Inherited retinal diseases (IRDs) are a group of rare conditions that result in severe vision loss at birth or gradually over time. Advances in treatments for IRDs have progressed dramatically over the past decade, with gene replacement therapy demonstrating the greatest success in recent history. Since the FDA approval of voretigene neparvovec-rzyl (Luxturna; Spark Therapeutics) for RPE65 mutation-related disease, the diversity and scope of gene therapy has begun to expand to other ophthalmic conditions. Specific studies listed on clinicaltrials.gov include those for choroideremia, retinitis pigmentosa (RLBP1, RPGR, PDE6A), Leber congenital amaurosis (RPE65), X-linked retinoschisis (RS1) and even neovascular age-related macular degeneration (AMD), all of which are being investigated as potential targets for subretinal gene therapy for the near future.
Due to the nascency of the field as well as a need for more evidence-based surgical techniques, no general consensus of standard best practice exists for the delivery of gene therapy for IRDs. The original clinical trial by Russell et al for the approval of Luxturna utilized a 2-surgeon approach for the delivery of subretinal drug.1 As such, the instruction manual for the commercially available Luxturna also suggests a 2-operator method. However, many other surgical techniques have been explored, including variations upon intravitreal and subretinal injections, which will be discussed herein.2-5
Intravitreal injections have become one of the most widely performed and safest ophthalmic procedures, with postinjection endophthalmitis rates as low as 0.056%.6 Therefore, it comes as no surprise that investigators have attempted to deliver gene vector to target retinal tissues via intravitreal injection.2
Initial preclinical studies of intravitreal injections of recombinant adeno-associated virus (AAV) vectors in animal models demonstrated limited transduction to the foveal and peripheral retinal ganglion cells, but appeared to be hindered by the vitreous and internal limiting membrane.7,8 In vitrectomized eyes or eyes in which a vitreous detachment had been chemically induced by microplasmin,8 investigators found that AAV vector more easily entered the retina, covering over a larger surface area of the retina.2
Although Tshilenge et al2 summarized that vitrectomy proved to be an efficient way to optimize intravitreal injections of viral vector gene, the need for vitrectomy prior to injection in this technique makes it almost as invasive as the subretinal approach. One important consideration for the selection of intravitreal injection as oppposed to subretinal injection of vector is the target cell. In certain IRDs in which retinal ganglion cells and inner retinal layers are affected, it may be efficacious to use an intravitreal approach; conversely, if the pathology lies within the layers of photoreceptors or retinal pigmented epithelium (RPE), subretinal delivery of the drug may make more sense.3 As such, in diseases such as Leber hereditary optic neuropathy, where the disease primarily targets retinal ganglion cells, clinical trials are currently being performed via intravitreal delivery.9-11
The prevailing method of gene vector delivery today is subretinal injection. After the FDA approval of Luxturna, subretinal delivery has become more widely used, but the same obstacles remain. Firstly, subretinal injections are not commonly performed by most vitreoretinal surgeons, and training and supervision may be required when surgeons first begin. Furthermore, gene therapy for IRDs is a nascent field, and the unchartered waters contain few published studies on best surgical practices. For example, aside from the original operator’s manual, few evidence-based, step-by-step surgical approaches have been published.1,5 Lastly, complications can often be devastating in this patient population. Over the past few years, some subretinal delivery techniques have become more accepted than others, and we will discuss them below.
In general, most preoperative steroid protocols are similar. The Luxturna operator manual instructs initiating oral prednisone of 1 mg/kg/day (maximum of 40 mg/day) for a total of 7 days starting 3 days before the first eye with a 10-day taper (repeat for second eye, if indicated).1 Other surgeons used oral prednisone 1 mg/kg/day up to 80 mg maximum dose, with gastric protection, starting 2 or 3 days prior to surgery, with up to a 21-day to 60-day taper after surgery.4,5
After a standard 23-gauge (g) or 25g vitrectomy is performed, a posterior vitreous detachment is induced, often with the assistance with dilute triamcinolone acetonide. A full vitrectomy is typically performed with a 360-degree depressed shave to avoid missing any peripheral breaks or detachments, which could compromise surgical outcomes.
In all methods of subretinal vector delivery, an extendible 23g/41g soft-tip cannula injector is used.1,4,5,12 Although the cannula is blunt-tipped, some surgeons prefer trimming the tip of the cannula at an angle to bevel the cannula for easier entry into the subretinal space.5 Others believe this can introduce intraretinal schisis in unwanted areas that can be difficult to navigate without the assistance of intraoperative OCT or even with OCT.
In the original Luxturna procedure, the vector was delivered by manual depression of the syringe plunger containing the drug by a second operator.1 Since then, surgeons have switched to the viscous fluid control (VFC) injector offered by most vitrectomy console systems.13 The VFC technology allows for a linear infusion of vector to create the subretinal bleb and is controlled by a foot pedal, thereby eliminating the chance of manual error during this step.
In the Luxturna operator’s manual, subretinal injection of vector is performed without a balanced salt solution (BSS) pre-bleb and with recommendation of only a single bleb.1 More recently, individual surgeons and investigators in preclinical trials13-15 have started raising a subretinal pre-bleb with BSS prior to injection of drug for several reasons. First, the pre-bleb initiates hydrodissection into the subretinal space without wasting any valuable vector and predissection of the correct target treatment location.3-5
However, the single-bleb delivery method is also frequently utilized1, 16 and also has its own advantages (Figure 1). Surgeons utilizing this method believe that the BSS pre-bleb can dilute the subretinal drug content and overstretch the retina, often then requiring multiple dilute subretinal blebs. Additionally, reinjection through the same retinotomy poses a surgical challenge not only in physically locating the retinotomy site (with or without the assistance of intraoperative OCT), but also in ensuring that the retinotomy site is not enlarged upon re-entry or a second adjacent retinotomy is not accidentally made. If this occurs, gene vector may be lost by egress through an enlarged retinotomy wound when injecting.
The preferred location and number of blebs is also a debated subject. In the original operator’s manual for Luxturna, a single subretinal injection along the superior vascular arcade, but avoiding vascular structures and areas of dense atrophy, at least 2 mm away from the foveal center is recommended, for a total of 0.3 mL of vector.1 There is no specific indication to detach the fovea. Other authors have suggested that in some diseases, intentional detachment of the fovea is desired.4,5,13 In these cases, when the overlying retina is thin and the risk of macular hole is high with bleb formation, multiple injections are recommended (Figure 2).4,5,13 Coalescence of the individual subretinal bleb sites may not be necessary but can occur naturally with fluid–air exchange. With either approach, the integrity of the bleb sites may be inspected with intraoperative OCT, if desired.
Avoidance of overstretching or thinning of the retina, macular hole, or excessive egress of the vector is paramount to the success of the procedure. Some surgeons prefer to use intraoperative OCT throughout the entire surgery, and others after subretinal injection to confirm successful subretinal delivery.
After satisfactory delivery of the vector, a fluid–air exchange is performed to remove any drug from the posterior segment to minimize postoperative inflammation. A standard closure is typically performed. Some surgeons will choose to injection sub-Tenon or retrobulbar triamcinolone acetonide. Depending on the age of the patient, postoperative positioning can be determined by the surgeon’s preference.
Gene therapy for IRDs is ushering the retinal physician into an era that was nearly unimaginable just a decade ago. As this exciting field rapidly widens its scope of application across the spectrum of inherited diseases, we as retinal physicians must know how best to deliver the gene therapy to its target tissues. Soon, gene therapy will be a part of the ophthalmologist’s regular armamentarium. Future studies will be critical to identifying the optimal surgical techniques for successful vector delivery and patient outcomes. RP
- Russell S, Bennett J, Wellman JA, et al. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet. 2017;390(10097):849-860.
- Tshilenge KT, Ameline B, Weber M, et al. Vitrectomy before intravitreal injection of AAV2/2 vector promotes efficient transduction of retinal ganglion cells in dogs and nonhuman primates. Hum Gene Ther Methods. 2016;27(3):122-134.
- Ochakovski GA, Bartz-Schmidt KU, Fischer MD. Retinal gene therapy: surgical vector delivery in the translation to clinical trials. Front Neurosci. 2017;11:174.
- Xue K, Groppe M, Salvetti AP, MacLaren RE. Technique of retinal gene therapy: delivery of viral vector into the subretinal space. Eye (Lond). 2017;31(9):1308-1316.
- Davis JL, Gregori NZ, MacLaren RE, Lam BL. Surgical technique for subretinal gene therapy in humans with inherited retinal degeneration. Retina. 2019;39(Suppl 1):S2-S8.
- Fileta JB, Scott IU, Flynn HW Jr. Meta-analysis of infectious endophthalmitis after intravitreal injection of anti-vascular endothelial growth factor agents. Ophthalmic Surg Lasers Imaging Retina. 2014;45(2):143-149.
- Ivanova E, Hwang GS, Pan ZH, Troilo D. Evaluation of AAV-mediated expression of Chop2-GFP in the marmoset retina. Invest Ophthalmol Vis Sci. 2010;51(10):5288-5296.
- Yin L, Greenberg K, Hunter JJ, et al. Intravitreal injection of AAV2 transduces macaque inner retina. Invest Ophthalmol Vis Sci. 2011;52(5):2775-2783.
- Feuer WJ, Schiffman JC1, Davis JL, et al. Gene therapy for Leber hereditary optic neuropathy: initial results. Ophthalmology. 2016;123(3):558-570.
- Zhang Y, Li X, Yuan J, et al. Prognostic factors for visual acuity in patients with Leber’s hereditary optic neuropathy after rAAV2-ND4 gene therapy. Clin Exp Ophthalmol. 2019;47(6):774-778.
- Wan X, Pei H, Zhao MJ, et al. Efficacy and safety of rAAV2-ND4 treatment for Leber’s hereditary optic neuropathy. Sci Rep. 2016;6:21587.
- Bennett J, Wellman J, Marshall KA, et al. Safety and durability of effect of contralateral-eye administration of AAV2 gene therapy in patients with childhood-onset blindness caused by RPE65 mutations: a follow-on phase 1 trial. Lancet. 2016;388(10045):661-672.
- Lam BL, Davis JL, Gregori NZ, et al. Choroideremia gene therapy phase 2 clinical trial: 24-month results. Am J Ophthalmol. 2019;197:65-73.
- Edwards TL, Jolly JK, Groppe M, et al. Visual acuity after retinal gene therapy for choroideremia. N Engl J Med. 2016;374(20):1996-1998.
- Dimopoulos IS, Hoang SC, Radziwon A, et al. Two-year results after AAV2-mediated gene therapy for choroideremia: the Alberta experience. Am J Ophthalmol. 2018;193:130-142.
- Maguire AM, Simonelli F, Pierce EA, et al. Safety and efficacy of gene transfer for Leber’s congenital amaurosis. N Engl J Med. 2008;358(21):2240-2248.
Editor’s note: This article is part of a special edition of Retinal Physician that was supported by REGENXBIO.