Charles G. Miller, MD, PhD, Jean Bennett, MD, PhD, and Albert M. Maguire, MD

Once upon a time, not so very long ago, when someone with an inherited retinal degeneration (IRD) went to an ophthalmologist, they were told “I’m sorry. There is nothing we can do. You should learn how to use a blind cane.” Although night blindness had been recognized in ancient Egypt, diverse retinal degenerations were still being lumped into broad categories into the 1990’s. Treatment attempts included placental implants, injection of ozone, atropine or bee sting venom.[1] These speak to the desperation of patients who are aware of the progressive loss of their vision and, many of whom, who have seen their relatives go blind. When in 1982, “super mice” were generated by injecting a fusion gene for growth hormone into the pro nucleus of newly fertilized mouse eggs,[2] the implications of gene transfer became tangible. Although very few disease-causing genes had been identified and cloned, a number of investigators started proposing gene therapy studies. The first approved study was initiated in 1990 using an ex vivo approach, delivering the normal adenosine deaminase gene to lymphocytes with a retrovirus, and then infusing those genetically modified autologous cells into the patient. This year also marked the identification of rhodopsin-based RP and the first cloning of rhodopsin.[3, 4] This fueled categorization of IRDs and the cloning of a number of disease-causing genes, a process that accelerated with the human genome project. It also served as the rationale for us to move forward to develop gene therapy, despite the fact that so many other pieces of the puzzle were missing (quantitative measures for evaluating disease progression, genetically defined animal models, gene transfer methods, etc.).

While the concept of gene therapy may seem simple, safe and efficient delivery of transgenes in vivo is challenging. Nucleic acids are highly charged and do not penetrate cell membranes easily. Thanks to support of the Associated Retinal Consultants at Beaumont Hospital (Royal Oak, MI) (who commented “I have no idea what they are talking about, but they seem pretty smart”; Ray Margherio), we were able todo experiments that demonstrated in vivo retinal gene transfer in rabbits using LacZ, the b-galactosidase-encoding gene. This was done using a commercially available lipid formulation for compacting DNA and rendering it permeable to the cell membrane. On the advice of Mark Blumenkranz, MD, Al Maguire, MD, adapted a surgical procedure that had been previously described for injecting fluid under the retina. This way we delivered the gene transfer cocktail in direct apposition to photoreceptors by causing a small “bleb,” i.e. a retinal detachment. This was our first glimmer that this approach could be effective, although expression was transient.

In 1992, as junior faculty at University of Pennsylvania (UPenn), we collaborated with Dr. James Wilson, who had developed a recombinant adenovirus containing LacZ. Because little was known about the safety of recombinant viruses, we worked in a Biohazard level 3 (BL3) facility in cubicles adjacent to animals harboring listeria, herpes and HIV. We delivered LacZ efficiently to retinal cells and expression persisted for months.[5, 6] We used a similar virus to develop the first proof-of-concept of retinal gene therapy – delay of photoreceptor cell death mediated by delivery of Pde6b to the neonatal rd mouse.[7] Further animal studies demonstrated elimination of expression at 2 weeks if that reagent was delivered intravitreally or systemically.[8, 9] Another recombinant virus, adeno-associated virus (AAV), became available with a far more favorable transduction and safety profile.[10-12] As we were carrying out gene transfer studies in canines with G. Acland at UPenn, Kristina Narfstrom identified a naturally occurring model with Leber congenital amaurosis due to RPE65 disease in Swedish Briard dogs.[13, 14] In collaboration with William Hauswirth, who generated an AAV containing the canine RPE65 cDNA (from G. Aguirre’s lab), our team treated 3 puppies with this congenital blinding disease using the subretinal injection technique that we had developed in several other studies (Figure1). The first people to recognize that this was successful were the animal technicians. We received a call from a technician saying “The puppies can see! They are now watching us walking around the facility!”  The puppies’ behavior had transformed: previously, they huddled in a corner; now they played together, avoided bumping into objects and were frisky. Measurable improvements were seen on testing.[15] The next question was, “Can we do this with blind children?” Varying the age of animal, AAV capsid serotype, promoter and dose helped to determine that efficacy was greatest in young animals and we determined the threshold dose.[15-17]

Unfortunately, the timing of the studies couldn’t have been worse as far as human trials. There was a death at our own institution (Jesse Gelsinger) in a study using a recombinant adenovirus vector and, over time, several cases of leukemia emerged in a trial using a retrovirus to treat severe combined immunodeficiency. Some of our collaborators moved the planned clinical trial out of state. We were approached by Dr. Katherine High at the Children’s Hospital of Philadelphia (CHOP) to run a pediatric gene therapy trial, which allowed us to continue the research. [When Jean told Jeremy Nathans (who had cloned rhodopsin) that she wanted to test gene therapy in humans, he said “You’ve got balls!”] We cloned the human cDNA, optimized the vector (Figure 1) and delivery procedures,[18] developing methods since incorporated into other AAV gene therapy trials. We had observed greatest efficacy in younger animals,[16] but gene therapy clinical trials for non-lethal disease had not previously included children. We were required by The NIH Recombinant DNA Advisory Committee (RAC),to justify the ethics of performing a phase 1 trial in children. The overwhelming evidence for a prospect for benefit in younger animals convinced the RAC and they voted unanimously to allow us to proceed.[19] We initiated a multi-center trial with a team in Italy, studies that were near-contemporaneous with two other Phase 1 trials (Figures 2, 3). All three Phase 1 trials reported positive results.[20-22]

There was no road map on how to proceed beyond Phase 1. Obstacles were numerous and included:

1) The FDA rejected our plan to compare the gene therapy-treated vs. the contralateral untreated eyes – an approach which we reasoned would give the best statistical outcomes in this rare patient population. They insisted that the contralateral eye should also be treated. We knew that in systemic diseases, readministration of gene therapy reagents had failed due to immune response after the initial delivery. We carried out studies in animal models to establish the safety and efficacy of sequential bilateral administration of AAV.hRPE65v2 [23] (Figure 4) and then cautiously tested readministration in the subjects who had already been treated unilaterally in our Phase 1 studies. Readministration was both safe and effective;[24, 25]  

2) The only outcome measures used previously to approve ophthalmic medications were irrelevant to the low vision subjects we planned to enroll. The FDA rejected our initial plan to use the objective measure of pupillary light reflex as a primary outcome measure, on the basis that the primary outcome should reflect functional vision. We therefore standardized and developed an exploratory outcome measure – navigation through a physical obstacle course – that we had implemented in phase 1 studies; [20, 26]

3) The FDA insisted that we run a randomized controlled Phase 3 study. We were concerned that if subjects risked randomization to a “no treatment” group, that no one would enroll. We addressed this by allowing control patients to cross to the intervention group after one year.[27] This also allowed us to collect data demonstrating that symptoms of the disease do not improve spontaneously;

4) The FDA insisted on a multi-center trial. Fortunately, we had a good relationship with Ed Stone and Steve Russell at University of Iowa who had been involved and were enthusiastic about a seminal gene therapy clinical trial;

5) Finally, there was a long process of finding a commercial sponsor, which eventually required the investigators at CHOP to leave the institution and form a biotech company, Spark Therapeutics, which then oversaw the project. [By the way, none of the authors have any financial interest in this product.]

The Phase 3 studies met all safety, primary and secondary endpoints, and improvements have been durable.[27-29] The FDA approved Voretigene Neparvovec-rzyl (Luxturna) on December 17, 2017. It was approved by the EMA in 2018. This reagent is now prescribed as a treatment for RPE65 disease in numerous Centers and countries around the world, thereby providing gene therapy experience to clinicians and hospitals. A commercialization approach was established, which accounts for a new paradigm for drug delivery –one where only a single treatment may be necessary (Figure 5).

The success of Luxturna, the first approved gene therapy in the USA and in Europe, unlocks the potential of the Human Genome Project and has paved the way for gene therapy to emerge as a viable approach for treating a broad spectrum of acquired and inherited retinal diseases. More than 3 dozen retinal gene therapy clinical trials are in progress, including several in Phase 3. The strategies include gene augmentation, gene editing, delivery of antibodies, decoy receptors and optogenetic molecules.

There are now dozens of biotechnology and established pharmaceutical companies that have invested in retinal gene therapy. Retina specialists now tell their IRD patients that there is something that they can do: They can be genotyped to see whether they might qualify for Luxturna treatment or participation in any of the ongoing or planned clinical trials. There is now a path for genetic treatments to blindness. While there are numerous obstacles ahead, it appears that gene therapy will be part of the retinal disease treatment armamentarium happily ever after.

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‍References 

1.         G. A.Fishman, A Historical Perspective on the Early Treatment of Night Blindness and the Use of Dubious and Unproven Treatment Strategies for Patients With Retinitis Pigmentosa. Surv Ophthalmol58, 652-663 (2013).

2.         R. D.Palmiter et al., Dramatic Growth of Mice That Develop From Eggs Microinjected with Metallothionein-growth Hormone Fusion Genes. Nature 300, 611-615 (1982).

3.         T. P.Dryja et al., A Point Mutation of the Rhodops in Gene in one Form of Retinitis Pigmentosa. Nature 343, 364-366(1990).

4.         G. J.Farrar et al., Autosomal Dominant Retinitis Pigmentosa: Linkage to Rhodopsin and Evidence for Genetic Heterogeneity. Genomics 8, 35-40 (1990).

5.         J.Bennett, J. Wilson, D. Sun, B. Forbes, A. Maguire, Adenovirus Vector-mediated In Vivo Gene Transfer Into Adult Murine Retina. Investigative Ophthalmology & Visual Science 35, 2535-2542 (1994).

6.         T. Li et al., In Vivo Transfer of a Reporter Gene to the Retina Mediated by an Adenoviral Vector. Investigative Ophthalmology and Visual Science 35, 2543-2549 (1994).

7.         J.Bennett et al., Photoreceptor Cell Rescue in Retinal Degeneration (rd) Mice by In Vivo Gene Therapy. NatureMedicine 2, 649-654 (1996).

8.         V.Anand et al., A Deviant Immune Response to Viral Proteins and Transgene Product is Generated on Subretinal Administration of Adenovirus and Adeno-associated Virus. Mol. Therapy 5, 125-132(2002).

9.         D.Budenz, J. Bennett, L. Alonso, A. Maguire, In Vivo Gene Transfer Into Murine Trabecular Meshwork and Corneal Endothelial Cells. Investigative Ophthalmology and Visual Science 36, 2211-2215 (1995).

10.       J.Bennett, D. Duan, J. F. Engelhardt, A. M. Maguire, Real-time, Noninvasive In Vivo Assessment of Adeno-associated Virus-mediated Retinal Transduction. Invest Ophthalmol Vis Sci 38, 2857-2863 (1997).

11.       J.Bennett, V. Anand, G. M. Acland, A. M. Maguire, Cross-species Comparison of In Vivo Reporter Gene Expression After Recombinant Adeno-associated Virus-mediated Retinal Transduction. Methods Enzymol316, 777-789 (2000).

12.       J.Bennett et al., Recombinant Adeno-associated Virus-mediated Gene Transfer to the Monkey Retina. Proc. Natl. Acad. Sci. USA 96, 9920-9925 (1999).

13.       G.Aguirre et al., Congenital Stationary Night Blindness in the Dog: Common Mutation in the RPE65 Gene Indicates Founder Effect. Mol. Vis. 4, 23 (1998).

14.       A.Veske, S. Nilsson, K. Narfstrom, A. Gal, Retinal Dystrophy of Swedish briard/briard-beagle Dogs is Due to a 4-bp Deletion in RPE65. Genomics 57, 57-61 (1999).

15.       G. M.Acland et al., Gene Therapy Restores Vision in a Canine Model of Childhood Blindness. Nat Genet 28, 92-95(2001).

16.       S. G.Jacobson et al., Identifying Photoreceptors in Blind Eyes Caused by RPE65 Mutations: Prerequisite for Human Gene Therapy Success. Proc Natl Acad SciU S A 102, 6177-6182 (2005).

17.       G. M.Acland et al., Long-term Restoration of Rod and Cone Vision by Single Dose rAAV-mediated Gene Transfer to the Retina in a Canine Model of Childhood Blindness. MolecularTherapy 12, 1072-1082 (2005).

18.       J.Bennicelli et al., Reversal of Blindness in Animal Models of Leber Congenital Amaurosis Using Optimized AAV2-mediated Gene Transfer. Mol Ther 16, 458-465 (2008).

19.       In Recombinant DNA Advisory (RAC) Committee.(Bethesda, MD, 2005).

20.       A. M.Maguire et al., Safety and Efficacy of Gene Transfer for Leber's Congenital Amaurosis. N Engl J Med 358,2240-2248 (2008).

21.       J. W.Bainbridge et al., Effect of gene therapy on visual function in Leber's congenital amaurosis. N Engl J Med 358, 2231-2239 (2008).

22.       W. W.Hauswirth et al., Treatment of Leber Congenital Amaurosis due to RPE65 Mutations by Ocular Subretinal Injection of Adeno-associated Virus Gene Vector: Short-term Results of a Phase I Trial. Hum Gene Ther 19, 979-990 (2008).

23.       D.Amado et al., Safety and Efficacy of Subretinal Re-administration of an AAV2 Vector in Large Animal Models: Implications for Studies in Humans. SciTransl Med 2, 21ra16 (2010).

24.       J.Bennett et al., AAV2 Gene Therapy Readministration in Three Adults with Congenital Blindness. Sci Transl Med 4, 120ra115 (2012).

25.       J.Bennett 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 388, 661-672 (2016).

26.       D. C.Chung et al., Novel Mobility Test to Assess Functional Vision in Patients with Inherited Retinal Dystrophies. Clin Exp Ophthalmol 46, 247-259. (2017).

27.       S.Russell 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 390, 849-860 (2017).

28.       A. M.Maguire et al., Durability of Voretigene Neparvovec for Biallelic RPE65-Mediated Inherited Retinal Disease: Phase 3 Results at 3 and 4 Years. Ophthalmology128, 1460-1468 (2021).

29.       A. M.Maguire 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 126,1273-1285 (2019).

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(Milestone essay published 2022)

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Read more on this topic

The Story Behind Luxturna: Retina Romance Leads to a Gene TherapyBreakthrough, Retina Times, Spring2018