Published in Retina

Gene Therapy 101 for Ophthalmology Residents

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15 min read

Discover how ophthalmologists can use gene therapy to treat inherited retinal disease. Also, check out the video on how to administer Luxturna surgically.

Gene Therapy 101 for Ophthalmology Residents
Gene therapy entails the deliberate modification of the genetic code to achieve therapeutic goals. In ophthalmology, most research in gene therapy has focused on retinal diseases. There are more than 300 mapped genes that, when mutated, can cause various forms of inherited retinal disease (IRD).1
For diseases caused by loss-of-function mutations, gene therapy generally aims to introduce a normal copy of the mutated gene so that a normal protein is expressed. For diseases caused by gain-of-function mutations, the aim generally is to replace, remove or modify the mutated gene, or downregulate its expression to prevent the corresponding abnormal protein from being expressed and causing the disease.2

Gene therapy delivery vectors

Supplying a normal copy of a gene, or modifying a gene or its expression, requires a delivery mechanism.3 To date, the most popular method is to package a gene therapy within a virus that already possesses the machinery to invade host cells and replicate.
The genes normally used by the virus to replicate are replaced with a normal copy of the mutated disease gene. The virus then enters a cell and expresses the normal gene copy. The types of viruses commonly used in gene therapy are adeno-associated viruses (AAVs), primarily because their genetic material usually does not integrate with the host genome.
Integration with the host genome carries the potential for dysregulated expression of the gene therapy and oncogenic mutagenesis. However, a major drawback of non-integrating viral vectors is their limited ability to carry large genes.

Determining which viral vector to use

There are many other factors to consider when selecting a viral vector.3 One is tropism, i.e., which is the type of cells the virus can infect. Tropism varies by the type of virus and cell within the retina. Another factor is the efficiency of infection (also called transduction).
If efficiency is low, very high numbers of the virus may be required to transduce enough retinal cells to achieve a therapeutic effect. This can be problematic because viruses are immunogenic, although variably so.
Inflammatory responses can negate the ability of a virus to transduce the gene therapy and damage the eye. Some of these barriers may be overcome with delivery strategies that do not involve viruses. For example, direct delivery of plasmid DNA or nanoparticles without an encapsulating viral vector has shown promise but remains experimental.2

Watch the surgical administration of Luxturna gene therapy


Luxturna Gene Therapy for RPE65-Associated Leber's Congenital Amaurosis

Watch the video for a step-by-step breakdown of how to administer Luxturna.

Surgical delivery strategies for gene therapy

Direct contact of the gene therapy agent with the transfection target maximizes transduction.2 The transfection target varies for different IRDs. To treat IRDs caused by outer retinal/retinal pigmented epithelium (RPE) dysfunction, the gene therapy agent likely requires delivery to the subretinal space.2
The gold standard surgical approach to gene therapy for conditions in which an IRD is caused by inner retinal dysfunction is intravitreal subretinal delivery.2 Additionally, transchoroidal subretinal delivery is a promising, unproven alternative.2
Some gene therapy agents, such as short interfering oligonucleotides, may be better able to diffuse across the retina. Gene therapy targeted at a secreted protein, such as vascular endothelial growth factor (VEGF), may also be achievable with intravitreal delivery.
The internal limiting membrane (ILM) is likely the greatest barrier to trans-retinal transduction after intravitreal delivery. It also constrains the spread of subretinally-injected agents into the vitreous cavity. Various means of modifying the ILM, such as pars plana vitrectomy (PPV) with ILM peeling, may facilitate trans-retinal transduction and obviate the need for delivery into the subretinal space, but this remains unproven.

Luxturna: A novel gene therapy

The only FDA-approved gene therapy in ophthalmology as of 2022 is Luxturna (voretigene neparvovec-rzyl).4 Luxturna treats patients with Leber's congenital amaurosis (LCA) or retinitis pigmentosa (RP) caused by biallelic mutations in the RPE65 gene.
RPE65 encodes a trans-isomerase that performs an essential enzymatic step in the visual cycle. Mutations in RPE65 impair the visual cycle, eventually causing the death of the RPE and photoreceptors. Luxturna comprises a normal copy of the RPE65 gene packaged in an AAV2 vector.
Intravitreal subretinal delivery of Luxturna restores the recycling of vitamin A, improving visual function as measured by pupillary light responses and mobility assays.4 Luxturna is a one-time treatment, with the effect persisting for at least 4 years (longer follow-up research is ongoing).5

How to administer Luxturna

The recommended surgical technique for Luxturna administration, which was followed in the phase III study that demonstrated functional efficacy,4 differs markedly from routine pars plana vitrectomy.2
Institutional protocols are essential to ensure timely thawing and dilution of the agent by a trained pharmacist and ready access to an operating room because the agent must be injected within 4 hours of dilution. In adults, retrobulbar anesthesia with monitored anesthesia sedation can be used; children require general anesthesia.
Either 23- or 25-gauge PPV instrumentation can be used. However, non-valved trocars, which are no longer routinely used in PPV, are recommended for Luxturna surgery because valves can damage the tip of the subretinal injection cannula. To minimize the risk of extraocular reflux of the gene therapy agent, sclerotomies should be sutured after the PPV; a limbal peritomy facilitates closure and sequestration of any refluxed material.
A core vitrectomy is performed. Unnecessary manipulations such as vitreous base shaving are discouraged. The goal of vitrectomy is only to provide a vitreous-free pathway for the injection cannula to traverse the distance between the sclerotomies and the posterior pole.2 Vitreous staining agents such as triamcinolone have not been tested for compatibility with Luxturna and should be avoided.2,4

Clinical pearls for inducing posterior vitreous detachment

Resistance to posterior vitreous detachment (PVD) is often less than anticipated, given the young age of most Luxturna patients. PVD induction can be performed safely without triamcinolone-assisted visualization by aspirating the posterior hyaloid with a soft tip cannula near the nerve and gently pulling it in a gradually enlarging spiral.
PVD should be propagated beyond the anticipated macular injection site(s) but need not be complete to the vitreous base. The peripheral retina is then examined with scleral depression for breaks, which are lasered if present. The surgeon then assembles the injection apparatus, which consists of a pharmacy-supplied syringe, extension tubing, and a 39- or 41-gauge injection cannula.
It is essential to utilize only certain types of these supplies that have been tested to ensure biocompatibility and minimal Luxturna adsorption.2 The initial subretinal injection site is typically along or just outside the superior arcade to treat the inferior and central macular fields used in ambulation and most near-visual tasks.
Also, this region is thicker than the fovea, and bullous subfoveal blebs can cause a full-thickness macular hole. The injection site is gently scraped with a Tano brush to ensure hyaloid removal and weakening of the ILM, which facilitates cannula insertion. The cannula is then inserted to indent the retina or traverse it. If the tip abuts the RPE, a faint blanch is observed.
The cannula is held steady while the assistant initiates a slow injection of 300 microliters. As the bleb elevates, the surgeon keeps the cannula centered at the injection site, typically with the tip remaining in the subretinal space. Sometimes, multiple attempts are required to raise a bleb successfully.

A fluid-air exchange is then performed to remove any refluxed viral particles in the vitreous cavity and to spread the bleb over a greater retinal surface area. The sclerotomies and overlying conjunctiva are sutured.

Post-operative treatment

After the procedure, patients remain supine for 4 hours to minimize exposure of the virus to the ciliary body (which is thought to promote inflammation). Systemic steroids, which should have been started 3 days before surgery, are continued for 3 days after and then tapered over the next 10 days.
When applicable, the second eye is typically treated within 1 to 2 weeks of the first eye to minimize the likelihood of serial immunogenic sensitization.2

Procedural alternatives

Anecdotally, many surgeons have reported deviating from aspects of the recommended protocol, including utilizing non-sutured sclerotomies, valved trocars, triamcinolone, ILM peeling, foot pedal-assisted injection, tapering of the cannula tip, “pre-blebs” of saline, multifocal subretinal blebs, a greater volume of the agent, intra-operative OCT, and so forth.6
These alternative surgical protocols have unproven functional efficacy.2 Post-marketing, retrospective studies have reported a high incidence of postoperative RPE atrophy, which may have resulted from deviations in technique since this complication was not observed in the phase 3 study when utilizing the recommended injection protocol.7,8

Gene therapies in the pipeline

Dozens of gene therapies are developing for IRDs, this article will briefly highlight a few therapies in active clinical trials.
  1. Stargardt disease is caused by biallelic mutations in ABCA4 and is the most common cause of IRD. The ABCA4 gene is too large to package into an AAV vector, making gene therapy for Stargardt disease challenging.
    1. A recent phase 1/2a study utilized the equine infectious anemia virus (a lentiviral vector) expressing ABCA4 to treat 22 patients in the worse-seeing eye with intravitreal subretinal delivery.9 The treatment was well-tolerated, but after 3 years, there were no significant functional changes, and in 6 patients, there was progressive RPE atrophy worse in the treated eye.
  2. Choroideremia is an X-linked recessive form of RP caused by mutations in the CHM gene, which encodes REP1, a protein involved in photoreceptor metabolism.
    1. The recent STAR phase 3 trial utilized AAV2-REP1 to treat 170 patients in one eye with intravitreal subretinal delivery.10,11 The study did not meet its primary endpoint of improved VA or demonstrate efficacy on secondary endpoints.
  3. X-linked retinitis pigmentosa (XLRP) is usually caused by mutations in the RPGR gene, which encodes a protein involved in photoreceptor cargo trafficking. RPGR-associated XLRP is the most common cause of recessive RP.
    1. A recent phase 1/2 trial utilized AAV2/5-RPGR to treat 49 patients in one eye with intravitreal subretinal delivery.12,13 At 6 months, improvements in visual acuity and functional assays were reported. A phase 3 trial is ongoing.
  4. X-linked retinoschisis (XLRS) is caused by mutations in RS1, a gene encoding Retinoschisin, which is a soluble protein that mediates the adhesion of cells within the retina.
    1. Two ongoing phase 1/2 trials are investigating AAV-mediated gene therapy to treat XLRS in one eye with intravitreal delivery.14,15 Preliminary results have indicated a favorable safety profile.

Obstacles to administering gene therapies

There are many obstacles to making gene therapy widely available. The most intimidating is cost; since each IRD is rare and requires a distinct therapy, the market for a given therapy is small. Pre-clinical development and clinical trials are laborious, resulting in high gene therapy price tags. Unfortunately, many conditions are too rare to induce such an investment and, therefore, have no potential gene therapies on the horizon.
Designing clinical trials that demonstrate the efficacy of gene therapy is challenging. To be considered for enrollment, patients must undergo rigorous genetic analysis that is typically not covered by insurance and not widely available. Despite extensive analysis, many families have no identifiable genetic cause.16
For molecularly-confirmed patients, it is difficult to decide whom to enroll. Those early in the disease course could benefit most by staving off disease progression but also could suffer harm from side effects. Conversely, those with advanced disease may have too much retinal damage to benefit from the therapy.
Most IRDs progress very slowly, so trials lasting many years or even decades may be required to detect statistically significant treatment effects. Variability in treatment responses may require large numbers of patients, which only increases costs.

The future of gene therapy

Treating everybody with gene therapy who needs it will require regulatory approval of scalable platforms, such as optimized vectors that can be readily modified to include one of a multitude of genes or even be modified to treat specific mutations in individual patients. Surgical delivery strategies will likely require individualization based on the primary site of retinal dysfunction.2
Some conditions may benefit from more diffuse subretinal delivery and others from re-treatments. Transchoroidal subretinal (or suprachoroidal) delivery may become viable, perhaps even allowing in-office administration.
More broadly, there are intense and ongoing investigations into the use of gene therapy for common disorders, such as age-related macular degeneration. Someday, we may find gene therapy a routine part of our armamentarium as ophthalmologists. The future is bright.

Make sure to check out the video breaking down step-by-step how to administer Luxturna!

  1. RetNet. Summaries of genes and loci causing retinal diseases. Updated January 10, 2023.
  2. Russell JF, Maguire AM, Russell SR. Surgery for Gene Therapy. Book Chapter in Pediatric Retina, 3rd edition. Ed. Hartnett ME. Wolters-Kluwer.
  3. Lee CS, Bishop ES, Zhang R, et al. Adenovirus-mediated gene delivery: potential applications for gene and cell-based therapies in the new era of personalized medicine. Genes Dis. 2017;4(2):43-63.
  4. 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.
  5. Maguire AM, Russell S, Chung DC, et al. Durability of voretigene neparvovec for biallelic RPE65-mediated inherited retinal disease: phase 3 results at 3 and 4 years. Ophthalmology. 2021;128(10):1460-1468.
  6. Russell, J. Author’s Unpublished Observations.
  7. Reichel FF, Seitz I, Wozar F, et al. Development of retinal atrophy after subretinal gene therapy with voretigene neparvovec. BR J Ophthalmol. 2022;321023.
  8. Gange WS, Sisk RA, Besirli CG, et al. Perifoveal chorioretinal atrophy after subretinal voretigene neparvovec-rzyl for RPE65-mediated leber congenital amaurosis. Ophthalmol Retina. 2022;6(1):58-64.
  9. Benoit P, Appelqvist T, Krahmer M, et al. Three-year safety results of SAR422 (EIAV-ABCA4) Gene therapy in patients with ABCA4-asociated Stargardt disease: an open-label dose-escalation phaseI/IIa clinical trial, cohorts 1-5. Am J Ophthalmol. 2022;10.1016.
  10. Biogen. Efficacy and safety of BIIB111 for the treatment of choroideremia (STAR). identifier: NCT03496012. Updated December 7, 2021.
  11. Biogen. Biogen announces topline results from phase 3 gene therapy in choroideremia. Published June 14, 2021.
  12. MeiraGTx UK II Ltd. Gene therapy for x-linked retinitis pigmentosa (XLRP) - retinitis pigmentosa GTPase regulator (RPGR). identifier: NCT03252847. Updated December 7, 2022.
  13. MeiraGTx. MeiraGTx announces positive top-line data from the MGT009 phase ½ clinical study demonstrating safety and improvement in multiple domains of vision in x-linked retinitis pigmentosa patients treated with botaretigene sparoparvovec (AAV-RPGR) compared to untreated randomized control. Published June 28, 2022.
  14. National Eye Institute. Study of RS1 ocular gene transfer for x-linked retinoschisis. identifier: NCT02317887. Updated June 10, 2022.
  15. Applied Genetic Technologies Corp. Safety and efficacy of rAAV-hRS1 in patients with x-linked retinoschisis (XLRS). identifier: NCT02416622. Updated July 20, 2022.
  16. Stone EM, Andorf JL, Whitmore SS, et al. Clinically focused molecular investigation of 1000 consecutive families with inherited retinal disease. Ophthalmology. 2017;124(9):1314-1331.
Jonathan F. Russell, MD, PhD
About Jonathan F. Russell, MD, PhD

Dr. Russell is a vitreoretinal surgeon at the University of Iowa as well as an assistant professor of Ophthalmology and Visual Sciences.

Jonathan F. Russell, MD, PhD
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