Update on gene therapy

A discussion of strategies, optimization, alternatives and administration.

Ocular gene therapy research has shown tremendous growth over recent years, especially after the 2017 FDA approval of voretigene neparvovec-rzyl (LUXTURNA, Spark Therapeutics), the first U.S. gene therapy for a genetic disease. The retina remains a lead focus for gene therapy research, given the number of monogenic disorders, accessibility to target cell delivery, the noninvasive ability to monitor for disease progression or therapeutic response and relative immune privilege, which limits inflammatory response.1-3

Our prior article in Ophthalmology Management provided an introduction to ocular gene therapy ( ).4 This article will provide an update with respect to gene augmentation, inactivation and editing strategies, optimization of viral vectors, alternatives to viral vector gene therapy and evolving ocular administration routes.


The major categories of gene therapy include gene augmentation (adding a gene to a cell), gene editing (revising the existing genetic code), gene inactivation (silencing a gene, often a dominant-negative one) and selective toxicity (introducing “suicide” genes and immune sensitization, as in chimeric antigen receptor [CAR] T Cells to recognize cancer cells).

Ocular gene therapy development strategies have mostly involved gene augmentation to introduce the normal copy of a gene to correct for loss of function, of which recessive single-gene disorders are most amenable. RPE65 mutation-associated inherited retinal disease (IRD) is one such recessive monogenic disorder, which results from total or near absence of functional protein. LUXTURNA, currently the only FDA-approved ocular gene therapy, reestablishes normal gene product and corrects the lost function in this disorder. Other recessive disorders currently undergoing gene augmentation clinical study include achromatopsia (AGTC, MeiraGTx), Stargardt disease (Oxford Biomedica/Sanofi) and Usher syndrome (Oxford Biomedica/Sanofi). Gene augmentation also is undergoing clinical investigation for X-linked recessive disorders, such as choroideremia (Biogen, Spark Therapeutics) and X-linked retinitis pigmentosa (AGTC, Biogen, MeiraGTx), as well as mitochrondrially-inherited disorders such as Leber hereditary optic neuropathy (Gensight), although a mitochrondrial targeting sequence is necessary to shuttle messenger RNA (mRNA) from the nucleus to the mitochondria.

On the other hand, toxic gain-of-function mutations in autosomal dominant conditions have been less responsive to treatment by gene therapy, as one copy produces an abnormal product that must be suppressed.5 For example, development for the most common autosomal dominant IRD, autosomal dominant retinitis pigmentosa (adRP), has been more challenging. One promising strategy involves short hairpin RNAs (shRNAs) using a “knockdown and replace” model. The shRNAs are noncoding RNA (ncRNA) that regulate mRNA expression and can be encoded into DNA to be introduced into the cell nucleus by a vector for continuous production. With this approach, the abnormal gene product is silenced using an shRNA targeting the causative mutated form of the rhodopsin (RHO) mRNA and is co-packaged with a resistant form of human RHO gene in a single vector.6 After promising results in canine models, further investigation is planned in human clinical trials (Iveric BIO).6,7

Gene editing, in contrast, can target autosomal dominant mutations by generating a precise DNA modification at a target locus to silence or edit the dysfunctional gene (Figure 2). Here, through the CRISPR/Cas9 system, the target locus is bound, and a double-stranded break is generated and then repaired by either non-homologous end joining, which can lead to insertion/deletion mutations and thus premature stop codons, or by homologous-directed repair using a template.8,9 The first human study of in-vivo CRISPR genome editing is currently in a Phase 1/2 clinical trial (Editas Medicine) to remove an abnormal splice site in the CEP290 gene which causes Leber congenital amaurosis 10,10 a severe autosomal recessive retinal dystrophy. Editas plans to develop genome editing therapies for Usher syndrome 2A and adRP.11

Figure 1. Adenovirus-mediated gene therapy (Image courtesy 331pten / CC BY-SA [ ])

In addition to treating IRDs by addressing genetic mutations in native genes, alternative strategies are attempting to treat multifactorial acquired conditions in a “biofactory approach.” Here, the goal is to convert cells into “biofactories,” producing and secreting non-native therapeutic proteins. For example, in neovascular AMD, gene therapies are in development to promote production and secretion of anti-vascular endothelial growth factor (VEGF) agents from retinal cells. There have been promising Phase 1/2a clinical trial results, either through subretinal delivery resulting in production of a ranibizumab-like anti-VEGF protein (REGENXBIO) or intravitreal delivery resulting in expression of aflibercept-like anti-VEGF protein (Adverum).12,13

Additionally, gene therapies are in development for AMD to target and inhibit components of the complement system, given its potential role.14 For example, Hemera Biosciences is developing HMR59 to continuously produce a soluble protein called CD59 (sCD59) that blocks the final step of the complement cascade. Hemera is conducting Phase 1 clinical trials in non-neovascular and neovascular AMD.15


Gene therapy requires the delivery and transfer of the genetic material to host cells, with a variety of viral and non-viral vectors developed for this purpose. Various factors influence the choice of the vector, including the target tissue, the cloning capacity of the vector and safety concerns (inflammatory responses and possibility of genotoxicity/insertional oncogenesis). Importantly, while the capsid antigens of vectors can invoke immunogenicity, the subretinal space provides a relative immune privilege, which limits inflammatory response.16

Originally studied with respect to corneal transplantation, immune privilege is thought to arise from immune ignorance (absence of lymphatic drainage, absence of MHC class-II expressing antigen presenting cells and angiostasis), anterior chamber-associated immune deviation (ACAID) and an intraocular immunosuppressive microenvironment.1 Additionally, murine studies have demonstrated immune deviation in the subretinal space.17

With respect to investigational retinal gene therapy, adeno-associated viruses (AAV) are the most utilized vector. AAV are small (approximately 25 nm) single-stranded DNA viruses of the parvovirus family that are non-pathogenic, non-replicating and non-integrating (Figure 1). Further, AAVs exhibit low immunogenicity and have demonstrated excellent safety in human trials.18-20 The AAV genome is packaged in an icosahedral capsid made of three structural proteins that contain variable loops leading to specific surface topologies for each AAV serotype, facilitating the interaction of the viral particle with the host.19

Figure 2. Illustration of gene editing. (Image courtesy Mariuswalter / CC BY-SA [ ])

More than 100 AAV variants have been identified, with multiple shown to display tropism for retinal tissue.21,22 Three of the naturally occurring serotypes (AAV2, AAV5, AAV8) have been most studied, demonstrating ability to efficiently transduce RPE when administered in the subretinal space.19 When administered in the vitreous cavity, however, use of these serotypes is limited due to inadequate transduction through the internal limiting membrane (ILM) and by potential preexisting neutralizing antibodies.23

Recently, second-generation AAVs have been in development to bypass limitations such as the ILM, cell specific tropism or immunogenicity. Advancement of these novel vectors is underway with two current approaches: rational design and directed evolution. Rational design works to introduce mutations in capsid proteins (e.g. capsid tyrosine to phenylalanine) to modify structure and function, reduce viral degradation, eliminate antibody binding epitopes or direct vector tropism for specific cell types.24 Directed evolution, in contrast, relies on the introduction of random genetic variability to generate large AAV libraries, which have undergone mutagenesis of capsid proteins. These vectors are then screened in vivo for transduction efficiency or specificity of a target tissue. In ocular gene therapy, for example, directed evolution has been utilized to develop a novel AAV variant, 7m8, that can more effectively cross the ILM and transduce retina following intravitreal administration.25


Several alternatives to viral vector gene therapy are also in development, with hopes of mitigating limitations associated with viral vectors. One example is naked plasmid DNA, which is less immunogenic, has a lower risk of insertional mutagenesis and has a larger cargo capacity. However, naked plasmid DNA also show limited cellular uptake and poor nuclear import.26

Strategies are in development to improve the ability of DNA to enter cells, with electrotransfection showing the most promise. Otherwise known as electroporation, this approach involves applying a local and short external electric field to a cell, modifying its permeability and facilitating entry of the DNA.26 First reported in skeletal muscle fibers, this strategy has been applied to ocular gene therapy. A Phase 1/2 clinical trial has demonstrated long-lasting intraocular expression and secretion of a protein that neutralizes the activity of TNF-alpha following electrotransfection of the ciliary muscle, using a biofactory approach in patients with chronic non-infectious uveitis (Eyevensys).27

Additionally, non-viral methods of delivery are being explored. DNA-compacted nanoparticles, for example, are single DNA molecules with carrying capacity larger than AAV that yield lower risk of immune response in patients with pre-existing viral immunity. In a Phase 1 clinical trial in patients with cystic fibrosis, nanoparticles have been shown to safely introduce the transmembrane conductance regulator lung cells following intranasal administration.28 Mouse models using these molecules have also shown promising results, including functional rescue of Stargardt disease following nanoparticle-mediated ABCA4 delivery.29

RNA-based therapy represents another form of gene therapy. The majority of RNA therapeutics in development are synthetic ncRNAs that regulate mRNA expression, are mutation specific (like gene editing but unlike gene augmentation), can be administered intravitreally and consequently have potential for pan-retinal effect. ncRNAs are inherent in the genome and carry essential regulatory functions, while disease states can lead to their differential expression, potentially contributing to disease development and progression. Antisense oligonucleotides (ASOs) are single-stranded synthetic RNAs (or DNAs) that have shown promise (Figure 3). ProQR, for example, is developing an ASO to address a common mutation that causes an aberrant splice site in ABCA4 in patients with Stargardt disease.30 Additional work by ProQR is underway to address mutations for conditions such as Leber congenital amaurosis 10, Usher syndrome type 2 and adRP.

Figure 3. Antisense DNA oligonucleotide (Image courtesy Robinson R / CC BY [ ])

RNA-based therapy could also address non-sense mutations, which lead to premature stop codons and resulting complete loss of functional protein. Current RNA-based treatment strategies include translational read-through inducing drugs, such as eukaryotic ribosomal selective glycosides (ERSG), to increase ribosomal read-through activity of mRNA with these non-sense mutations and enable production of full-length proteins.31 Eloxx Pharmaceuticals is developing subcutaneously administered ESRGs to address cystic fibrosis and cystinosis, with plans to apply this strategy to IRDs caused by non-sense mutations, with initial focus on Usher syndrome.31


Delivery of the vector to the target retinal tissue involves several potential approaches. Most commonly, delivery of genetic material to the RPE is achieved through pars plana vitrectomy, followed by retinotomy with subsequent injection of the vector in the subretinal space.

While this procedure creates a temporary retinal detachment, it allows for direct delivery of the genetic material to the RPE or photoreceptors. The viral particle then enters these cells, leading to expression of the protein through the host’s translational machinery. Subretinal administration is still an evolving procedure, with new optimizing technologies such as smaller subretinal cannulas, precision infusion pumps and intraoperative OCT to precisely monitor proper bleb formation.

Less invasive administration procedures with potential for fewer procedure-related complications are being sought. Injections into the vitreous cavity, for example, may provide a feasible alternative to subretinal delivery for some disorders. Intravitreal injection, however, is considered more immunogenic compared to subretinal injection32,33 given the subretinal space’s tight blood-ocular barrier, which limits systemic dissemination and exposure of vector to neutralizing antibodies.16,34

Furthermore, the ILM hinders penetration of the viral vector to underlying retinal layers, although, as discussed above, techniques such as “directed evolution” of viral vectors aim to enhance this intravitreal delivery.25,35 For example, Adverum’s ADVM-022 employs a directed evolution-derived viral vector (AAV.7m8) that facilitates intravitreal delivery, with promising results in early clinical trials in neovascular AMD.13 Additionally, suprachoroidal delivery of vectors is a potential nonsurgical in-office technique. REGENXBIO plans to assess suprachoroidal delivery in their ongoing RGX-314 clinical trials in neovascular AMD and ultimately in diabetic retinopathy, after promising preclinical studies with this delivery route.36


Gene therapy continues to evolve rapidly, and it is important to be aware of the basics given the potential treatments on the horizon. Novel gene inactivation and editing strategies may address rare autosomal dominant hereditary retinal disorders, while evolving biofactory approaches may address common multifactorial disorders, such as AMD and diabetic retinopathy. Additionally, optimization of viral vectors may enhance immune tolerance, cell specific tropism and intraocular delivery possibilities, while alternative gene therapies, including RNA therapeutics, as well as evolving administration techniques, could further expand therapeutic possibilities.

Technologic advances continue to drive innovative treatment strategies for the potential benefit of numerous retina patient populations. OM


  1. Streilein JW. Ocular immune privilege: therapeutic opportunities from an experiment of nature. Nat Rev Immunol. 2003; 3:879-889.
  2. Takahashi VKL, Takiuti JT, Jauregui R, Tsang SH. Gene therapy in inherited retinal degenerative diseases, a review. Ophthalmic Genet, 2018;39:560-568.
  3. Boye SE, Boye SL, Lewin AS, Hauswirth WW. A comprehensive review of retinal gene therapy. Mol Ther. 2013;21:509-519.
  4. Ciulla, TA. Introduction to gene therapy. A review of genetic testing and basics of gene therapy. Ophthalmology Management. July 2019:30-32, 47.
  5. Sengillo JD, Justus S, Tsai YT, Cabral T, Tsang SH. Gene and cell-based therapies for inherited retinal disorders: An update. Am J Med Genet C Semin Med Genet. 2016;172:349-366.
  6. Cideciyan AV, Sudharsan R, Dufour VL, et al. Mutation-independent rhodopsin gene therapy by knockdown and replacement with a single AAV vector. Proc Natl Acad Sci USA. 2018;115:E8547-E8556.
  7. Gene Therapy ­­- IVERIC bio. IVERIC bio. . Accessed May 7, 2020.
  8. Long C, McAnally JR, Shelton JM, et al. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science. 2014;345:1184-1188.
  9. Ziccardi L, Cordeddu V, Gaddini L, et al. Gene Therapy in retinal dystrophies. Int J Mol Sci. 2019;20:5722.
  10. Maeder ML, Stefanidakis M, Wilson CJ, et al. Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nat Med. 2019:25:229-233.
  11. Research and Pipeline - Editas Pipeline. Editas Medicine. Available from: . Accessed May 20, 2020.
  12. RGX-314 - REBENXBIO. REGENXBIO. . Accessed May 7, 2020.
  13. Pipeline - Adverum Biotechnologies. Adverum Biotechnologies. . Accessed May 7, 2020.
  14. Mullins RF, Schoo DP, Sohn EH, et al. The membrane attack complex in aging human choriocapillaris: relationship to macular degeneration and choroidal thinning. Am J Pathol. 2014;184: 3142-3153.
  15. Clinical Trials: Hemera Biosciences. Hemera Biosciences. . Accessed May 12, 2020.
  16. Seitz IP, Michalakis S, Wilhelm B, et al. Superior retinal gene transfer and biodistribution profile of subretinal versus intravitreal delivery of AAV8 in nonhuman primates. Invest Ophthalmol Vis Sci. 2017;58:5792-5801.
  17. Wenkel H, Streilein JW. Analysis of immune deviation elicited by antigens injected into the subretinal space. Invest ophthalmol vis sci. 1998;39:1823-34.
  18. Carter PJ, Samulski RJ. Adeno-associated viral vectors as gene delivery vehicles. Int J Mol Med. 2000;6:17-27.
  19. Day TP, Byrne LC, Schaffer DV, Flannery JG. Advances in AAV vector development for gene therapy in the retina. Adv Exp Med Biol. 2014;801:687-693.
  20. Daya S, Berns KI. Gene therapy using adeno-associated virus vectors. Clin Microbiol Rev. 2008;21:583-593.
  21. Ong T, Pennesi ME, Birch DG, Lam BL, Tsang SH. Adeno-associated viral gene therapy for inherited retinal disease. Pharm Res, 2019;36:34.
  22. Srivastava A. In vivo tissue-tropism of adeno-associated viral vectors. Curr Opin Virol. 2016;21:75-80.
  23. Calcedo R, Vandenberghe LH, Gao G, Lin J, Wilson JM. Worldwide epidemiology of neutralizing antibodies to adeno-associated viruses. J Infect Dis. 2009;199:381-390.
  24. Bartel MA, Weinstein JR, Schaffer DV. Directed evolution of novel adeno-associated viruses for therapeutic gene delivery. Gene Ther. 2012;19:694-700.
  25. Dalkara D, Byrne LC, Klimczak RR, et al. In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous. Sci Transl Med. 2013;5:189ra76.
  26. Bordet T, Behar-Cohen F. Ocular gene therapies in clinical practice: viral vectors and nonviral alternatives. Drug Discovery Today, 2019;24:1685-1693.
  27. Pipeline - EYS606. Eyevensys. . Accessed May 7, 2020.
  28. Konstan MW, Davis PB, Wagener JS, et al. Compacted DNA nanoparticles administered to the nasal mucosa of cystic fibrosis subjects are safe and demonstrate partial to complete cystic fibrosis transmembrane regulator reconstitution. Hum Gene Ther. 2004;15:1255-69.
  29. Han Z, Conley SM, Makkia RS, et al. DNA nanoparticle-mediated ABCA4 delivery rescues Stargardt dystrophy in mice. J Clin Inves. 2012;122:3221-3226.
  30. ProQR R&D Day Presentation. ProQR. . Accessed May 7, 2020.
  31. Pipeline - Eloxx Pharmaceuticals. Eloxx Pharmaceuticals. . Accessed May 7, 2020
  32. Reichel FF, Peters T, Wilhelm B, et al., Humoral immune response after intravitreal but not after subretinal AAV8 in primates and patients. Invest Ophthalmol Vis Sci. 2018;59:1910-1915.
  33. Li Q, Miller R, Han PY, et al. Intraocular route of AAV2 vector administration defines humoral immune response and therapeutic potential. Mol Vis. 2008;14:1760-1769.
  34. Kotterman MA, Yin L, Strazzeri JM, et al. Antibody neutralization poses a barrier to intravitreal adeno-associated viral vector gene delivery to non-human primates. Gene Ther. 2015;22:116-126.
  35. Dalkara D, Kolstad KD, Caporale N, et al. Inner limiting membrane barriers to AAV-mediated retinal transduction from the vitreous. Mol Ther. 2009;17:2096-2102.
  36. Ding K, Hen J, Hafiz Z, et al. AAV8-vectored suprachoroidal gene transfer produces widespread ocular transgene expression. J Clin Invest, 2019;129:4901-4911.

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