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Medicine

Intravitreal Injections in the Ovine Eye

Published: July 5, 2022 doi: 10.3791/63823

Abstract

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.

Introduction

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 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 (≤100 µL) of therapeutic viral vector to the ovine eye.

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Protocol

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 control. Electroretinography and pathology data were compared to historical healthy and affected control data. The viral vector used in this study was a self-complementary adeno-associated virus serotype 9, containing the chicken beta action (CBh) promoter and codon-optimized ovine CLN5 (scAAV9/CBh-oCLN5opt). The viral vector was provided by the University of North Carolina Vector Core, NC, USA.

1. Presurgery

  1. Autoclave the surgical kit (Figure 1).
  2. Fast the sheep for 24 h before the surgery.
  3. Record live weights prior to surgery.

Figure 1
Figure 1: Intravitreal surgery kit. Instruments required for IVT surgery include (1) a speculum to hold the eyelids open and (2) a pair of curved-nose forceps to grasp the bulbar conjunctiva and rotate the eye. (3) A straight nose hemostat is also included as an alternative instrument to grip the bulbar conjunctiva and hold the eye in place if it has rolled back into the eye orbit. This kit is autoclaved prior to surgery. Please click here to view a larger version of this figure.

2. Surgical procedure

  1. Restrain the animal and, using electronic clippers, shave the wool from one side of the neck over the jugular vein.
  2. Occlude the jugular vein by applying pressure at the base of the jugular groove and visualize the raised vein.
  3. Draw up the appropriate amount of diazepam (0.3 mg/kg) and ketamine (7.5 mg/kg) into a sterile syringe and attach a sterile 20 G needle. Insert the needle into the jugular vein and gently draw back on the plunger to ensure blood enters the hub and the needle is inside the vein. Once confirmed, induce through intravenous (jugular) administration.
  4. Immediately after induction, place the animal in dorsal recumbency, extend the neck, and hold the tongue up and forward, using a laryngoscope to visualize the larynx. Perform endotracheal intubation by gently inserting an endotracheal tube (size 6.0-9.0 depending on the size of the sheep) between the vocal cords when the animal exhales. Inflate the endotracheal cuff immediately and secure the tube with a tie around the lower jaw. Confirm air flow through the tube.
  5. Transfer the sheep to the surgical table and place it in lateral recumbency.
  6. Immediately connect the endotracheal tube to the hoses of the anesthetic machine for delivery of isoflurane in 100% oxygen. Initially commence with 3%-4% isoflurane and then reduce to 2%-3% for maintenance. Observe the spontaneous ventilation of the sheep.
  7. Monitor heart (pulse) rate, respiratory rate, oxygen saturation, end-tidal CO2 levels, and rectal body temperature throughout the procedure. See Table 1 for physiological values for these parameters in anesthetized sheep (variable, but use as guidance).
  8. Place a large, sterile, square drape on a surgical operating cart, followed by the sterile instruments.
  9. Position a sterile, fenestrated surgical drape over the eye to be injected.
  10. Aseptically disinfect the eye using a sterile 20 mL syringe to irrigate the eye with 10% povidone-iodine solution.
  11. Fit a Nopa Barraquer-Colibri eye speculum (10 mm) to the eyelids to hold the eye open.
  12. Grasp the bulbar conjunctiva on the dorsolateral aspect of the eye with forceps, and rotate the eye globe ventromedially.
Conscious Anesthetized Recommended critical intervention point
Heart rate (beats/min) 50-80 (rest) to 280 (active) 50-80 <50, >100
Respiratory rate (breaths/min) 15-40 (rest) to 350 (overheated)  10-30 <8, >40
Oxygen saturation (mm Hg) 95-100 98-100 <90
End-tidal CO2 (mm Hg) 35-45 35-45 >55
Body temperature (˚C) 38.5-39.5 38.5-39.5 <36, >40

Table 1: Physiological values of parameters to be monitored in anesthetized sheep.

3. Viral preparation

  1. Store AAV vector aliquots at −80 ˚C until use.
  2. On the day of surgery, thaw the required number of vials for IVT delivery on ice.
  3. Immediately prior to administration, vortex the viral vector aliquot and centrifuge at 400 × g for 10 s to collect the contents.
  4. Dilute each viral vector aliquot in sterile-filtered 1x phosphate-buffered saline (PBS) to the desired dose in a final volume of 100 µL. Prepare vector dilutions in a sterile 1.5 mL low-protein-binding microcentrifuge tube using sterile filter pipette tips. Dispose of all consumables that have been in contact with the viral vector in disinfectant solution (see the Table of Materials).
    NOTE: In the original publication15 the dose of the therapeutic agent (AAV9.CLN5) was 1.9 x 1010 viral genomes. The recommended dosage will vary depending on the therapeutic agent being administered; therefore, a dosage has not been included in the standard protocol presented here.
  5. Draw the full 100 µL of the AAV vector preparation into a sterile, low-dead space 1 mL syringe with a permanently attached 28 G x 1/2 in needle for immediate injection. Ensure the length of time from preparation to injection is less than 2 min.

4. Viral administration

  1. Insert the needle approximately 7 mm posterior to the sclera on the lateral aspect of the eye and angled posteriorly to avoid the lens (Figure 2 and Figure 3). Administer the single injection of 100 µL as a bolus as close to the retina as possible without disturbing the retinal surface.
  2. Rinse the eye with approximately 10-15 mL of 10% povidone-iodine solution followed by 10 mL of saline before removal of the speculum and drape.
  3. Turn the sheep over and repeat with the other eye if required.

Figure 2
Figure 2: Ventromedial rotation of the eye globe. (A) Grasp the bulbar conjunctiva with nontoothed forceps and (B) rotate ventromedially (i.e., down and towards the snout) to expose the dorsolateral surface of the eye for injection. Abbreviations: V = ventral, D = dorsal, M = medial, L = lateral. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Injection location and depth. The needle is injected on the dorsolateral aspect of the eye globe, and the full length of the needle shaft (0.5 in/12.7 mm) is inserted into the eye. Note the angle of the needle toward the posterior of the eye to avoid the lens and inject as close to the retina as possible. Please click here to view a larger version of this figure.

5. Postoperative management

  1. On completion of the procedure, stop isoflurane gas inhalation anesthesia, flush the line with 100% oxygen, disconnect the hose from the endotracheal tube, and transfer the sheep to the recovery room.
  2. Place the sheep in sternal recumbency, with legs tucked underneath, and monitor until full recovery. Ensure the animal's mouth is clear of any obstructions.
  3. When the swallowing reflex is observed, partially deflate the cuff of the endotracheal tube and gently remove the tube from the mouth.
  4. Administer an intramuscular nonsteroidal anti-inflammatory into the biceps femoris muscle of the hind limb, subcutaneous antibiotics on the side of the neck or behind the shoulder, and 0.5% chloramphenicol eye drops to the surface of the eye globe.
  5. Provide water and food (lucerne pellets and chaff) once the sheep can stand unassisted.
  6. Administer 0.5% chloramphenicol eye drops 2-3 per day for 7 days post surgery.
  7. Keep the sheep indoors overnight before returning to the outdoor paddock approximately 24 h post surgery.
  8. Record rectal temperatures daily for 3 weeks. Monitor for any changes in pulse or respiratory rate, food consumption, neurobehavior, body temperature, weight, posture, eye health, and clinical signs of ill-health. Seek appropriate veterinary treatment if there are any indications of adverse events.

6. Assessing efficacy in vivo

  1. If the goal of the IVT injection is to preserve vision, monitor efficacy in vivo by methods such as maze testing or electroretinography (ERG) to assess retinal cell function or optical coherence tomography (OCT) to assess retinal structure.
    ​NOTE: These efficacy measures have been well described following IVT gene therapy11,15,16.

7. Postmortem tissue analysis

  1. Perform sheep euthanasia by an approved method at an appropriate endpoint following intravitreal injection surgery.
    NOTE: Suggested euthanasia methods, such as intravenous veterinary euthanasia drugs or a penetrating captive bolt to the cervical spine followed by rapid exsanguination, are detailed elsewhere15,16.
  2. Harvest sheep eye globes using surgical sharp/blunt curved scissors. Cut the lateral and medial canthus to increase the eye socket opening and then systematically cut through the conjunctival folds, connective tissue, muscles, and optic nerve to free the eye globe from the socket.
  3. Immersion-fix intact, enucleated eye globes in 10% formalin for 2 h, followed by postfixation in Bouin's solution for 4 h, making a small (0.5 cm) cut in the sclera to allow sufficient perfusion. Alternatively, immersion-fix the eye globes in Davidson's solution for 48 h.
  4. Process sections of eye tissue via routine paraffin wax embedding and sectioning at 3-5 µm.
    NOTE: Staining procedures for hematoxylin and eosin (H&E) staining and immunohistochemical analysis have been described previously15,16.
  5. Assess the efficacy in postmortem tissue by measures such as total retinal thickness, retinal layer thickness, counts of outer nuclear layer cellular rows, and immunohistochemical staining for retinal cell types, retinal glia, or proteins of interest.
    NOTE: For protocols for these analyses, see previous publications15,16.

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Representative Results

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 µL 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). Postmortem analysis of retinal histology was performed on treated and untreated eyes, as well as age-matched healthy and CLN5-affected controls.

Electroretinography (ERG) analysis demonstrated preserved retinal function in the treated eye, while the untreated eye declined in a similar manner to CLN5-affected animals (Figure 4)15. Retinal histology was near-normalized in the treated eye, with a total retinal thickness comparable to healthy control animals in the central retina. In contrast, the thickness of the untreated retina was comparable to CLN5-affected animals (Figure 5)15. Lysosomal storage, a hallmark pathological feature of NCL, was not observed in the treated eye but was present in the untreated eye15. These results demonstrate that the gene therapy vector delivered via IVT injection was able to halt disease pathogenesis in the CLN5-affected sheep eye. The expression of glial fibrillary acidic protein (GFAP), a marker of retinal stress and astroglia, was lower in treated eyes than in untreated eyes, indicating that disease-associated inflammation was attenuated following treatment (Figure 6)15.

Figure 4
Figure 4: Dark-adapted ERG responses of CLN5-/- sheep following intravitreal delivery of AAV9.CLN5. (A) Mean (± SEM) ERG amplitudes over time in the treated (dark green, n = 3) and untreated (light green, n = 3) eyes of CLN5-/- sheep, as well as healthy control (blue, n = 6) and CLN5-affected (red, n = 6) sheep. (B) Representative ERG traces from the treated and untreated eyes and healthy controls and affected sheep at 5 (black line) and 17 (grey line) months of age. * indicates P < 0.05. This figure reproduced is from Murray et al.15 with permission from Elsevier. Abbreviations: ERG = electroretinography; AAV = adeno-associated virus. Please click here to view a larger version of this figure.

Figure 5
Figure 5: Retinal thickness of CLN5-/- sheep following intravitreal delivery of AAV9.CLN5. Representative photomicrographs of H&E histological staining in the treated and untreated eyes of CLN5-/- sheep compared to age-matched controls. Images and thickness measurements were taken in two locations; central retina (A-E) and peripheral retina (F-J). (E) Mean (± SEM) retinal thickness (µm) in the central retina of the treated (dark green, n = 3) and untreated (light green, n = 3) eyes compared with healthy control (blue, n = 4) and CLN5-affected (red, n = 4) retina. (J) Mean (± SEM) retinal thickness (µm) in the peripheral retina of the treated and untreated eyes of CLN5-/- sheep compared with healthy control and CLN5 affected retina. * indicates P < 0.05, **** indicates P < 0.0001. Scale bars = 50 µm. This figure is reproduced from Murray et al.15 with permission from Elsevier. Abbreviations: NFL = nerve fiber layer; GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; OPL = outer plexiform layer; ONL = outer nuclear layer; IS/OS = inner and outer segments of photoreceptors; RPE = retinal pigment epithelium. Please click here to view a larger version of this figure.

Figure 6
Figure 6: GFAP immunoreactivity in the retina of CLN5-/- sheep following intravitreal delivery of AAV9.CLN5. Representative confocal images of GFAP immunoreactivity in the treated and untreated eyes of CLN5-/- sheep compared to controls. (A-D) GFAP immunoreactivity, (E-H) DAPI nuclear marker, (I-L) Merged images of the two channels. Scale bar = 20 µm. This figure is reproduced from Murray et al.15 with permission from Elsevier. Abbreviations: NFL = nerve fiber layer; GCL = ganglion cell layer; INL = inner nuclear layer; ONL = outer nuclear layer; IS/OS = inner and outer segments of photoreceptors; GFAP = glial fibrillary acidic protein; DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.

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Discussion

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.

The protocol for small-volume IVT injections into a sheep eye is relatively straightforward and noninvasive, readily reproducible, and easy for a non-expert to learn. The success depends on the target cells within the retina, the therapy being delivered, and the location and direction of the injection itself. Although IVT injections are often thought to be most effective at targeting inner retinal layers, many researchers have demonstrated the efficacy of IVT injections in diseases where the outer retina is the primary location of disease pathogenesis15,17,18,19. The differences in functional and pathological outcomes following IVT injections also likely relate to the type of therapy given. For example, gene therapy to deliver genes encoding soluble proteins (e.g., CLN5) has been shown to be much more effective than gene therapies to deliver genes encoding intracellular or membrane-bound proteins (e.g., CLN6)15. Regardless of the target and type of therapy, it is critical to get the injection site and angle correct to maximize efficacy. As described in the protocol, the injection site for sheep should be approximately 7 mm posterior to the sclera on the lateral aspect of the eye and angled posteriorly to target the posterior vitreous. The angling of the needle is both to avoid the lens and to direct the injected drug as close to the retina as possible. The use of a safety syringe with a permanently attached 28 G (or smaller) x 0.5 in low-dead space needle is crucial to minimize injection-related discomfort and dead volume remaining in the needle or hub. This length needle can be fully inserted into the sheep eye at a posterior angle without a surgical microscope and/or the risk of puncturing the retina. Researchers who have access to a surgical microscope can use this to provide an added level of certainty around avoiding retinal disruption. Otherwise, using safety syringes and being aware of the dimensions of the sheep eye globe at the age being treated is sufficient to perform this procedure safely15.

When grasping the eye to rotate it medially and expose the injection site, it is essential to use nontoothed atraumatic instruments to avoid damaging the delicate tissue of the eye. If the eye is positioned centrally, it is relatively straightforward to grasp the bulbar conjunctiva at the border of the sclera and the iris and rotate with one hand, while injecting with the other hand. However, if the eye has rotated off-center, which can occur under general anesthesia, it is often necessary to use a hemostat to clamp onto the bulbar conjunctiva and rotate the eye into position, leaving the hemostat in place to continue with the procedure.

Sheep eyes are robust and recovered well following IVT treatment with AAV9 carrying ovine CLN5, with only one sheep developing uveitis in the treated eye 1 week post injection15. In this case, the uveitis resolved within 1 week and had no long-term impact on vision. Aside from this one case, no negative effects of IVT injections were reported in the first published study15, or in the >30 additional animals in the research program injected at 3 months, 6 months, or 9 months of age following the protocol described here. However, there are additional quantitative measures of injection safety that researchers may want to consider adding to their postoperative assessments. These include measures of intraocular pressure (IOP), fundus imaging, or OCT. Images of the fundus before and after injection can highlight if there has been any disruption of the retina because of the injection and, in the long-term, can provide an overview of retinal health in general.

In the case where a fluorescent marker is injected, fluorescence imaging of the fundus can assist with visualizing the spread of the injected marker16,20. OCT can be used to visualize the retina in cross section in vivo to identify any potential structural damage following injection and measure the thickness of the retina over time in response to treatment. Post-injection visual function can also be assessed by ERG or maze testing15,16. In the case of IVT viral-mediated gene therapy, the impact of the immune response and the presence of neutralizing antibodies to AAV vectors should be considered. Although not part of the protocol outlined here for sheep, it is suggested that subjects are tested for the presence of anti-AAV neutralizing antibodies prior to gene therapy, regardless of the route of administration, to increase transduction efficiency16,21. Readers are referred to a more comprehensive discussion of the issue of immune responses to AAV by Whitehead et al.22.

A common complication during IVT procedures is subconjunctival hemorrhage (SCH)23, which can occur if the capillaries in the bulbar conjunctiva are punctured during needle insertion. Fortunately, SCH is generally harmless and resolves within a few days; however, it is best to avoid conjunctival capillaries when inserting the needle. Following IVT injection, it is important to treat the injected eyes with antibiotic eye drops (e.g., chloramphenicol) and monitor the eyes for any signs of infection or inflammation (uveitis). Another common event during IVT procedures is an increase in IOP. These increases are most commonly transient spikes in pressure in the minutes following injection and cause no long-lasting damage24,25. However, there are cases where IOP should be considered and monitored. When pre-existing ocular conditions such as glaucoma are present, or when higher volumes (≥100 µL) are being injected, IOP should be closely monitored, and prophylactic anterior chamber paracentesis should be considered to reduce pressure in the eye globe4,16. In addition, repeated spikes in IOP in the case of repeated IVT injections can be a concern and should be attenuated as above4. Here, we are demonstrating IVT for the purposes of a single AAV-mediated gene therapy; therefore, the long-term consequences of repeated injections are not a major consideration.

Limitations of IVT injections include the need to penetrate anatomical barriers and the lower target specificity compared to more invasive methods. First, the injected substance will be diluted in the vitreous and then has a distance to diffuse through the vitreous cavity and retinal tissue to the target cells. This means that the inner retina is more readily transduced than the outer retina following IVT injection, and higher doses may be required to counteract dilution26. The protocol described here emphasizes the importance of injecting as close to the retina as possible to mitigate the effects of dilution and diffusion through the vitreous body. Therefore, the full length of the 0.5 in/12.7 mm needle shaft is inserted into the eye. The additional advantage of inserting the full needle length is the reduced chance of fluid reflux during injection4,27.

Although this current protocol omits it, it is recommended to delay the removal of the needle for several minutes post injection to further reduce the chances of fluid reflux. In addition, the injection should be done slowly to ensure that the jet of injected fluid does not disrupt the retina or cause a rapid spike in IOP, and an increased speed of injection does not affect diffusion rates through the vitreous28,29. The inner limiting membrane (ILM) is the primary barrier between the vitreous and the retina, which functions to restrict the movement of molecules into the retina30. However, the permeability of the ILM can be increased by digestion or surgical peeling and is likely increased in the diseased eye, making the penetration of therapeutic molecules easier.

Regarding target specificity, IVT administration has the least compared to other intraocular routes such as subretinal and suprachoroidal, as discussed above and elsewhere10. However, new generations of AAVs are routinely being developed to contain modifications, which enhance targeting to particular cell types or more efficiently overcome barriers such as the ILM31. The use of such modified capsids has increased transduction efficiency following IVT administration in a number of model species5,32,33.

The aim of the gene therapy detailed by Murray et al.15 was to deliver a functional copy of the CLN5 gene; therefore, one measure of efficacy is the presence of transduced cells expressing CLN5 protein. We have attempted to use immunohistochemistry to detect CLN5-transduced cells in the retina, as we routinely do in sheep brain tissue; however, the antibody that we typically use in free-floating brain tissue does not work in paraffin-embedded retinal tissue. Troubleshooting and investigating alternative ways of detecting the gene or protein of interest in the retina are underway to add to the assessment of efficacy. One potential way to achieve this is by injecting a viral vector containing a reporter gene (such as green fluorescent protein; GFP) and assessing GFP expression via immunohistochemistry. Another way to assess efficacy is using quantitative PCR to assess levels of transgene expression.

Developing protocols for IVT injections in large animals is a crucial step toward treating degenerative diseases of the retina, particularly diseases with a genetic component, as IVT gene therapy is a promising potential therapeutic. For degenerative diseases where the retina is already fragile, IVT treatment poses less risk of retinal detachment or tear. Given the similarities in size and structure of the sheep and human eyes, optimizing the dose and volume of IVT injections in sheep is a relevant step toward translation to the clinic. This paper details the protocol for IVT injection into the sheep eye, which is safe and shows a very low rate of ocular inflammatory responses. This method also demonstrates the efficacy of AAV9-mediated ocular gene therapy to address the retinal component of NCL in sheep.

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Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgments

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 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.

Materials

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

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References

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Murray, S. J., Mitchell, N. L. Intravitreal Injections in the Ovine Eye. J. Vis. Exp. (185), e63823, doi:10.3791/63823 (2022).More

Murray, S. J., Mitchell, N. L. Intravitreal Injections in the Ovine Eye. J. Vis. Exp. (185), e63823, doi:10.3791/63823 (2022).

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