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Will experience support use of first-ever retinal gene therapy?


Recent reports of retinal atrophy have raised concerns on potential long-term safety.

Voretigene neparvovec (Luxturna, Spark Therapeutics) is the first causal treatment for biallelic RPE65 mutation–associated retinal disease, which regularly progresses to legal blindness. The one-time gene therapy aims to deliver the correct coding sequence of the human RPE65 gene to the retinal pigment epithelium and is performed via subretinal injection following vitrectomy.

The therapy was approved by both the European Medicines Agency (in 2018) and US Food and Drug Administration (in 2017) after data from the supporting pivotal phase 3 study revealed statistically significant functional vision improvement in patients in terms of increased light sensitivity. The findings also showed improved ability to navigate a mobility course at different levels of environmental illumination.

New gene therapies such as these raise the hope of treating a previously incurable disease with a favorable adverse effect profile. However, as with any new therapeutic product, there are limited real-world data, so it is natural that uncertainties regarding the durability and benefit-risk ratio exist.

Principle of application of gene therapy with voretigene neparvovec.(Images courtesy of LMU Eye Hospital)

Principle of application of gene therapy with voretigene neparvovec.(Images courtesy of LMU Eye Hospital)

Recent reports of retinal atrophy development in the postoperative course of the disease have led to concerns that voretigene neparvovec could lead to potentially devastating consequences in the long-term. It is therefore necessary to closely follow-up treated patients with multimodal imaging approaches in order to assess retinal morphology and gain further knowledge on the factors possibly contributing to atrophy development.

RPE65 mutation–associated disease

The RPE65 gene encodes a key enzyme in the retinoid cycle and is responsible for the regeneration of the light-sensitive component of rhodopsin, our visual purple.1 When light enters the eye, it hits the photoreceptors in the retina leading to a conversion of the light signal into a chemical signal.2

Mode of action of AAV-mediated gene therapy. (Images courtesy of LMU Eye Hospital)

Mode of action of AAV-mediated gene therapy. (Images courtesy of LMU Eye Hospital)

This so-called photoisomerization of the vitamin A derivative 11-cis-retinal (to all-trans-retinal) cannot take place unless there is sufficient functional 11-cis-retinal available. Because 11-cis-retinal decays after initiation of the visual process, it must be perpetually regenerated by specific metabolic processes in the retinal pigment epithelium to initiate and maintain the visual process.3

Mutations in the RPE65 gene result in deficiency or severely functionally impaired isomerohydrolase activity, causing a severe rod-cone dystrophy.4 The clinical courses of RPE65-associated retinal dystrophy are thought to result from different residual activity of the enzyme.

Clinically, the 2 most common forms of RPE65-related retinal disease are Leber congenital amaurosis (LCA) and early-onset severe retinal dystrophy. In both forms, visual impairment is first noticed at birth and during the first years of life, respectively, and worsens over time, eventually leading to complete blindness.5

LCA is considered the most severe form of early childhood blindness and was first described by Theodor Leber in 1869.6 Affected infants usually show lack of eye contact and nystagmus and/or present with conspicuous pressing of their eyeballs with fingers, fists, or toys (oculo-digital phenomenon).

Parents may report that their child frequently trips over objects or bumps into obstacles, especially in dim light. Early-onset severe retinal dystrophy that manifests after infancy also has a very poor prognosis and, like LCA, usually leads to blindness in the third to fifth decade of life.

To date, it is known that LCA can be caused not only by alterations in the RPE65 gene, but also by mutations in a further 24 genes (see: https://sph.uth.edu/retnet/sum-dis.htm#A-genes). However, mutations in the RPE65 gene might also manifest in the form of retinopathia pigmentosa (RP20).7

In the subtype of RP20, a noticeable deterioration of visual acuity usually occurs in young adulthood or adolescence while concentric loss of visual field is already advanced.7 Common to all forms of RPE65 mutation–associated retinal disease is a pronounced night blindness, which presents as one of the earliest and most characteristic symptoms of the disease, and a progressive, irreversible retinal degeneration.

The night blindness can be explained by the functional impairment of the rods, which are already affected at the earliest disease stages. Rods, unlike cones, are completely dependent on 11-cis-retinal regeneration through the retinoid cycle of the retinal pigment epithelium. Cones are less affected in early stages of the disease because they can rely on 11-cis-retinal from other sources, such as Müller cells, which explains their better function in early disease stages.8-10

Gene therapy surgery and mechanism of action

Voretigene neparvovec consists of the capsid of an adeno-associated viral vector serotype 2 (AAV2) containing a correct coding sequence (cDNA) of the human RPE65 gene and regulatory elements.11 This is provided in the form of frozen concentrate, which must be prepared into a vector solution by trained personnel.

Subsequently, the therapy is provided in a syringe containing the vector solution that must be applied within 4 hours after preparation. Following vitrectomy, delivery of the vector solution is performed by using a small injection cannula by placing it onto the retina and applying slight pressure to create a retinotomy through which the fluid can pass into the subretinal space (see Figure 1).

The injection may be performed manually with the help of an assisting surgeon or using a foot pedal–controlled injection device. Patients receive a single dose of 1.5 x 1011 vector genomes of voretigene neparvovec in each eye; the intended target volume is 300 µl.

The injection forms 1 or more fluid-filled bubbles under the retina (blebs). These are reabsorbed within 24 to 48 hours after subretinal delivery, as the drug is taken up by the target cells, the retinal pigment epithelium. Uptake into the target cells is receptor mediated.

Once in the nucleus, the single-stranded DNA is transcribed into double-stranded DNA, and the mRNA is subsequently translated in the cytosol into the functional protein, the enzyme isomerohydrolase (illustrated in Figure 2).

Reports of atrophy development following administration

Data from the clinical studies leading to approval of voretigene neparvovec showed that there are certain risks associated with the gene therapy procedure. However, most of the treatment-related adverse events were transient and mild. These included elevated IOP (18%), cataract formation (18%), ocular inflammation (8%), retinal tears (8%), dellen (8%), and retinal deposits (8%).12,13

The previously undescribed complication of chorioretinal atrophy development following treatment with voretigene neparvovec was recently reported by Gange and colleagues.14 Eighteen eyes of 10 patients developed perifoveal chorioretinal atrophy; in 80% of the cases this was seen bilaterally.

Atrophy was first identifiable anywhere between 1 week and 1 year postoperatively at an average of 4.7 months after treatment (follow-up period, 4-18 months). In 10 eyes, the atrophy occurred within and outside the area of the subretinal bleb, whereas in 7 eyes, it formed exclusively within the bleb’s area.

One eye showed atrophy only outside the bleb area. Despite atrophy development, functional results remained stable or improved in the majority of patients. Twelve of 18 eyes showed improved visual acuity (VA), whereas in 3 of the eyes VA did not change.

VA decreased in a further 3 eyes. After statistical analysis, no significant change in mean VA was found pre- versus postoperatively (P = .45). Although all 13 eyes with reliable Goldmann visual field tests showed improvement (expansion or gain of isopters), paracentral scotomas caused by the atrophy were seen in 3 eyes.

Another recent publication reported progressive atrophy development in 13 eyes of 8 patients.15 All eyes developed atrophy within the bleb area and 3 patients additionally developed atrophic changes outside the bleb.

The mean duration of the patients’ follow-up period was 15.3 months (range, 6-27). First signs of developing atrophy as detected by reduced autofluorescence were identifiable in 5 of 8 eyes as early as 2 weeks after treatment, which represented the earliest postoperative visit.

At month 3 following therapy, all 13 eyes showed areas of retinal atrophy. Notably, the atrophy area enlarged over time and in 6 of 7 eyes with existing follow-up data after 1 year, atrophy development progressed even after 1 year. Functional improvement shown by increased light sensitivity and perimetry seemed to be overall stable over the observational period despite atrophy development.

What could be the cause of atrophy?

Possible explanations for the development of atrophy include immune reactions against the vector genome (eg, promotor sequence as the CAG promotor in voretigene neparvovec, the expressed transgene, or manufacturing-related impurities) or against the capsid. Manufacturing-related factors could also include subtle deviations in the preparation of the gene therapy shortly before therapy administration at the respective treatment center.

Surgical factors may also play an important role. Mechanical stress and/or damage at the injection site as well as shear stress within the bleb may directly lead to damage of the retina or trigger deleterious stress responses. This may be particularly relevant in the setting of RPE65-related retinal disease and other inherited retinal dystrophies in which dystrophic changes and a more fragile, thinned retina may predispose to damage.

Patient-related factors should be considered as well. Age, gender, stage of disease, and immune status of the individual patient could influence the functional outcome and morphological state of the retina after treatment.


Early detection of inherited retinal diseases is becoming increasingly important, as earlier diagnoses enable more timely initiations of therapy and thus potentially lead to better prognoses for affected patients. Symptoms such as increased sensitivity to light (photophobia), night blindness, or nystagmus may indicate an inherited retinal disease and require a thorough ophthalmological work-up within a specialized ophthalmogenetic department.

If there is reasonable suspicion, molecular genetic testing should be carried out. This is crucial to determine whether therapy with voretigene neparvovec is applicable. Gene therapy with this product must only be performed in individuals with confirmed biallelic RPE65 mutation–associated retinal dystrophy and sufficient viable retinal cells.

Limited real-world data initially confirmed the tolerable safety profile seen in marketing authorization trials.16 However, the recent reports of progressive atrophy development following therapy are undoubtedly concerning.

In summary, currently available data are insufficient to draw definite conclusions about the causes of atrophy development and their functional consequences in the long-term. Experiences of other treatment centers, including reports of the exact surgical procedure and patient details as well as longer follow-up periods, are necessary to reach a more accurate picture on possible long-term effects of the therapy. This could have important implications for the selection of patients and will help in predicting the expectable treatment benefit for eligible patients.

Maximilian J. Gerhardt, MD

Stylianos Michalakis, PhD

Günther Rudolph, MD

Claudia Priglinger, MD

Siegfried Priglinger, MD*

E: siegfried.priglinger@med.uni-muenchen.de

Drs Gerhardt, Michalakis, Rudolph, Priglinger, and Priglinger are based at the Department of Ophthalmology, Ludwig-Maximilians-University Munich in Germany.

*Corresponding author


1. Redmond TM, Yu S, Lee E, et al. Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle. Nat Gen. 1998;20(4):344-351. doi:10.1038/3813

2. Palczewski K. G protein–coupled receptor rhodopsin. Annu Rev Biochem. 2006;75:743-767. doi:10.1146/annurev.biochem.75.103004.142743

3. Kiser PD, Golczak M, Palczewski K. Chemistry of the retinoid (visual) cycle. Chem Rev. 2014;114(1):194-232. doi:10.1021/cr400107q

4. Takahashi Y, Chen Y, Moiseyev G, Ma JX. Two point mutations of RPE65 from patients with retinal dystrophies decrease the stability of RPE65 protein and abolish its isomerohydrolase activity. J Biol Chem. 2006;281(31):21820-21826. doi:10.1074/jbc.M603725200

5. Weleber RG, Michaelides M, Trzupek KM, Stover NB, Stone EM. The phenotype of Severe Early Childhood Onset Retinal Dystrophy (SECORD) from mutation of RPE65 and differentiation from Leber congenital amaurosis. Invest Ophthalmol Vis Sci. 2011;52:292-302.

6. Leber T. Über Retinitis pigmentosa und angeborene Amaurose. Archiv für Ophthalmologie. 1869;15:1-25.

7. Morimura H, Fishman GA, Grover SA, Fulton AB, Berson EL, Dryja TP. Mutations in the RPE65 gene in patients with autosomal recessive retinitis pigmentosa or Leber congenital amaurosis. Proc Natl Acad Sci U S A. 1998;95:3088-3093.

8. Wang JS, Kefalov VJ. An alternative pathway mediates the mouse and human cone visual cycle. Curr Biol. 2009;19:1665-1669.

9. Sato S, Frederiksen R, Cornwall MC, Kefalov VJ. The retina visual cycle is driven by cis retinol oxidation in the outer segments of cones. Vis Neurosci. 2017;34:E004.

10. Das SR, Bhardwaj N, Kjeldbye H, Gouras P. Muller cells of chicken retina synthesize 11-cis-retinol. Biochem J. 1992;285(pt 3):907-913.

11. European Medicines Agency. Summary of product characteristics: Luxturna. Accessed 7 June 2022. https://www.ema.europa.eu/en/documents/product-information/luxturna-epar-product-information_en.pdf

12. 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;390:849-860.

13. Maguire AM, Russell S, Wellman JA, et al. Efficacy, safety, and durability of voretigene neparvovec-rzyl in RPE65 mutation–associated inherited retinal dystrophy: results of phase 1 and 3 trials. Ophthalmology. 2019;126:1273-1285.

14. 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;6:58-64.

15. Reichel FF, Seitz I, Wozar F, et al. Development of retinal atrophy after subretinal gene therapy with voretigene neparvovec. Br J Ophthalmol. Published online May 24, 2022. doi:10.1136/bjophthalmol-2021-321023

16. Deng C, Zhao PY, Branham K, et al. Real-world outcomes of voretigene neparvovec treatment in pediatric patients with RPE65-associated Leber congenital amaurosis. Graefes Arch Clin Exp Ophthalmol. 2022;260:1543-1550.

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