Gene therapy using viral vectors is a technology to complement or repair dysfunctional genes. The eye has three characteristics—immune privilege, the blood-retinal barrier, and terminally differentiated cells—making it particularly suitable for gene therapy.
Immune privilege is a mechanism by which the eye limits inflammatory responses to maintain visual function. Immunomodulatory molecules suppress inflammatory cells and reduce immune responses to the transgene. The blood-retinal barrier limits systemic exposure of drugs, reducing side effects. Because the eye is composed of terminally differentiated cells (photoreceptors and RPE cells), the risk of chromosomal integration and insertional mutagenesis is inherently low.
Over 270 gene mutations causing inherited retinal diseases (IRDs) have been discovered, primarily affecting photoreceptors or RPE cells. Since many IRDs are monogenic, they are ideal targets for gene replacement therapy.
In December 2017, the FDA approved voretigene neparvovec (brand name: Luxturna), developed by Spark Therapeutics. The EMA also approved it in 2018 5). This is the world’s first ophthalmic gene therapy product for IRDs (LCA2 and retinitis pigmentosa) associated with biallelic mutations in the RPE65 gene.
The eye has characteristics such as immune privilege, the blood-retinal barrier, and a closed space, allowing small amounts of vector to efficiently reach target cells. Additionally, since the retina consists of non-dividing terminally differentiated cells, genes are stably expressed over the long term. These factors accelerate gene therapy in ophthalmology.
The symptoms of hereditary retinal diseases targeted by gene therapy vary by disease, but the main symptoms are as follows.
Fundus Findings in LCA2
Bone spicule pigmentation: Black granular deposits around retinal vessels and in the mid-periphery. Classic finding in RP.
Retinal vascular attenuation: Arteries become narrower with progression.
Optic disc pallor: Observed in advanced stages.
Reduced FAF: Autofluorescence is diminished due to visual cycle impairment. Characteristic of RPE65-IRD.
Comorbid Findings After VN Treatment
Chorioretinal atrophy (CRA): Reported in 13–28% of cases postoperatively. It can also occur outside the retinotomy site 2).
Subretinal bleb formation: A temporary detachment of photoreceptors and RPE after subretinal injection.
Vitritis: An inflammatory reaction that mainly occurs early after treatment.
Posterior subcapsular cataract: Reported in long-term cases1).
The main viral vectors used in ophthalmic gene therapy are AAV, adenovirus, and lentivirus. Their characteristics are shown below5).
| Vector | Nucleic acid | Capacity | Immunogenicity | Insertional mutagenesis |
|---|---|---|---|---|
| AAV | Single-stranded DNA | ~4.7 kb | Low | Low (episomal) |
| Adenovirus | Double-stranded DNA | Up to 37 kb | High | Low (episomal) |
| Lentivirus | Single-stranded RNA | 8–10 kb | Medium | Yes (chromosomal integration) |
AAV is a non-enveloped, single-stranded DNA virus belonging to the Parvoviridae family. It is currently the most widely used vector in retinal gene therapy5).
The advantages of AAV are as follows5):
Thirteen distinct serotypes have been identified, with AAV2, AAV4, AAV5, and AAV8 being primarily used in ophthalmology. AAV2 successfully infects the inner retina from the vitreous side, but its reach to the outer retina is limited. Subretinal administration of AAV2 and AAV8 shows effective transduction of RPE and photoreceptors, respectively.
Approximately 70% of the general population has pre-existing antibodies against AAV2, and 38% have antibodies against AAV8. Since these neutralizing antibodies are associated with reduced gene expression, AAV8-based vectors may be more effective than AAV2.
The packaging capacity is limited to approximately 4.7–4.8 kb, making it unable to accommodate large genes such as ABCA4 (Stargardt disease) and MYO7A (Usher syndrome)5).
Adenoviruses are non-enveloped double-stranded DNA viruses that can carry genes up to 37 kb. They have the rapid characteristic of starting expression within 48 hours after injection. However, they trigger strong immune responses, and severe side effects such as fever, liver damage, systemic infection, and death have been reported. Therefore, in ophthalmology, they are currently used only in a single trial for retinoblastoma research5).
Lentiviruses are single-stranded RNA retroviruses derived from HIV, equine infectious anemia virus (EIAV), etc. They can carry genes of 8–10 kb and are effective for transducing RPE cells, but cannot efficiently target photoreceptors. Unlike AAV and adenovirus, they integrate complementary DNA into the chromosome, posing a risk of insertional mutagenesis5). EIAV vectors have been used in clinical trials for Stargardt disease and Usher syndrome (USH1B).
Because the capacity of AAV is limited to approximately 4.7–4.8 kb, diseases requiring coding of large genes exceeding this size, such as ABCA4 (Stargardt disease), MYO7A (Usher syndrome), and EYS, cannot be addressed with AAV5). In such cases, lentiviral vectors become an option, but they carry the risk of chromosomal integration.
There are three main routes of administration for ophthalmic viral gene therapy. The characteristics and indications of each route are shown below.
Intravitreal Injection
Invasiveness: Least invasive. Can be performed as an outpatient procedure under local anesthesia.
Target Area: Primarily the inner retina. The inner limiting membrane (ILM) is a major barrier to reaching the outer retina (RPE and photoreceptors)5).
Immune Response: Humoral immune response is likely to be strong. Neutralizing antibodies may be produced, affecting administration of the same vector to the contralateral eye.
Complications: Endophthalmitis, retinal detachment (incidence <1%).
Subretinal Injection
Invasiveness: Most invasive. Requires pars plana vitrectomy in the operating room.
Target site: Direct access to the outer retina (RPE and photoreceptors). Voretigene neparvovec is administered via this route 5).
Immune response: Minimal humoral immune response. Less inflammation than intravitreal injection 5).
Complications: Macular hole, subretinal hemorrhage, fibrosis, retinal detachment.
Suprachoroidal administration is a relatively new route that can broadly target the peripheral RPE and choroid, potentially avoiding direct manipulation of the macula 5).
Performed in the operating room under retrobulbar anesthesia (or general anesthesia). After disinfecting the ocular surface with iodine, a 23- or 25-gauge pars plana vitrectomy is performed. Then a 41-gauge tip is placed into the subretinal space, a bleb is formed by injecting intraocular irrigation solution (BSS), and the viral vector is administered. Intraoperative OCT is useful for localization and improving injection accuracy.
Postoperatively, the patient is instructed to remain supine for 2–24 hours to keep the fluid in the posterior pole. Oral prednisone is administered for 21–61 days postoperatively, tapering after the first 2 weeks. Follow-up includes assessment of visual acuity, visual field, microperimetry, ERG, OCT, fundus photography, and autofluorescence (AF).
Voretigene neparvovec (Luxturna) is an ophthalmic viral gene therapy product approved by the FDA (2017) and EMA (2018) for IRD associated with biallelic mutations in the RPE65 gene5). It incorporates the RPE65 transgene into AAV2 and is delivered to surviving RPE cells via subretinal injection.
In a phase III trial, 29 patients aged 3 years and older with visual acuity of 20/60 or worse participated, and functional vision improvement was demonstrated on the multi-luminance mobility test (MLMT) (mild inflammation occurred in 2 patients, but no serious complications).
Eligibility criteria are as follows:
| Disease | Gene | Vector | Route of Administration |
|---|---|---|---|
| X-linked retinoschisis (XLRS) | RS1 | AAV | Intravitreal |
| Stargardt disease | ABCA4 | EIAV (lentivirus) | Subretinal |
| Choroideremia | CHM (REP1) | AAV2 | Subretinal |
| Retinitis pigmentosa (X-linked) | RPGR | AAV8/AAV9 | Subretinal |
| Achromatopsia | CNGA3/CNGB3 | AAV | Subretinal |
| Neovascular age-related macular degeneration | aflibercept | AAV2 | Intravitreal |
| LHON | ND4 | AAV2 | Intravitreal |
Currently, more than 30 clinical trials of ophthalmic gene replacement therapy are underway.
The indication is based on the presence of biallelic pathogenic mutations in the RPE65 gene and the presence of viable retinal cells that can be treated 5). Since complete blindness with near-zero visual function is unlikely to benefit, treatment is important when residual function is confirmed.
RPE65 (retinoid isomerohydrolase) is an enzyme highly expressed in RPE cells and plays a role in converting all-trans-retinyl esters to 11-cis-retinol in the visual cycle. Deficiency of RPE65 leads to a shortage of 11-cis-retinal (a vitamin A derivative that is a precursor of the photopigment in photoreceptor cells), impairing phototransduction in photoreceptors.
The AAV capsid is formed by a beta-barrel structure and surface-exposed loops, and the surface loops determine tissue tropism 5). Since the cells contacted by the capsid differ depending on the administration route, which cells are transduced is determined by the combination of administration route and capsid.
The RPE and photoreceptor tropism by serotype is as follows:
Foreign proteins and foreign DNA are known as inflammatory inducers and can cause inflammation even in the immune-privileged intraocular environment. With intravitreal administration, the vector spreads throughout the vitreous cavity, making humoral immune responses more likely. In contrast, subretinal administration provides better shielding of the vector from the immune system, resulting in fewer inflammatory reactions 5).
The capsid specifically induces intravitreal inflammation, while the genetic material is involved in inflammation of both the anterior and posterior segments. Empty capsids have also been confirmed to cause vitritis after intravitreal administration.
The mechanism of CRA after VN treatment is not fully understood, but multiple factors are thought to be involved.
Kolesnikova et al. (2022) reported an 8-year follow-up of a patient who received VN treatment at age 11 and developed chorioretinal atrophy at age 194). Autofluorescence was almost absent before treatment, but at 6- and 8-year follow-ups, autofluorescence was detected in the parafoveal area, indicating continued function of the visual cycle. At 8 years, visual acuity in both eyes returned to baseline values before treatment, and the disease was stable.
Merle et al. (2025) reported short-term outcomes of VN treatment in 4 children (aged 6–12 years) with mild RPE65-IRD3). All cases showed rod rescue effects with improved full-field stimulus threshold (FST). Three cases had chorioretinal atrophy at the retinotomy site, but no progressive enlargement was observed. Early intervention at a stage with less retinal degeneration may reduce the risk of rapidly expanding chorioretinal atrophy.
Ku et al. (2024) reported 4 cases (initial treatment at ages 6–11, VN administration at ages 12–21) where VN was later administered to the contralateral eye that had been previously treated with a different AAV vector (rAAV2-CB-hRPE65)2). Three of the 4 cases developed chorioretinal atrophy in the VN-treated eye (onset 5–22 months). Although FST improvement was confirmed in all cases, the incidence of chorioretinal atrophy was significantly higher at 15–75% than usual. The authors proposed a combined mechanism of immune sensitization and metabolic overload. Careful consideration is needed when administering different vectors to different eyes.
Directed evolution: By artificially optimizing the AAV capsid, development of vectors that can pass through the thick inner limiting membrane of primates and reach the outer retina is progressing5). This may enable gene delivery to the outer retina even with intravitreal injection.
RdCVF (rod-derived cone viability factor): It is being investigated as a neuroprotective strategy to promote cone survival5). It could be a common treatment for multiple genetic subtypes.
Gene editing (CRISPR/Cas9): Preclinical research on AAV-mediated genome surgery is advancing, particularly for applications in age-related macular degeneration and diseases caused by dominant mutations5).
Suprachoroidal administration: Research is progressing on this new route that may enable broad treatment of the peripheral RPE and choroid5).
Many reports indicate that even in eyes with CRA, visual acuity (BCVA) is maintained and FST improvement tends to persist 4). However, if CRA extends to the fovea, there is concern about the impact on visual acuity. The growth pattern of CRA varies greatly among individuals, and long-term imaging monitoring is necessary.
