The technique draws on personal experience with gene augmentation therapy by subretinal injection in patients with choroideremia, RPE65 mutation-associated retinal dystrophy, and X-linked retinitis pigmentosa. Only 2 surgeons initially performed RPE65 gene therapy in the Phase 3 clinical trial leading to drug approval in the United States and the European Union.1 As new surgeons begin to use the commercially available product voretigene neparvovec-rzyl for RPE65 mutation–related inherited retinal dystrophy (IRD) or to participate in clinical trials, there is a need for education in best practices for subretinal injection. Clinical trials are currently listed on clinicaltrials.gov for choroideremia,2–5 X-linked retinitis pigmentosa (RPGR genotype), autosomal recessive retinitis pigmentosa (MERTK genotype), achromatopsia (CNGA3 and CNGB3), Stargardt disease (ABCA4), Leber congenital amaurosis (CEP290), and Usher syndrome type 1B (MY07A), with several additional subretinal gene therapies in the pipeline.
There are few options with currently available technology for delivery of gene therapy for IRD other than subretinal injection. Intravitreal injection of gene therapy is simpler to perform but is predicted to be useful only for optic nerve or retinal diseases affecting primarily the retinal nerve fiber layer, ganglion cell layers, and inner retinal layers (e.g., Leber hereditary optic neuropathy and X-linked retinoschisis RS1) due to barriers presented by the internal limiting membrane and the retinal tissue.6,7 Microcatheters advanced into suprachoroidal space of rabbits achieved transfection with adeno-associated virus 2 (AAV2) vector as demonstrated by GFAP expression8; however, there is concern that therapy delivered to the suprachoroidal space would be more difficult to target, require higher doses, be more likely to disseminate to unwanted systemic sites, or fail to penetrate outer blood retinal barrier at the level of the retinal pigment epithelium and Bruch membrane. Therefore, subretinal injection is likely to remain the procedure of choice for the near future. Technological innovations may facilitate surgical delivery of vector to the subretinal space.9–12
Although previous publications discuss surgical technique for gene therapy surgery, they do not provide a complete, surgeon-oriented surgical plan that is easy to follow in the operating room. The surgeon's manual for administration of voretigene neparvovec-rzyl intentionally gives general instructions with few details to allow for surgical discretion. The current protocol may help experienced vitreoretinal surgeons new to gene therapy adapt their own procedures and operating room setting to facilitate safe and efficient surgery.
Our methods of subretinal injection were refined during multiple surgeries at Bascom Palmer Eye Institute involving patients enrolled in Phase II to III clinical trials, but also patients treated with commercial products. Many steps are routine best practice procedures for vitreoretinal surgery adapted to a comprehensive plan for gene therapy surgery. The interventions described here were approved by the Human Subjects Research Office of the University of Miami when they involved patients enrolled in clinical trials. All patients signed standard consents for surgery as required by our hospital.
Patient Selection and Preparation
- Select candidates by phenotype.
- Use CLIA-certified genetic testing to confirm the pathogenic genetic variant.
- Document retinal function with visual acuity, visual fields, and electrophysiology; retinal anatomy with fundus photography, optical coherence tomography (OCT), and autofluorescence.
- Use microperimetry to assess the favored fixation point.
- Obtain informed consent emphasizing risks of subretinal surgery including blindness, decreased vision, scarring, disease progression, injury to surrounding structures, inflammation, macular hole, and retinal detachment. Gene therapy surgery now may limit access to future clinical trials.
- Consider pretreatment with oral corticosteroids at up to 1 mg/kg for 3 days with postoperative taper. Doses higher than 60 to 80 mg daily may increase the risk of systemic complications.
- Order gene vector product with sufficient lead-time for surgery. Coordinate time of surgery with vector prep time to ensure injection is within the viable use period.
- Have pharmacists prepare the vector using sterile protective gear in a negative pressure room and biohazard hood or as specified by protocol.
- Inspect vector for signs of contamination and verify that it is the intended product for the patient. Note expiration time.
Preparation for Surgery
- Supplemental Digital Content 1 (see Table, http://links.lww.com/IAE/B27) lists commonly used instruments and supplies.
- Review photographs. Assess target zone and proposed injection sites.
- Plan the injection site(s) to cover the target zone without overstretching the retina or exceeding the allowed volume.
- Select injection sites that are recognizable by retinal vascular landmarks but avoid excessive atrophy, epiretinal membranes, papillomacular bundle, or thick nerve fiber layer. Most injection sites will be somewhere along the temporal vascular arcades.
- Perform a core vitrectomy.
- Inject diluted triamcinolone acetonide to visualize the residual vitreous.
- Elevate cortical vitreous with any combination of aspiration with vitrector, soft-tip needle, or membrane scraper. Confirm complete removal of adherent vitreous to target sites using a second triamcinolone injection if needed. Remove contracted or thick epiretinal membranes. Do not remove the internal limiting membrane.
- Use judgment-regarding peripheral vitreous removal anterior to the equator to reduce risk of retinal breaks in thin peripheral retina. Vitreous texture and adhesions vary, and the retina is usually thinner in IRD (see Video, Supplemental Digital Content 2, http://links.lww.com/IAE/B28, demonstrates removal of cortical vitreous after staining with diluted triamcinolone acetonide and careful trimming of the peripheral vitreous to anterior to the equator).
Prepare for Pre-bleb (If Elected)
- Pour balanced salt solution (BSS) into the 10 cc Viscous Fluid Control (VFC) syringe without turbulence.
- Place the bung in the syringe barrel and remove air including any visible small bubbles.
- Mount an extendible 23 g/41 g subretinal injector on the VFC and manually prime, watching for bubbles.
- Attach the syringe to the vitrectomy console using the VFC tubing.
- Adjust injection pressure to 20 mmHg and re-prime the needle using foot pedal control. Lower injection pressure to 12 mmHg. Adjust pressure to achieve moderate drip without jetting. Maximum injection pressure is usually in the range of 12 to 18 mmHg.
- Bevel the fiber tip at 45°, watching for shards and re-prime (see Video, Supplemental Digital Content 2, http://links.lww.com/IAE/B28).
- Replace the 25-gauge superior cannula for the dominant hand with a 23-gauge valved or nonvalved cannula if needed (see Video, Supplemental Digital Content 2, http://links.lww.com/IAE/B28).
- Retract fiber tip and insert needle. Re-prime in mid-vitreous to expel small air bubbles.
- Switch to a narrow angle lens. Focus at highest magnification to achieve parfocality with the assistant surgeon. Switch to intraoperative Microscope-Integrated OCT (MI-OCT) and fine focus using the remote monitor (if used). Take an initial MI-OCT cube of the central macula.
Balanced Salt Solution Pre-bleb
- Plan the pre-bleb size to match the treatment goal. Appreciate that the fovea is both the most difficult to detach and the most likely to rupture from excessive stretching. Use a larger pre-bleb if needed to detach a sticky target zone (e.g., choroideremia), but a smaller one if the fovea detaches easily to allow more room for vector (e.g., retinitis pigmentosa) (see Video, Supplemental Digital Content 3, http://links.lww.com/IAE/B29, shows the relative size of the pre-bleb planned for a large-volume injection. The puncture site is visible in the MI-OCT scans and self-seals after surgery.).
- Position MI-OCT scan close to the injection site and start scanning. Surgeon loses control of the microscope X-Y control when MI-OCT is on but can control the location of the MI-OCT with the foot pedal joystick and turn MI-OCT off and on.
- Place the fiber tip in contact with the retina. Lessen pressure if there is blanching or compression of choroid on OCT (Figure 1A).
- Adjust technique to achieve penetration. Consider increasing injection pressure or shortening the fiber needle tip to increase stiffness.
- Confirm retinal elevation with MI-OCT (Figure 1B). Check for suprachoroidal injection and reposition needle in the subretinal space (Figure 2A). Choroidal hydration is common, and outer retinal hydration is common (Figure 2A lower panel and Figure 2B) (see Video, Supplemental Digital Content 4, http://links.lww.com/IAE/B30, shows bleb elevation occurring as the subretinal space is entered with the needle tip while infusing BSS. Simultaneous MI-OCT confirms retinal elevation).
- If it is intended to detach a foveal target zone, reposition MI-OCT over the fovea, and continue injection until it is detached. Monitor the foveal thickness and stop infusion immediately if the foveal contour inverts or is overstretched. Assessment may be difficult in very high bleb without vertical repositioning (see Video, Supplemental Digital Content 5, http://links.lww.com/IAE/B31, shows positioning the MI-OCT during bleb creation to confirm foveal detachment and then monitor it for thinning during injection).
- Create one or more additional blebs if needed to cover the target zone (Figure 3). High blebs can be allowed to flatten slightly before proceeding with vector injection.
Preparation of Vector Injection Syringe
- For vectors supplied in vials or syringes not intended for direct injection: Surgeon or assistant surgeon mounts a filterless 18- or 19-gauge fill needle on a 1-mL supply syringe and withdraws vector. Avoid turbulence. Some vectors are supplied in sterile syringes suitable for manual injection.
- For machine injection, attach an empty sterile injection syringe and needle to the VFC with an adaptor. Check for free movement with foot pedal control. Remove needle.
- Fill the injection syringe through the narrow tip from the supply syringe. Replace the fiber-tipped injecting needle on the injection syringe. Prime the syringe and needle holding the syringe upright, so that air is not trapped in the needle hub around the base of the fiber. The vector can be moved gently back and forth over the hub to dislodge bubbles.
- Set machine injection pressure at 12 mmHg and check drip rate. A steady drip rate without jetting is desirable. Note volume on syringe barrel after priming.
- For manual injection, attach the subretinal injection needle to short, high-pressure microdose tubing approximately 15 cm, which is then attached to the sterile injection syringe. The system is primed until the vector product is observed to drip from the tip of injection needle. Inspect needle and tubing for air bubbles. The skilled surgical assistant injects for the lead surgeon.
- Collect excess product released during priming in a small container to reduce contamination of the surgical field.
- Place the plastic fiber-tipped needle carefully into the vitreous cavity.
- Adjust magnification and focus. Flat macular lenses and narrow angle lenses used with inversion systems are good choices. Adjust MI-OCT focus for the narrow angle lens.
- Take initial MI-OCT to include pre-bleb and the fovea.
- Foveal detachment is desirable in most cases. Begin infusion with the MI-OCT vertical or horizontal line passing through the fovea.
- For cases with pre-bleb, use the same retinal hole for the vector injection needle (see Video, Supplemental Digital Content 6, http://links.lww.com/IAE/B32, shows vector injection into the pre-bleb. Initially, there is compression of the choroid which impedes elevation. Because of lack of foveal detachment perhaps related to interference from the inadvertent air bubble, a second pre-bleb is created inferiorly to ensure coverage of the target zone. The OCT scan is moved to check the boundaries of the bleb.). For cases without a pre-bleb, proceed as for 6.3 above, using vector injection to detach the retina (see Video, Supplemental Digital Content 7, http://links.lww.com/IAE/B33, shows an atraumatic pre-bleb formation with BSS in real time. There is initial compression from the beveled fiber-tipped needle. Pressured is lessened, and the fluid hydrodissects under the retina, visualized by the surgeon as graying and elevation of the retina. MI-OCT confirmed retinal elevation and detachment of the foveal target zone [not shown]. In the second half of the video, the blunt fiber-tipped needle of the vector injection syringe makes contact with the exact point of the injection site. Small bubbles confirm subretinal injection, as does the simultaneous MI-OCT [not shown]. The needle tip does not need to deeply penetrate the retina as long as there is good contact with an area of ILM disruption. The video clip is in real time. The injections were judged to be excellent without enlargement of the retinal hole and no reflux.).
- Monitor bleb height during injection to confirm that subretinal injection is effective (see Video, Supplemental Digital Content 8, http://links.lww.com/IAE/B34, shows vector injection into the inferior pre-bleb created in Supplemental Digital Content 5, http://links.lww.com/IAE/B31. The superior bleb and inferior bleb are connected by a thin channel allowing for some mixing. There was no loss of height to suggest reflux.). The surgeon should suspect reflux if the bleb fails to elevate, the injection hole enlarges, or hyperreflective material appears on the MI-OCT (see Video, Supplemental Digital Content 9, http://links.lww.com/IAE/B35, shows tissue particles refluxing from a small dehiscence in the fovea after large air bubbles rapidly entered the subretinal space during vector injection, creating shadows on the MI-OCT. The air tamponaded the rupture, and a second inferior bleb was created to treat the inferior portion of the target zone with vector.). If multiple coalesced blebs are used, reflux may occur through the other penetration sites. Monitor the fovea continuously for thinning (Figure 4, A and B).
- Assistant surgeon monitors the amount of vector injected up to the predetermined maximum dose.
- Surgeon and assistant visualize the extent of the final injection bleb. MI-OCT scans confirm the boundaries of the bleb.
- Inspect peripheral retina for retinal tears. Avoid high-risk trimming of vitreous base.
- Wash with BSS infusion and aspiration, or perform fluid–air exchange.
- Dispose of excess vector and material that has contacted vector in biohazard trash.
- Document bleb location and injection sites on fundus photographs and archive.
- In general, position patient supine for 2 to 24 hours, even if the eye is air-filled.
- In general, continue oral prednisone for 21 to 60 days, tapering after the first 2 weeks of high-dose therapy. Prescribe routine topical antibiotics and adjust topical corticosteroids for intraocular inflammation, which may vary among patients.
- Obtain macular OCT on postoperative Day 1 in fluid-filled eyes and on Day 7 in air-filled eyes. Assess for resolution of submacular fluid with sequential OCT examinations.
- Assess visual acuity, visual field, microperimetry, and electroretinogram as indicated to judge response. Monitor OCT, fundus photographs, and autofluorescence.
Results Using This Protocol
Because most patients treated have been in clinical trials for which it is not possible to report partial data, we refer to our results in general terms that are relevant to the surgical procedure.
In our typical protocol, the neurosensory retina is first elevated with BSS, and then the viral vector is injected into the pre-bleb while confirming expansion with MI-OCT.
Using this general protocol, we have delivered subretinal gene therapy more than 50 times in clinical trials and for RPE65-mediated IRD. Most steps are standard for vitreoretinal surgery with some modification. The intraoperative OCT is important to guide viral vector injection in real time to cover the target zone, to avoid suprachoroidal injection, and to avoid complications such as overstretching the fovea and macular hole formation.
Subretinal gene therapy is effective based on available literature. The Phase III study of voretigene neparvovec-ryzl injection for RPE65-mediated IRD1 without pre-bleb or triamcinolone visualization led to FDA approval. Pilot studies for REP1 therapy of choroideremia with a pre-bleb reported beneficial effects on vision.2–4,13 There are forthcoming results from ongoing trials. The principal criteria for surgical success are delivering the planned volume of vector to the prespecified zone and absence of operative complications such as retinal holes. Ultimate success is whether retina treated with vector functions better or longer than natural history.
The procedure induces variable amounts of inflammation, some of which may be subretinal and might jeopardize outcomes. Consensus is that oral prednisone 1 mg/kg up to 80 mg per day can be given starting 3 days before surgery with tapering starting after about 2 weeks. Total duration of treatment can be 21 to 60 days. Different viral vectors seem to induce variable amounts of inflammation and may require different regimens. The inflammation can present as anterior chamber or vitreous cells. Outer retinal changes and exudation are possible, which are more concerning.
The critical, novel surgical step is creating a bleb for injection into the subretinal space. Using a pre-bleb of physiological fluid (BSS) avoids wasting vector if the retina does not immediately elevate.14 The bleb is created with a combination of pressure to access the subretinal space and hydrodissection.14,15 Ease of elevation may be influenced by patient factors or characteristics of the IRD. If the retina detaches easily, small BSS pre-blebs may avoid overstretching and dilution. If the fovea is in the target zone and difficult to detach, larger pre-blebs may help ensure coverage. The procedure is best conceptualized as a breach in the ILM with hydrodissection under the retina rather than an insertion of the tip under the retina, which may lead to hole enlargement.
One-milliliter syringe adaptors for the VFI function allow foot pedal control and reduce the need for a skilled assistant to inject. Even with surgeon control of the force and timing of injection and surgical experience, bleb expansion can be unpredictable as variable subretinal adhesion influences whether the retina will stretch vertically or expand horizontally, and in which direction. Multiple blebs can improve target zone coverage by increasing surface area or by approaching the fovea from two sides to detach it. Creating a second bleb is also a good strategy when the initial attempt produces a very high bleb or a bleb that tracks away from the fovea (see Video, Supplemental Digital Content 10, http://links.lww.com/IAE/B36, shows a bleb tracking superiorly away from the fovea. Continued injection did not lead to detachment of the fovea and a second pre-bleb was created). Air–fluid exchange may help two blebs coalesce.
After bleb creation, additional challenges are to inject the desired amount of vector. Not injecting the maximum dose may not reduce effectiveness much because the vector is dosed in log units. Safety is paramount. Some small blebs and target zones may only accept a small portion of the planned treatment dose. Reflux can be difficult to see. It should be suspected if there is enlargement of the hole around the injection needle. It is difficult to judge how much the bleb should rise for a given duration of injection at a certain pressure. The amount injected can only be estimated: surgeons overestimated the amount of a cloudy solution injected subretinally into cadaver porcine eyes by 36% based on objective measurements made with MI-OCT.16 In addition, there was a 10% variance in the weight of 50-μL doses dispensed by them from a 1-mL syringe.16
Subretinal air is undesirable. Meticulous attention to filling the BSS and the vector syringe without turbulence and priming the injection needles can help. A few small bubbles in the subretinal space are likely not harmful. A large bubble that creates volume issues can be removed with the BSS injection needle in extrusion mode. MI-OCT monitoring for foveal thinning can help avoid macular hole formation.17 Macular hole may interfere with injecting the full dose or permit egress of the vector from the subretinal space. Foveal stretching without hole may lead to subsequent thinning postoperatively and decreased vision.5
The surgery is expensive, requiring many resources, and exposes patients to all the usual risks of vitreoretinal surgery including retinal detachment with vision loss and cataract. Retinal tears were reported in 2 of 20 eyes in the Phase III voretigene neparvovec-rzyl clinical trial and were treated with laser retinopexy without sequelae.1 That trial reported one macular hole. It is anticipated that adults who undergo this surgery will require cataract surgery sooner than if the eye had not been operated. There are other patient-centered issues related to travel to special centers for treatment and time off from work or school. Damage to the retina from brief detachment does not seem to be a major problem. Inflammation limited the benefits of treatment in two published cases.3,5 High doses of corticosteroids seem necessary to control inflammation stimulated by the surgery.
The current technique allows treatment of only a relatively small portion of the retina in retinal degenerative disorders that are usually diffuse. There is as yet no way to smoothly apply vector over large target zones or the entire subretinal space. Treatment of the subretinal space with gene therapy by this method is also limited by the ability to detach atrophic retina.
Additional limitations relate to the complexity of the retina and retinal pigment epithelium.18 Preferential binding to different cell types may depend on capsid design. The promotors packaged with the gene may preferentially express in one cell type. Interphotoreceptor matrix proteins can prevent spread of the vector to the cell bodies of the photoreceptors.19 Even precise surgical targeting cannot control the cellular targeting of the vector or its transduction. The limitations of both the surgical and medical aspects of subretinal gene therapy have recently been reviewed.18
The method described here may benefit from technological advances, but access to the subretinal space is likely to continue to require direct surgeon control, even if robotically enhanced.12 The internal limiting membrane and Bruch isolate the subretinal space unless the inner and outer barriers are physically breached, making intravitreal injection ineffective for most IRD.
Linda Cernichiaro, MD, Sulaiman Alhumaid, MD, and Jila Noorikolouri, MD, were helpful in developing the intraoperative OCT protocols. Dr. Linda Cernichiaro was helpful in preparing edited video segments of gene therapy surgeries. Lipton Gonzalez, PharmD, and Camille Gil, PharmD, prepared voretigene neparvovec-rzyl for subretinal administration. Mr. Achely Joseph, senior surgical technician, and Uriel O Gonzalez, RN, circulating nurse, participated in most of the gene therapy surgeries.
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