park, I., Park, H. 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Intravitreal injections are one of the most common surgical procedures in human ophthalmology and have proven effective in delivering AAV-mediated gene therapies to the retina of sheep. We had previously demonstrated the efficacy of AAV9.CLN5 gene therapy delivered intravitreally in attenuating retinal dysfunction and degeneration in sheep with CLN5 NCL15. It is hoped that the translation of this route of administration to human NCL patients will also prove beneficial.
...

Several methods can be used to deliver therapeutic agents to the retina, including intravitreal (IVT), subretinal, suprachoroidal, periocular, or topical administration. Each route of administration involves overcoming barriers such as the blood-retina barrier or the inner and outer limiting membranes and has varying rates of efficacy depending on the drug being delivered and the specific retinal target1,2.
IVT drug delivery involves an injection into the vitreous humor of the eye, a gelatinous substance that occupies the posterior chamber of the eye. The primary function of the vitreous humor is to maintain the shape of the eye globe and keep ocular tissues, such as the lens and retina, in place. The vitreous humor is composed largely of water, with small amounts of collagen, hyaluronic acid, and other noncollagenous proteins3. IVT injection is a simple and common procedure used routinely to treat a wide&#160;range of ocular conditions, including age-related macular degeneration, diabetic macular edema, diabetic retinopathy, retinal vein occlusion, and several inherited retinal dystrophies4,5.
Neuronal ceroid lipofuscinoses (NCL; Batten disease) are a group of fatal lysosomal storage diseases that cause severe degeneration of the brain and retina. There are currently 13 known variants of NCL resulting from mutations in different genes (CLN1-8, CLN10-14) that predominantly affect children but have varying ages of onset and disease severity6. The NCLs share common progressive symptoms, including cognitive and motor decline, seizures, and loss of vision. There is no cure for NCL; however, brain-directed enzyme-replacement therapy is currently in clinical trials for CLN2 disease7,8, and AAV-mediated gene therapy has shown great promise in preclinical studies, with a clinical trial for CLN5 gene therapy expected to begin in 20229,10.
Many other species develop naturally occurring forms of NCL, including cats, dogs, sheep, and cows. Two ovine models of NCL are currently under active study in New Zealand: a CLN5 disease model in Borderdale sheep and a CLN6 disease model in South Hampshire sheep. Affected sheep exhibit many of the clinical and pathological features of the human disease, including retinal atrophy and loss of vision10,11. Although brain-directed CLN5 gene therapy in sheep with CLN5 disease can prevent or halt brain atrophy and clinical decline, the treated sheep do still lose their vision9. This highlighted the need to treat the retina to preserve vision and maintain a better quality of life, leading to the establishment of a protocol for ocular gene therapy in sheep.
The sheep eye represents a good model of the human eye due to its similarity in eye globe dimensions, vitreous volume, and retinal structure10,12,13. This paper details the surgical protocol for IVT delivery of a small volume (&#8804;100 &#181;L) of therapeutic viral vector to the ovine eye.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL low dead-space safety syringe with permanently attached 0.5 inch needle</td>
			<td>Fisher Scientific, Auckland, New Zealand</td>
			<td>05-561-28</td>
			<td>Covidien Monoject Tuberculin Safety syringe or similar</td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Sigma Aldrich</td>
			<td>HS4323</td>
			<td>Autoclave tubes to sterilise prior to use</td>
		</tr>
		<tr>
			<td>Anesthesia machine with gas bench and monitor&#160;</td>
			<td>Hyvet Anesthesia, Christchurch, New Zealand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic eye drops&#160;</td>
			<td>Teva Pharma Ltd, Auckland, New Zealand</td>
			<td></td>
			<td>Commercial name: Chlorafast (0.5% chloramphenicol)</td>
		</tr>
		<tr>
			<td>BrightMount plus anti-fade mounting medium</td>
			<td>Abcam, Cambridge, United Kingdom</td>
			<td>ab103748</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#8242; ,6-diamidino-2-phenylindole dihydrochloride)</td>
			<td>Sigma Aldrich, St. Louis, Missouri, United States</td>
			<td>10236276001</td>
			<td></td>
		</tr>
		<tr>
			<td>Diazepam sedative</td>
			<td>Ilium, Troy Laboratories Pty Ltd, Tauranga, New Zealand</td>
			<td></td>
			<td>5 mg/mL</td>
		</tr>
		<tr>
			<td>Endotracheal tubes</td>
			<td>Flexicare Medical Ltd, Mountain Ash, United Kingdom</td>
			<td></td>
			<td>Standard, cuffed. Sizes 7, 7.5, or 8 depending on sheep size</td>
		</tr>
		<tr>
			<td>Eye speculum</td>
			<td>Capes Medical Ltd, Tauranga, New Zealand</td>
			<td>KP151/14</td>
			<td>Nopa Barraquer-Colibri (10 mm)</td>
		</tr>
		<tr>
			<td>Fenestrated surgical drape</td>
			<td>Amtech Medical Ltd, Whanganui, New Zealand</td>
			<td>DI583</td>
			<td>Or similar&#160;</td>
		</tr>
		<tr>
			<td>Filter Tips</td>
			<td>Interlab, Auckland, New Zealand</td>
			<td></td>
			<td>10, 200, and 1,000 &#181;L&#160;</td>
		</tr>
		<tr>
			<td>Formaldehyde solution (37%)</td>
			<td>Fisher Scientific, Auckland, New Zealand</td>
			<td>AJA809-2.5PL</td>
			<td>Make up to 10% in distilled water with 0.9% NaCl</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa Fluor 594</td>
			<td>Invitrogen Carlsbad, CA, USA</td>
			<td>&#160;A-11012</td>
			<td>Use at a dilution of 1:500</td>
		</tr>
		<tr>
			<td>Isoflurane anesthetic</td>
			<td>Attane, Bayer Animal Health, Auckland, New Zealand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine HCl anesthetic/analgesic</td>
			<td>PhoenixPharm Distributors Ltd, Auckland, New Zealand</td>
			<td></td>
			<td>100 mg/mL</td>
		</tr>
		<tr>
			<td>Laryngoscope (veterinary)</td>
			<td>KaWe Medical, Denmark</td>
			<td></td>
			<td>Miller C blade, size 2</td>
		</tr>
		<tr>
			<td>Needles&#160;</td>
			<td>Capes Medical Ltd, Tauranga, New Zealand</td>
			<td>302025</td>
			<td>BD Hypodermic Needles, or similar</td>
		</tr>
		<tr>
			<td>Non-steroidal anti-inflammatory</td>
			<td>Boehringer Ingelheim (NZ) Ltd, Auckland, New Zealand</td>
			<td>49402/008</td>
			<td>Commercial name: Metacam 20 (20 mg/mL meloxicam)</td>
		</tr>
		<tr>
			<td>Non-toothed forceps</td>
			<td>Capes Medical Ltd, Tauranga, New Zealand</td>
			<td>AB864/16</td>
			<td>Or similar&#160;</td>
		</tr>
		<tr>
			<td>Non-toothed hemostat</td>
			<td>Capes Medical Ltd, Tauranga, New Zealand</td>
			<td>AA150/12</td>
			<td>Or similar&#160;</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Thermo Fisher Scientific, Christchurch, New Zealand</td>
			<td>16210072</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen (medical)</td>
			<td>BOC Gas, Christchurch, New Zealand</td>
			<td></td>
			<td>D2 cylinder, gas code 180</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline&#160;</td>
			<td>Thermo Fisher Scientific, Christchurch, New Zealand</td>
			<td>10010023</td>
			<td>Sterile, filtered</td>
		</tr>
		<tr>
			<td>Povidone-Iodine solution</td>
			<td>Capes Medical Ltd, Tauranga, New Zealand</td>
			<td>005835</td>
			<td>Commercial name: Betadine (10% povidone-iodine)</td>
		</tr>
		<tr>
			<td>Rabbit anti-cow glial fibrillary acidic protein (GFAP)</td>
			<td>Dako, Glostrup, Denmark</td>
			<td>Z0334</td>
			<td>Use at a dilution of 1:2,500</td>
		</tr>
		<tr>
			<td>Self-complementary adeno-associated virus serotype 9, containing the chicken beta action (CBh) promoter and codon-optimized ovine CLN5</td>
			<td>University of North Carolina Vector Core, NC, USA.</td>
			<td>scAAV9/CBh-oCLN5opt</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride 0.9% IV Solution</td>
			<td>Capes Medical Ltd, Tauranga, New Zealand</td>
			<td>AHB1322</td>
			<td>Commercial name: Saline solution&#160;</td>
		</tr>
		<tr>
			<td>Subcutaneous antibiotics</td>
			<td>Intervet Schering Plough Animal Health Ltd, Wellington, New Zealand</td>
			<td></td>
			<td>Commercial name: Duplocillin LA (150,000 IU/mL procaine penicillin and 115,000 IU/mL benzathine penicillin)</td>
		</tr>
		<tr>
			<td>Surgical sharp blunt curved scissors&#160;</td>
			<td>Capes Medical Ltd, Tauranga, New Zealand</td>
			<td>SSSHBLC130</td>
			<td></td>
		</tr>
		<tr>
			<td>Terumo Syringe Luer Lock</td>
			<td>Amtech Medical Ltd, Whanganui, New Zealand</td>
			<td>SH159/SH160</td>
			<td>Sterile syringes; 10 mL for drawing up induction drugs, 20 mL for drawing up saline</td>
		</tr>
		<tr>
			<td>Virkon Disinfectant Powder</td>
			<td>EBOS Group Ltd, Christchurch, NZ</td>
			<td>28461115</td>
			<td></td>
		</tr>
	</tbody>
</table>

intravitreal injections in the ovine eye

There are several methods for the delivery of therapeutic agents to the retina, including intravitreal (IVT), subretinal, suprachoroidal, periocular, or topical administration. IVT drug delivery involves an injection into the vitreous humor of the eye, a gelatinous substance that fills the posterior chamber of the eye and maintains the shape of the eye globe. Although the IVT route is less specifically targeted than subretinal delivery, it is much less invasive and is widely used in clinical settings for a range of ocular diseases. 
We previously demonstrated the efficacy of intravitreal delivery of an adeno-associated virus (AAV)-mediated gene therapy product (AAV9.CLN5) in sheep with a naturally occurring CLN5 form of neuronal ceroid lipofuscinosis (NCL). Affected sheep received IVT gene therapy in one eye, with the other untreated eye serving as an internal control. Retinal structure and function were maintained in the treated eye up to 15 months after treatment, while the untreated eye displayed progressively declining function and severe atrophy during postmortem examination. Based on the sheep studies, the CLN5 gene therapy product was cleared as a candidate investigational new drug (IND) by the United States Food and Drug Administration in September 2021. This paper details the surgical protocol for IVT delivery of a therapeutic viral vector to the ovine eye.

The efficacy of IVT delivery of a CLN5 gene therapy vector in attenuating retinal dysfunction and degeneration in sheep with CLN5 NCL has previously been demonstrated by this research group15. Affected sheep received a single 100 &#181;L IVT injection of CLN5 packaged in an AAV serotype 9 (AAV9) vector (AAV9.CLN5) into one eye, with the contralateral eye serving as an untreated internal control. Vision was assessed monthly from the age at injection (3 months) to end-stage disease (18 months). Post...

Watch this Scientific Journal Video about Intravitreal Injections in the Ovine Eye at JoVE.com

Intravitreal Injections in the Ovine Eye

Intravitreal injections were performed in the sheep eye with the aim of delivering viral-mediated gene therapy to the retina.

There are several methods for the delivery of therapeutic agents to the retina, including intravitreal (IVT), subretinal, suprachoroidal, periocular, or ...

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