The basic concept of gene therapy for inherited retinal disease (IRD) is simple: replace a defective gene with a normal copy to treat disease. In vivo gene therapy involves the delivery of genetic material directly to living organisms, while ex vivo gene therapy delivers the genetic material to cultured cells, which are then transplanted into the host. In vivo gene therapy employs a viral vector to insert genetic material into host cells. The goal is to achieve stable expression of the inserted genetic material in the host cell while avoiding complications from inflammation or deleterious off-site effects. In theory, ophthalmic disorders are especially attractive targets for in vivo gene therapy, because local therapy can easily be delivered to the eye and inflammatory responses may be limited because the eye is an immune-privileged organ. However, despite this advantage, the vast majority of gene therapy trials to date target nonophthalmic disorders and many employ ex vivo approaches. Recently, greater focus has been placed on using gene therapy to treat ocular disease in vivo largely due to the success of voretigene neparvovec-rzyl (Luxturna; Spark Therapeutics) for the treatment of RPE65-associated retinal dystrophy. This review will focus on current gene therapy trials aimed at treating retinal disease (Table 1), with a focus on IRDs.

INSERTING GENETIC MATERIAL INTO LIVING CELLS

Current methods to deliver genetic material in vivo can be categorized as viral and nonviral methods. Viruses, by nature, infect cells and insert their genetic material into the host cell’s nucleus. After removing pathogenic viral genes, viral vectors can be used to insert a desirable gene (or cDNA) into the host. Viral vectors either integrate their genetic material directly into the host’s genome (lentivirus and retrovirus) or insert their genetic material as stable extrachromosomal episomes (adenovirus, herpes virus, and adeno-associated virus).¹ Nonviral methods of gene delivery rely on chemical or physical methods to insert naked DNA into a cell’s nucleus. These methods include electroporation, iontophoresis, liposomes, and nanoparticles, such as cell-penetrating peptides.² Nonviral methods have the benefits of production scalability, low immunogenicity, and the ability to deliver large genes. However, they are less efficient than viral vectors at targeting cells in vivo.

RETINAL DISEASES TARGETED FOR GENE THERAPY

Currently, IRDs and other retinal diseases targeted for gene therapy rely on gene augmentation. A normal copy of a gene is inserted by a recombinant virus to reestablish normal cell function. This transgene exists in extrachromosomal DNA as an episome, and there is no direct replacement of the abnormal gene. This strategy will work for loss-of-function mutations, but not for dominant gain-of-function alleles. This article will focus on reviewing the gene therapies that are currently targeting inherited and rare retinal diseases. The sidebar presents a brief overview of research into gene therapy for AMD, which, although it is not an inherited or rare disease, is relevant to the overall discussion of clinical trials in ocular gene therapy, as the number of trials studying gene therapy for AMD grows.

In 2017, the FDA approved the first in vivo gene therapy for a genetic disease when it approved Luxturna for RPE65-associated retinal dystrophy. Patients with pathogenic variants in both alleles of RPE65 typically present clinically with autosomal recessive Leber congenital amaurosis (LCA) or retinitis pigmentosa (RP). Vision loss is progressive and variable and may be severe in early childhood. The RPE65 gene is expressed in RPE and is one of the critical enzymes in the visual cycle, necessary for converting all-trans-retinol to 11-cis-retinal required for proper photoreceptor health and function. Human RPE65 cDNA is delivered using an AAV2 vector injected into the subretinal space. Spark Therapeutics manufactures and markets AAV2-hRPE65 as Luxturna for commercial use. The phase 3 trial that led to its approval showed significant improvements in navigation in dim light, full-field light sensitivity threshold, and visual field in the treated group.³

Choroideremia is an X-linked disorder caused by a defective CHM gene, which encodes the rab escort protein 1 (REP-1). Loss-of-function REP-1 results in progressive retinal, retinal pigment epithelium, and choroidal atrophy. Nyctalopia and peripheral field loss initially develop in childhood, eventually progressing to severe vision loss. Central vision is usually well preserved early in the disease process. Two clinical trials are under way using AAV2 to deliver human CHM cDNA via subretinal injections. Spark Therapeutics is sponsoring a phase 1/2 dose-escalating trial (NCT02341807), and Biogen (formerly Nightstar Therapeutics) is enrolling patients in a phase 3 randomized controlled efficacy and safety trial (NCT03496012). Reports of the prior phase 2 Nightstar trial showed good safety profile, with no serious adverse event. Although there were no statistically significant changes in visual acuity, 2 eyes in 6 treated patients demonstrated a gain of 5 letters and 10 letters.⁴

Retinoschisis is an X-linked disorder caused by a LOF RS1 gene. The RS1 gene is expressed in photoreceptors and bipolar cells, is believed to be important for cell-to-cell interactions, and is responsible for the structural integrity of the retina. Macular schisis is seen early in the disease process and may cause severe central vision loss in childhood. Patients may present with vitreous hemorrhage and/or retinal detachment. The schisis cavities can extend into the periphery, and vision loss may progress. There are currently 2 gene therapy trials utilizing AAV vectors with different cDNA constructs utilizing different promoters. In contrast to the other retina gene therapy trials described above, the vector is delivered with an intravitreal injection instead of a subretinal injection. A phase 1/2 study sponsored by Applied Genetic Technologies Corporation utilized an AAV2 vector and a ubiquitous chicken beta-actin promoter with cytomegalovirus (CMV) enhancer driving the expression of RS1 (NCT02416622). Unfortunately, due to unfavorable results, this study will likely be suspended. A second ongoing study sponsored by the National Eye Institute uses AAV8 with a modified human RS1 promoter augmented by an interphotoreceptor retinoid binding protein enhancer element (NCT02317887). Preliminary data suggest the vector is safe but little clinical efficacy was seen in all but 1 patient, who showed a transient decrease in schisis cavities in the study eye.⁵

The most common cause of X-linked RP is mutations in RPGR. The gene product’s function is unknown, but is thought to regulate ciliogenesis in photoreceptors. Loss-of-function variants cause photoreceptor degeneration and clinically present as either RP, cone, or cone/rod dystrophy. There are currently 3 different clinical trials targeting RPGR retinal dystrophy.⁶ A phase 1/2 open-label dose-escalation study sponsored by AGTC utilizes an AAV2 vector and rhodopsin kinase promoter to drive expression of RPGR cDNA in photoreceptors (NCT03316560). A second early phase dose-escalation trial is sponsored by MeiraGTx using an AAV2/5 vector and rhodopsin kinase promoter to drive the expression of RPGR (NCT03252847). The third clinical trial is sponsored by Biogen and uses an AAV8 vector with a codon-optimized RPGR cDNA (NCT03116113). Biogen is currently expanding the study into a phase 2/3 safety and efficacy trial with a control group.

Achromatopsia is caused by a loss of cone photoreceptor function. In complete achromatopsia, all 3 axes of color (red, green, blue) are impaired. Patients present with central vision loss, nystagmus, photophobia, and absent color discrimination from birth. Incomplete achromatopsia is less severe and could present with a partially functional cone subtype. Achromatopsia is a recessive disorder caused by LOF mutations in a variety of genes. Loss-of-function mutations in 2 of the most common genes affected, CNGA3 and CNGB3, is currently targeted for gene therapy.⁷ AGTC is sponsoring 2 phase 1/2 dose-escalation trials for CNGA3 (NCT02935517) and CNGB3 (NCT02599922) using subretinal injections of AAV2 vectors. The vectors use an engineered cone opsin promoter driving the expression of CNGA3 or CNGB3. MeiraGTx is sponsoring a similar phase 1/2 dose-escalation trial for both CNGA3 (NCT03758404) and CNGB3 (NCT03001310). As of September 2019, the CNGA3 trial is enrolling in the United States and United Kingdom, but the CNGB3 trial is only available to patients in the United Kingdom. The vector used is AAV2/8, and CNGB3 is driven by the human cone arrestin promoter, whereas CNGA3 uses an engineered cone opsin promoter.

Stargardt macular dystrophy is the most common macular dystrophy, causing progressive central and color vision loss as well as prolonged dark adaptation. Mutations in the ABCA4 gene are the most common cause. The ABCA4 gene encodes a membrane transporter important for phototransduction and also removes toxic byproducts from the phototransduction cascade. The ABCA4 transcript size is approximately 6.8 kb, which is too large to package into AAV vectors. Sanofi is studying a lentivirus with a larger capacity to deliver ABCA4 in patients with Stargardt macular dystrophy. In addition to a larger carrying capacity, lentiviruses integrate their genetic material into the host genome, theoretically resulting in a more stable transfection. The Sanofi trial uses an equine infectious anemia virus, which is unable to cause disease in humans (NCT01367444). The expression of ABCA4 is driven by the ubiquitous CMV promoter.⁸ The use of lentivirus (and other retroviruses), however, has its drawbacks, including a risk of deleterious effects from viral DNA integration. This is best illustrated by the development of T-cell leukemia in patients with severe combined immunodeficiency syndrome.

Usher syndrome 1B is a ciliopathy caused by mutations in the myosin 7A (MYO7A) gene. Patients with Usher syndrome 1B present with profound congenital hearing loss, vestibular dysfunction, and early progressive vision loss due to retinitis pigmentosa. Myosin 7A functions as a molecular motor and is important for melanosome trafficking and the translocation of key phototransduction cascade proteins, such as RPE65 and alpha transducing.⁹ Similar to ABCA4, the MYO7A transcript (~7kb) is larger than the 4.8kb limit for AAV vectors. Using the same equine lentivirus vector for the Stargardt trial, Sanofi is currently sponsoring an Usher 1B phase 1/2 open label dose-escalation trial (NCT01505062). The expression of MYO7A is driven by the CMV promotor and the vector is delivered into the subretinal space.

FUTURE DIRECTIONS

Current gene therapy strategies involve gene augmentation, which works by supplying a functional gene product. This strategy, however, is ineffective for disorders involving gain-of-function variants. Inhibition of the gene product would be necessary. One strategy would be delivery of RNAi or microRNAs, which act to silence expression of genes. Both viral and nonviral delivery approaches can be used. Gene editing technology using the CRISPR/CAS9 system would also be well suited to correcting dominant gain-of-function mutations. A CRISPR/CAS9-based approach would involve editing out the pathogenic variant in a targeted fashion. A viral vector could in theory deliver a guide RNA and the CAS9 nuclease. However, low efficiency and off-target effects would need to be overcome before CRISPR/CAS9 can be used in vivo.

The bridge between basic science advancements and translation into clinical applications is expanding with each successful gene therapy trial. As therapeutic options increase, the need for molecular diagnosis will become increasingly important for IRDs. RP

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Editor’s note: This article is part of a special edition of Retinal Physician that was supported by REGENXBIO.