The recent approval of voretigene neparvovec (Luxturna®) for patients with biallelic RPE65 mutation-associated inherited retinal dystrophy with viable retinal cells represents an important step in the development of ocular gene therapies. Herein, we review studies investigating the episomal persistence of different recombinant adeno-associated virus (rAAV) vector genomes and the preclinical and clinical evidence of long-term effects of different RPE65 gene replacement therapies. A targeted review of articles published between 1974 and January 2021 in Medline®, Embase®, and other databases was conducted, followed by a descriptive longitudinal analysis of the clinical trial outcomes of voretigene neparvovec. Following an initial screening, 14 publications examining the episomal persistence of different rAAV genomes and 71 publications evaluating gene therapies in animal models were included. Viral genomes were found to persist for at least 22 months (longest study follow-up) as transcriptionally active episomes. Treatment effects lasting almost a decade were reported in canine disease models, with more pronounced effects the earlier the intervention. The clinical trial outcomes of voretigene neparvovec are consistent with preclinical findings and reveal sustained results for up to 7.5 years for the full-field light sensitivity threshold test and 5 years for the multi-luminance mobility test in the Phase I and Phase III trials, respectively. In conclusion, the therapeutic effect of voretigene neparvovec lasts for at least a decade in animal models and 7.5 years in human subjects. Since retinal cells can retain functionality over their lifetime after transduction, these effects may be expected to last even longer in patients with a sufficient number of outer retinal cells at the time of intervention.

Gene Therapy for Ocular Disorders: Overview and Progress to Date

Gene augmentation therapy addresses the gene defect causing particular clinical phenotypes by delivering additional correct DNA coding sequences (transgenes) of the mutated gene directly to the host cell nucleus using a vector [1, 2]. Considerable progress has been made since the first human gene transfer experiment in 1989 [3, 4]. The main area of improvement has been the development of suitable vectors through an array of methods for transferring DNA and mRNA into mammalian cells both ex vivo and in vivo [5]. After initial setbacks in the development of suitable viral vectors, the evolution of gene therapy was marked by successes mainly in two therapy areas, inherited retinal diseases (IRDs) and primary immune deficiencies. Understanding the genetic basis of IRDs has helped elucidate the diagnosis, inheritance pattern, and prognosis of these disorders [6, 7].

The eye is ideally suited as a target organ for gene therapy: its highly compartmentalized and accessible anatomy enables the precise delivery of transfer vectors to target sites, with minimal risk of systemic dissemination or side effects [8‒10]. Over 270 different genes are responsible for IRDs, including ABCA4, CEP290, CNGA3, CNGB3, MERTK, ND4, PDE6B, RLBP1, REP1, RPE65, RPGR, RS1, etc. [11]. There are currently >30 ongoing clinical trials (www.clinicaltrials.gov) investigating gene therapies for IRDs, including those which target the RPE65 gene, sponsored by different research groups. The RPE65 gene is expressed in the retinal pigment epithelium (RPE) and is responsible for encoding retinoid isomerohydrolase (also known as RPE-specific protein 65 kDa) [12]. This RPE65 enzyme is critical for recycling the visual chromophore involved in the visual cycle (Fig. 1). Mutations in the RPE65 gene impair the formation of visual pigments [13]. The accumulation of opsin apoprotein in photoreceptors and toxic retinyl esters in the RPE results in loss of photoreceptor function and their progressive degeneration [14].

Fig. 1.

Role of the RPE65 enzyme in the visual cycle. ABCA, ATP-binding cassette subfamily A group 4; LRAT, lecithin retinol acyltransferase; RDH, retinol dehydrogenase; RPE, retinal pigment epithelium.

Fig. 1.

Role of the RPE65 enzyme in the visual cycle. ABCA, ATP-binding cassette subfamily A group 4; LRAT, lecithin retinol acyltransferase; RDH, retinol dehydrogenase; RPE, retinal pigment epithelium.

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In 2017, voretigene neparvovec (VN)-rzyl (Luxturna® Spark Therapeutics Inc.) became the first US Food and Drug Administration (FDA)-approved ocular gene therapy and later in 2018 was approved by the European Medicines Agency (EMA, voretigene neparvovec [Luxturna® Novartis]), for the treatment of patients with IRD due to biallelic mutations in the RPE65 gene who have retained viable retinal cells. This marked an important milestone in the evolution of this treatment modality [15]. VN is delivered to the RPE cells by injection in the subretinal space. It is composed of human RPE65 cDNA along with a cytomegalovirus (CMV) enhancer and a hybrid chicken β-actin (CBA) promoter incorporated in the recombinant adeno-associated virus (rAAV) vector [15, 16]. Initial results with this vector showed clear therapeutic benefit in animal models and later in a human clinical setting [17, 18].

Viral Vectors and Episomal Persistence

The ideal vector for a particular genetic condition should have sufficient carrier capacity, mediate tissue specific, therapeutically relevant, sustained gene expression with minimal immunogenicity [1]. A variety of different viruses have been used for investigational gene delivery, including lentivirus, adenovirus, and adeno-associated virus (AAV).

AAV is a nonpathogenic, predominantly episomal vector (i.e., not integrated into the host cell genome) that can infect both dividing and nondividing cells [19]. Episomal vectors are less likely than integrating vectors to perturb the normal expression of host genes and are thus safer to use. rAAV vectors are the platform of choice for in vivo retinal gene therapy due to their transduction efficiency for a broad range of target ocular tissues [1, 9, 20]. The rAAV vectors produce a minimal systemic immune response following subretinal administration, unlike vectors based on adenovirus or the herpes simplex virus [1].

Transduction of postmitotic or slowly replicating cells with rAAV vectors was found to result in stable gene expression and persistence of vector genomes for several years up to a decade in muscle and liver cells, with little or no evidence of genomic integration [20]. However, since the AAV genome mainly persists in an episomal form in the transduced cells, it can be lost during cell proliferation (such as liver growth), which may limit its efficacy [20]. In fact, episomal persistence has recently been questioned in a study of 9 dogs with hemophilia A [21]. The effect of rAAVs can be diluted in dividing cells; however, this is not a significant issue in the retina as RPE cells have limited capacity for mitosis. Current limitations of AAV-mediated gene transfer also include the potential genotoxicity of integrated genomes; however, these findings were in nonocular tissue [22].

For the purposes of this review, episomal persistence is defined as persistence of rAAV-derived genetic material in episomal form in the transduced cells. While the episomal persistence of viral vectors is distinct from persistence of effect, the two are nevertheless linked: persistence of the vector in episomes is a necessary but not sufficient condition for a clinical efficacy signal, which also assumes improvement in cellular physiology.

Persistence of Therapeutic Effect: A Key Question in Ocular Gene Therapy

In early studies, the ability to create vectors with sustained transgene expression such as rAAVs opened the possibility of targeting progressive retinal diseases following a single administration [10, 23]. A number of vectors targeting the RPE65 gene have been developed and tested in various animal and disease models by multiple research groups (Table 1), including safety and efficacy studies in a large animal model of childhood blindness (the RPE65−/− dog) [24, 25], and safety of an alternative ocular rAAV2-RPE65 in nonhuman primates [26].

Table 1.

A selection of studies highlighting different vectors developed by different research groups, targeting the RPE65 gene

 A selection of studies highlighting different vectors developed by different research groups, targeting the RPE65 gene
 A selection of studies highlighting different vectors developed by different research groups, targeting the RPE65 gene

Supplying functional RPE65 gene using the rAAV vector platform was shown to improve preclinical and clinical outcomes for RPE65-associated IRDs, with durable effects [20, 27, 28]. For example, sustained clinical benefits were reported for up to 3 years after a single injection (rAAV2) in patients with Leber congenital amaurosis type 2 (LCA2) [29].

However, estimating the long-term (years to decades) persistence of effect following gene therapy remains a challenge, particularly as separate early Phase I-II trials varied in design, vector, and with varying degrees of visual improvements [10, 15, 30]. One study with a rAAV2 vector revealed a decline in therapeutic effect following an initial peak at 6–12 months post-injection [31], while another showed evidence of a fast and slow phase of therapeutic effect with ongoing degeneration and eventual localized loss of visual function after therapy [32]. Meanwhile, outcomes from clinical studies using VN (rAAV2) showcased stable visual function over 4 years in the Phase I follow-on study [17, 18] and at 5 years posttreatment in the Phase III study [33]. Long-term evidence is being collected via post-approval safety studies, which may potentially provide further information around durability of treatment response in a real-world setting [34, 35]. It should, however, be noted that early phase clinical trials invariably use a dose escalation strategy, which would result in underdosing and progressive degeneration.

Objectives

In the present work, we review the literature to date examining the episomal persistence of different rAAV vectors and the preclinical and clinical evidence of long-term effects of different RPE65 gene replacement therapies and factors affecting vector durability. We further focus on VN and evaluate the durability of treatment response in animal models and clinical trials. We also present the latest thinking and consensus opinion on a number of topics related to durability of effect and highlight implications for future clinical practice. For transparency, when discussing gene therapies and/or vectors, VN is referred to directly. Where not specified, this indicates non-VN gene therapies and/or vectors.

Literature Review – Preclinical Data

We conducted a targeted literature review of articles published between 1974 and January 2021 in Medline®, Embase®, Medline in-process, and other nonindexed databases to identify the relevant studies examining the episomal persistence of rAAVs and the durability of treatment response in animal models. The bibliography of included studies was also screened to find additional potential studies for final inclusion. Inclusion criteria for the screening of retrieved citations from the databases are presented in Table 2.

Table 2.

Inclusion criteria for the screening of retrieved citations on episomal persistence of rAAV-mediated gene therapy

 Inclusion criteria for the screening of retrieved citations on episomal persistence of rAAV-mediated gene therapy
 Inclusion criteria for the screening of retrieved citations on episomal persistence of rAAV-mediated gene therapy

Clinical Data for VN

A descriptive longitudinal analysis of Phase I (101 and 102) and Phase III (301) clinical trial data was carried out to assess the durability of effect for VN, the only product approved by regulatory authorities (FDA and EMA) for the treatment of RPE65-related IRDs.

Literature Review: Episomal Persistence of rAAVs in Preclinical Studies

Early work conducted mostly in muscle tissue found that the vector genome is concatemerized and circularized after conversion of the single-stranded rAAV genome into double-stranded DNA [36]. In rodent skeletal muscle, rAAV vector genomes are maintained mainly as extrachromosomal forms and gene expression in this tissue derives predominantly from episomal forms [36, 37]. Findings from a variety of animal models have confirmed that rAAV episomal genomes are remarkably stable and persist mainly as supercoiled monomeric and concatemeric circles, in a chromatin-like structure [38, 39].

This review identified a total of 14 publications evaluating episomal persistence for final inclusion. Out of this, 13 publications were included based on the screening of 41 citations, and one study was identified from the bibliography of the included studies. All studies were in nonretinal cells with genes other than RPE65. In the preclinical studies reviewed here, the rAAV genome persisted for up to 22 months (the longest study follow-up) as transcriptionally active episomes in muscle cells [39]. Long-term transgene expression after intramuscular administration of rAAV in primates indicated that the rAAV vector genome was functionally stable for at least 5 years [39].

Preclinical Evidence of Vector Durability

This review identified a total of 71 publications evaluating gene therapies in animal models for final inclusion. Out of this, 66 publications were included from the screening of 362 citations and five studies were identified from the bibliography of included studies. From the 71 included publications, two evaluated VN (AAV2-hRPE65v2) [40, 41], 24 assessed alternative RPE65 gene therapy vectors, and the remaining 45 evaluated other gene therapies (CNGA3, CNGB3, CNGB1a, PDE6A, PDE6B, RLBP1, RPGR). The duration of efficacy in various animal models is summarized in Table 3.

Table 3.

The duration of efficacy (longest follow-up) of RPE65 and other gene therapies in animal models

 The duration of efficacy (longest follow-up) of RPE65 and other gene therapies in animal models
 The duration of efficacy (longest follow-up) of RPE65 and other gene therapies in animal models

Mouse Models

In mouse studies, Lai et al. injected rAAV-mediated RPE65 gene therapy at postnatal day 5 up to 1 year of age. Gene expression lasted for as long as 18 months postinjection when administered at 3 weeks of age and in the absence of any histological evidence of photoreceptor degeneration [42]. In a study by Muhlfriedal et al. [43] in CNGA3 knockout mice injected at either 2 weeks or 3 months of age, therapeutic effects were reported for 12 months post-treatment, but the response was greater in the group receiving early treatment. Another study by Wu et al. [44] of RPGR knockout mice of 6 weeks to 1 year of age found a high level of preservation of [44] retinal structure and function at 18 months post-injection [44]. These results are significant in light of a maximum life span of 4 years for mice [45].

Dog Models

Acland et al. and Narfstrom et al. showed that rAAV-derived episomal transgenes maintained their therapeutic effect for at least 3 years after initial gene therapies of different vector types in RPE65−/− dogs [46, 47]. Some investigators observed a response to the gene therapy in dogs as soon as 15 days [48] and up to 4–6 weeks [47] post-injection.

RPE65-mutant dogs are known to show congenital visual dysfunction, later followed by retinal degeneration. One study by Cideciyan et al. [49] examined the consequences of a gene therapy on retinal degeneration in RPE65-LCA canines and human subjects. In this study, early administration of gene therapy (at ∼3 months of age) led to sustained functional gains up to 9.4 years (which can be life-long in animal models) in the dog that was followed longest but did not prevent photoreceptor degeneration in dogs that received treatment in later stages, i.e., post ∼5 years of age [49]. However, a more recent study in this dog model by Gardiner et al. demonstrated that rAAV2-mediated RPE65 gene therapy (VN) initiated in later stages of disease (e.g., mid-life in RPE65-mutant dogs) can halt disease progression at 4–5 years [41]. The authors of this study further observed that treated locations with over 63% of normal photoreceptors showed robust treatment-related retention of photoreceptors during long-term follow-up [41].

In a separate study by Beltran et al. [50], RPGR mutant (X-linked progressive retinal atrophy 2 [XLPRA2]) dogs received treatment with gene therapy as early as 5 weeks to as late as 6 years of disease [50]. In the younger dogs (aged around 5 weeks), prior to treatment, structural abnormalities of photoreceptors were observed in early-stage disease, with no significant loss of retinal outer nuclear layer [50]. Meanwhile, older XLPRA2 dogs showed pre-treatment signs of severe degeneration, ∼50–60% at the age of 26 weeks, depending on the dog species [51]. The rescue effect in XLPRA2 dogs at mid- and late-stage disease (due to RPGR gene mutation) was found to be stable for more than 2 years following intervention in late-stage disease [50].

VN: Clinical Basis for Durability of Effect

The clinical trial data with VN add to the body of evidence for long-term durability of effect (Fig. 2). The clinical development program for VN consisted of two Phase I studies and one Phase III trial, for which follow-up data are available for up to 7.5 and 5 years, respectively. In the Phase I trial, 12 subjects received VN (three different, escalating doses: 1.5 × 1010 (150 µL), 4.8 × 1010 (150 µL), and 1.5 × 1011 (300 µL) vector genomes) in their worse-seeing eye (study 101 [52]). Of these, 11 received VN (1.5 × 1011 vector genomes) in a total subretinal volume of 300 μL in their second eye as part of study 102 [53]. The Phase III pivotal trial of VN enrolled 31 patients, randomly assigned to the original intervention (OI) group (n = 21) or the delayed intervention (DI) group (n = 10); one participant from each group withdrew after consent [17]. In this trial, patients from the DI group were crossed over to the treatment arm after 12 months. The change score in bilateral multi-luminance mobility test (MLMT) [54] was selected as the primary endpoint. Secondary endpoints were the full-field light sensitivity threshold (FST) test, MLMT of the first eye injected, and visual acuity (VA), averaged over both eyes. The kinetic and static visual field (VF) tests were chosen as exploratory endpoints [17]. Russell et al. presented follow-up data available in 26 patients that indicated durability of improvement for up to 5 years, with mean MLMT bilateral score changes of 1.6 light levels at year 5 in the OI group (n = 18) and 2.4 light levels at year 4 in the DI group (n = 8) (Fig. 3a) [33]. Durable improvements for up to 5 years were also recorded for the FST endpoint (Fig. 3b); observations are ongoing. In addition, Maguire et al. showed that mean change in VF sum total degrees averaged over both eyes, measured at 4-year follow-up for OI patients and 3-year follow-up for DI patients, was 197.7 and 157.9, respectively. Mean change from injection baseline in VA was −0.003 and −0.06 logMAR, respectively, with the score in the OI group impacted by a retinal detachment in 1 patient. These findings show durability of initial improvement at 3 and 4 years [55].

Fig. 2.

Episomal persistence of AAV vectors and durability of treatment effect with RPE65gene therapies: increasing evidence from preclinical to clinical studies. AAV, adeno-associated virus; FST, full-field light sensitivity threshold; MLMT, multi-luminance mobility test.

Fig. 2.

Episomal persistence of AAV vectors and durability of treatment effect with RPE65gene therapies: increasing evidence from preclinical to clinical studies. AAV, adeno-associated virus; FST, full-field light sensitivity threshold; MLMT, multi-luminance mobility test.

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Fig. 3.

Mean (±SE) multi-luminance mobility test (MLMT) lux scores (a) and mean (±SE) full-field light sensitivity threshold (FST) white light averaged over both eyes (b), over time from baseline in the original intervention (OI, green) and control/delayed intervention (DI, blue) patient groups in the Phase III clinical trial of VN [33]. For the DI group, change in MLMT is relative to injection baseline after year 1; n represents the number of patients at year 5. BL, baseline; D, day; n, number of patients; SE, standard error; X, crossover; Y, year.

Fig. 3.

Mean (±SE) multi-luminance mobility test (MLMT) lux scores (a) and mean (±SE) full-field light sensitivity threshold (FST) white light averaged over both eyes (b), over time from baseline in the original intervention (OI, green) and control/delayed intervention (DI, blue) patient groups in the Phase III clinical trial of VN [33]. For the DI group, change in MLMT is relative to injection baseline after year 1; n represents the number of patients at year 5. BL, baseline; D, day; n, number of patients; SE, standard error; X, crossover; Y, year.

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A post hoc analysis of the clinical trial data by Chung et al. [18] indicated a high correlation between the MLMT and FST endpoints. Given the absence of reliable MLMT data due to the use of a nonvalidated early version of the MLMT in the Phase I trial, an analysis using FST as outcome was performed to evaluate the durability of response in patients enrolled in the Phase I trials [56]. The analysis showed sustained improvements in FST for up to 7.5 years (Fig. 4a) and 4 years (Fig. 4b), in the first and second injected eyes, respectively, although a certain degree of scatter is evident due to low patient numbers in the analysis, along with decreasing numbers with longer follow-up [56]. Furthermore, a post hoc analysis of 40 subjects who received VN in the Phase I add-on and Phase III trials confirmed sustained improvement in MLMT and FST at 4 years [18]. The results of this Phase I trial analysis of FST are consistent with those of the Phase III trial and 5-year follow-up analysis [18, 33]. Given the high correlation between MLMT and FST, these results collectively suggest a sustained improvement in patients’ functional vision up to 7.5 years. Patients from this clinical trial are being monitored for 15 years in a follow-up study.

Fig. 4.

Mean (±SE) full-field light sensitivity threshold (FST) white light for the first injected eye (eye with greatest impairment; pooled data of the three different doses: 1.5 × 1010, 4.8 × 1010, and 1.5 × 1011 vector genomes; a) and the second injected eye (the contralateral previously uninjected eye; dose: 1.5 × 1011 vector genomes; b) in the Phase I clinical trials of VN. The numbers within the boxes represent the numbers of patients at each time point; time points with less than three observations and without baseline data were excluded from the analysis [56]. SE, standard error.

Fig. 4.

Mean (±SE) full-field light sensitivity threshold (FST) white light for the first injected eye (eye with greatest impairment; pooled data of the three different doses: 1.5 × 1010, 4.8 × 1010, and 1.5 × 1011 vector genomes; a) and the second injected eye (the contralateral previously uninjected eye; dose: 1.5 × 1011 vector genomes; b) in the Phase I clinical trials of VN. The numbers within the boxes represent the numbers of patients at each time point; time points with less than three observations and without baseline data were excluded from the analysis [56]. SE, standard error.

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The clinical surrogate for long-term persistence of RPE65 transgene in humans is the durability of improved FST/MLMT scores. Patients treated with VN in 2010 in the Phase I dose escalation study are being reviewed annually as part of the long-term follow-up. An independent case report of a patient with LCA2 treated with VN describes evidence of therapeutic effect at 6 years based on autofluorescence measurements [57], although it should be noted that RPE65-IRDs can be associated with an abnormal autofluorescence pattern [58].

Persistence of Effect with Viral Vectors

The studies reviewed herein revealed that rAAV-derived genetic material can persist for years in episomal form in the transduced cells, underlining the suitability of these vectors for retinal gene therapy. The preclinical evidence for long-term durability of effect of rAAV vectors in the retina is limited to about 18 months in rodents, due to their short life span [42] and 9.4 years (considered late stage of life) in canine models [49]. Based on animal studies, it should be noted that the presence of rAAV episome in the cell does not necessarily predict whether the RPE65 enzyme will be expressed, and the potential decrease and eventual cessation of 11-cis retinal production are determined by several other factors, which are yet to be elucidated.

Most of the RPE65 animal studies reviewed here used vectors with different promoters and proteins, which can affect the durability of expression [59, 60]. For example, the use of the CMV promoter, which may undergo methylation-based silencing, is a potential cause for declining AAV transgene expression [30, 61].

Future research should also establish how the data on episomal persistence relate to humans and which factors modulate episomal persistence and transgene expression in patients undergoing gene therapy. As yet, there is no evidence to indicate that episomal genetic material would not persist in these patients, and there are also no studies excluding rare integration events that might occur if there are double-stranded breaks.

Factors Affecting Vector Durability

Target Cells

RPE cells form early in the development of the organism and subsequently undergo minimal proliferation throughout life. Retinal cells in the posterior segment of the eye are postmitotic, allowing for sustained gene expression without the need for genomic integration of the transgene [28]. While RPE cells can replicate to a limited extent when photoreceptor contact is lost, they can also regenerate normal homeostasis following the introduction of a functional RPE65 gene, which restores function by reducing the accumulation of toxic retinyl esters. Thus, if the functional gene is successfully incorporated, it is anticipated that RPE cells will remain functional over their lifetime; this has implications on the duration of effect that can be expected for retinal gene therapies like VN, which target RPE cells.

Vector Production

Challenges related to the longevity of effect of gene therapies are also being addressed through the development of rAAV vectors that provide high levels of expression with low particle numbers, thus minimizing the risk of inflammation or toxicity [62]. However, the rAAV vector profiles also differ on a number of levels, all of which can potentially affect durability of treatment effect: capsid composition; number of functional viral particles per exposed cell; full-to-empty capsid ratio; frequency of reverse packaging; and specific regulatory sequences surrounding the transgene of interest. In addition, the vector purification process and factors determining formulation and vialing can also impact treatment effects [28, 63].

Timing of Gene Therapy

The timing of intervention in relation to disease course may impact long-term therapeutic outcomes. The success of treatment depends on the number of retinal cells as well as on cell viability, which tend to decrease with disease duration [64]. While some preclinical studies suggest gene therapy achieves optimal results in younger animals [46, 49, 63, 65], other research indicates that gene therapy can be also successfully used in later stages of disease if the animals have retained a sufficient amount of viable photoreceptors at the time of treatment [41, 66]. In terms of clinical data, older patients treated with VN who had a sufficient number of outer retinal cells at the time of intervention have shown improvements with gene therapy [17, 33]. In addition, no significant difference between patients <10 and ≥10 years of age was found 1 year posttreatment with VN for MLMT, FST, VF, or VA outcomes [67].

Surgical Procedure

The details of the surgical procedure, such as injection speed and pressure, bleb site, and volume play an essential role in determining treatment outcomes. They should be optimized to ensure effective delivery of the product with minimal risk and complications associated with vitreoretinal surgery. For example, investigations to evaluate an optimized injection system for retinal gene therapy showed that a slow injection, small volume, and a retinotomy site greater than 2 mm from the fovea would minimize the biomechanical stress to the neuronal tissue, increasing the chance of survival for the transduced cells and thus contributing to persistence and vector efficacy [68]. Developments in viral vector capsids that allow spread beyond the bleb and achieve transduction of outer retinal cells with minimal number of particles are important in this regard [69‒71]. The surgical procedure also impacts the number of viral particles per cell (depending on the size of the bleb) and consequently the gene expression levels in the transduced cells, which are important for determining the optimal viral dose and the durability of response.

Viral Dose and Risk of Inflammation

Multiple injections can potentially treat larger areas of the retina than a single injection; however, more retinotomies carry associated risks, such as reflux. While single volumes up to 1,000 µL have been injected in clinical studies [31, 72], it is largely agreed that higher injection volumes come with a greater risk of long-term retinal detachment and damage. In addition, increasing the total viral dose with the volume given could lead to elevated risks of immune response. AAV vector capsids that allow spread beyond the bleb packaged with promoters allowing high-level expression specific to target cells are expected to have an impact in mitigating such surgical and immunological risks [70, 71]. Further research should identify ways of improving the total surface area treated with minimal immune response and better functional gain for the patient. Better knowledge of the immune responses to AAV-mediated ocular gene therapy will also help understand how they can be regulated in order to gain the full benefits of therapy, while minimizing risk [73].

Cell Viability of Nontransfected Cells

Gene augmentation therapies like VN require sufficient viable retinal cells [4, 15]. The effect of chimeric therapeutic gene expression on the surrounding cells has been investigated in mouse models (PDE6B) of retinitis pigmentosa [66, 74]. It is postulated that supplying a functional RPE65 protein at sufficient expression levels and early time points may have an indirect consequence on the nontransfected cells, which might benefit from the increased health and survival of the transduced cells.

Degeneration of Retinal Cells after Gene Augmentation Therapy

Experience with many clinical conditions, including nonheritable conditions and genetically complex diseases such as (dry) age-related macular degeneration, shows that RPE degeneration inevitably leads to photoreceptor degeneration, reflected in a loss of retinal sensitivity [75]. While data on this topic are limited, there is preclinical and clinical evidence to suggest that the areas of untreated RPE cells continue to degenerate in patients receiving gene therapy for RPE65-associated retinal dystrophy [76]. The rods immediately overlying the degenerate RPE cells are expected to die, but those overlying the successfully transduced RPE cells would survive. It is unclear whether there is a minimum number of RPE cells that need to be transduced in a given area for degeneration to stop.

Findings from an animal study with a rAAV2 vector in RPE65-associated retinal dystrophy suggest that the rate of photoreceptor loss in transduced cells is the same as the natural rate of disease progression when gene therapy is initiated after the onset of degeneration [49]. The authors of the study conclude that a two-pronged approach is required, which should address visual dysfunction in the short term and retinal degeneration in the long term [49]. However, these results, using an experimental rAAV2 vector gene therapy, are limited to a specific disease model (LCA2), where the natural rate of retinal degeneration is expected to be slow.

It is unclear whether gene therapy can prevent the progression of retinal degeneration in human patients [15]. It has been shown that photoreceptors continue to degenerate despite early treatment and that targeting retinal locations with retained photoreceptors is a prerequisite for successful gene therapy in humans with RPE65 mutations [77]. Thus far in human patients, retinal gene therapy (RPE65, CHM, XL-RP) has not yet been shown to halt or slow down photoreceptor degeneration outside the transduced area (as reflected by OCT). In the LCA2 study described above, photoreceptor degeneration was also found to progress in the transduced area in adult human patients, despite treatment with an experimental rAAV2 vector gene therapy [49]. In contrast, however, the first report of the effects of an AAV8 RPGR gene therapy in a clinical trial showed reversal of some signs of degeneration, as evidenced by improved microperimetry associated with outer retinal structural changes, which were consistent with regrowth of outer segments [78]. In a multicenter retrospective chart review, Gange et al. [79] identified 10 patients with LCA who underwent subretinal injection of VN and subsequently developed chorioretinal atrophy in either one or both eyes. Following identification at a mean 4.7 months after surgery, atrophy was progressive to a mean follow-up of 11.3 months; however, there was consistent improvement in visual function from baseline, indicating a successful response to treatment. This is in line with findings from clinical studies.

While the question of whether gene augmentation prevents further degeneration of the retina remains an area of debate, it should be noted that the results of clinical trials have demonstrated a stable, sustained improvement in retinal function [33, 64]. Functional magnetic resonance imaging results of patients with LCA2, who underwent treatment in their worse-seeing eye in the Phase I trial of VN and received a subretinal injection in their contralateral eye in the follow-on trial, show maintenance of visual function gains at least 3 years posttreatment [80].

Visual Function Tests and Patients’ Perception of Vision

In general, classic visual function tests such as best-corrected VA (BCVA) may not be the optimal measures of treatment effect due to the variability of individual responses. In addition, BCVA measurements may not capture the degenerative and progressive nature of IRDs as it is solely based on foveal cone function; patients with IRDs can lose a large amount of RPE photoreceptor complexes before showing a sizeable decline in BCVA. Alternative tests such as FST and Goldmann kinetic perimetry quickly capture improvement in visual function posttreatment with gene therapy and/or can also show loss of function as a result of photoreceptor-RPE cell loss. Clinical studies conducted with VN indicate that FST may also show a more lasting improvement and correlates with improvements in the MLMT score [17, 56].

Potential Loss of Visual Function

In nontreated individuals with RPE65-associated IRDs, the peripheral VF is gradually lost and central vision (e.g., BCVA) also becomes worse over time [81]. Clinical studies with VN showed that while MLMT does not have a linear correlation with VA or VF, it does have a threshold that corresponds to pass and fail. However, an “off-cliff effect” is not expected in these patients, whether or not they had been treated with VN. Furthermore, VA and the amount of photoreceptor loss as well as progression of disease vary significantly between patients, even in those from the same family [82, 83]. Thus, if a reduction in treatment effect was to occur, the decrease in visual function would also be affected by the natural history of the disease in that particular patient.

Persistence of Effect in Clinical Studies

A number of studies with long-term follow-up data, using different vectors for RPE65 mutation-associated IRDs, have been published in the past few years [18, 80, 84]. In the case of VN, a 4-year post hoc analysis of the Phase I, Phase I add-on, and Phase III data confirmed the long-term durability of effect of the drug [18]. Visual outcomes, as measured via change in FST, in patients enrolled in the VN Phase I trials were maintained for up to 7.5 years; the study is ongoing [56]. More recently, a follow-up of patients treated with VN in the Phase III study showed improvements in ambulatory navigation (MLMT), light sensitivity, and VF are maintained for at least 5 years [33]. In studies with a different RPE65 gene therapy, investigators reported efficacy outcomes from a 5-year Phase I/II study of patients with RPE65-mediated LCA and severe early childhood onset retinal degeneration, treated with an rAAV2-RPE65 gene therapy, supporting the long-term persistence of the RPE65 transgene in RPE cells [84]; this study further reported that a younger age was associated with better visual function outcomes 5 years after treatment, which was also found in preclinical studies.

The conclusions around persistence of therapeutic effect are strengthened by the clinical outcomes of patients treated in the VN clinical studies and their ongoing follow-up. However, differences related to the gene therapy vector, administration, and surgical procedure can all have an impact on long-term response to treatment in clinical practice.

Other Considerations

Part of the challenge when analyzing the long-term data of VN is that the earliest trials were Phase I dose escalation studies and the first patients received over a log unit lower dose than is currently accepted to be efficacious according to the FDA and EMA approvals. Hence, if only 10% of the retinal cells were successfully transduced, it is logical to assume that the remaining 90% would degenerate. However, this degeneration is not a consequence of the longevity of AAVs but rather a question of not receiving a sufficiently high dose. Furthermore, the early studies included recruitment of patients who, in addition to sufficient viable retinal cells, had advanced disease [30]. Therefore, the real question of longevity in humans is likely to be answered by the long-term follow-up of patients from the Phase III study and, so far, this is looking promising [33].

Gene therapies have brought about a change in the treatment paradigm for genetic diseases by providing lasting therapeutic effects with a single intervention. In preclinical animal models of ocular diseases, there have been a number of important findings: in one study, gene therapy demonstrated a sustained treatment effect of almost a decade (9.4 years) [49]. Other investigators found that gene therapy can have more pronounced effects with early intervention [50]. However, it has also been demonstrated that later stage gene therapy was also effective long term in canines, provided the treated area had a sufficient number of viable retinal cells at the time of treatment [41]. In the case of VN, the clinical outcomes are consistent with findings from these animal models. Taken together, they support the long-term durability of treatment effect of VN, with up to 7.5 years of sustained FST results from Phase I trials and 5 years of sustained ambulatory navigation (MLMT), light sensitivity (FST), and VF outcomes from the Phase III trial [33, 56].

Although of immense clinical benefit, gene therapies present a challenge for the different healthcare stakeholders, in particular due to specificities related to the one-time nature of the treatment and uncertainty around the duration of effect. This study highlights the evidence around episomal persistence of AAV vectors and the long-term durability of therapeutic efficacy of different vectors in animal models and human subjects. As the target cells for this therapy are largely postmitotic, it is anticipated that the successfully transduced RPE cells will maintain functionality, leading to sustained visual function over their lifetime. Despite the limited long-term clinical evidence, it is biologically plausible that the treatment effect of VN will continue for decades in patients with a sufficient number of viable retinal cells at the time of intervention. By optimizing important factors concerning the durability of vector activity, such as the design of the expression cassette and capsid structure, vector production and purification steps, and surgical delivery, we can strive to achieve similar long-term treatment effects with future IRD gene therapies.

The authors thank Vinay Preet Kaur (Novartis Healthcare Pvt. Ltd.) for her assistance with the literature search and collation of studies for inclusion in this review. Writing assistance in the preparation of this article was provided by Ileana Stoica, PhD, and Carol Crawford, PhD (Novartis Ireland Ltd.), and was funded by Novartis Pharma AG.

Bart P. Leroy is or has recently been a consultant to Akouos, Alia Therapeutics, Bayer, Biogen, GenSight Therapeutics, IVERIC Bio, Novartis, ProQR Therapeutics, Spark Therapeutics, REGENXBIO, Vedere Bio, and ViGeneron. He has received, receives, or will soon receive trial support from Biogen, GenSight Therapeutics, MeiraGTx, Novartis, and ProQR Therapeutics. Travel support has come from GenSight Therapeutics, IVERIC Bio, Novartis, ProQR Therapeutics, and Spark Therapeutics. Neither Bart P. Leroy nor any of his close family members have any financial interests in any of aforementioned companies. Bart P. Leroy is also supported by grants from the Research Foundation – Flanders, Belgium (Senior Clinical Investigator 1803821N), and the Concerted Research Action of the Special Research Fund Ghent University (BOF20/GOA/023). M. Dominik Fischer is on the advisory board of and/or consulting and/or receiving honoraria/grant money/travel support from following companies: Adelphi Values, Advent France Biotechnology, AlphaSights, Atheneum, Axiom Healthcare Strategies, Biogen, Decision Resources, Dialectica, Frontera Therapeutics, Janssen Research & Development, Navigant, Novartis, REGENXBIO, Roche, Sirion, and STZ eyetrial. M. Dominik Fischer is the director of Fischer Consulting Limited and holds a patent (50%) on a gene therapy product for X-linked retinitis pigmentosa. John G. Flannery is an inventor on a patent of adeno-associated virus with variant capsid and methods of use thereof. John G. Flannery is a founder of Vedere Bio and a paid consultant to Novartis. Robert E. MacLaren is or has recently been a consultant to Arctos, Spark, Novartis, Biogen, and Gyroscope Therapeutics. He is also a scientific advisor of the UK National Institute for Health and Clinical Excellence (NICE) retinal gene therapy committee. Robert E. MacLaren receives funding from the NHS for gene therapy through the NIHR Oxford Biomedical Research Centre. Robert E. MacLaren is listed as a named inventor on several patents owned by the University of Oxford. Deniz Dalkara is an inventor on a patent of adeno-associated virus with variant capsid and methods of use thereof with royalties paid to Avalanche Biotech (WO2012145601 A2). Deniz Dalkara is a founder of Gamut Tx (now SparingVision) and part-time CSO of SparingVision. Hendrik P.N. Scholl is supported by the Swiss National Science Foundation (Project funding: “Developing novel outcomes for clinical trials in Stargardt disease using structure/function relationship and deep learning” #310030_201165, and National Center of Competence in Research Molecular Systems Engineering: “NCCR MSE: Molecular Systems Engineering (phase II)” #51NF40-182895), the Wellcome Trust (PINNACLE study), the Translational Research Acceleration Program Award by the Foundation Fighting Blindness (“Cone-based optogenetics for vision restoration” #TA-NMT-0621-0805-TRAP), and the Foundation Fighting Blindness Clinical Research Institute (ProgStar study). Hendrik P.N. Scholl is a member of the Scientific Advisory Board of Apellis Switzerland GmbH, ARCTOS medical AG; Astellas Pharma Global Development, Inc./Astellas Institute for Regenerative Medicine; Biogen MA Inc.; Boehringer Ingelheim Pharma GmbH & Co; Gyroscope Therapeutics Ltd.; Janssen Research & Development, LLC (Johnson & Johnson); Novartis Pharma AG (CORE); Okuvision GmbH; Pharma Research & Early Development (pRED) of F. Hoffmann-La Roche Ltd; reVision Therapeutics, Inc.; Stargazer Pharmaceuticals, Inc.; and Third Rock Ventures, LLC. Hendrik P.N. Scholl is a paid consultant of Gerson Lehrman Group; Guidepoint Global, LLC; and Tenpoint Therapeutics Limited. Hendrik P.N. Scholl is a member of the Data Monitoring and Safety Board/Committee of Belite Bio (CT2019-CTN-04690-1), ReNeuron Group Plc/Ora Inc. (NCT02464436), and F. Hoffmann-La Roche Ltd (VELODROME trial; NCT04657289) and member of the Steering Committee of Novo Nordisk (FOCUS trial; NCT03811561). Hendrik P.N. Scholl is a co-director of the Institute of Molecular and Clinical Ophthalmology Basel (IOB) which is constituted as a nonprofit foundation and receives funding from the University of Basel, the University Hospital Basel, Novartis, and the government of Basel-Stadt. These arrangements have been reviewed and approved by the University of Basel (Universitätsspital Basel, USB) in accordance with its conflict-of-interest policies. Hendrik P.N. Scholl is a principal investigator of grants at the USB sponsored by the following entities: Kinarus AG; Okuvision GmbH; and Novartis Pharma AG. Grants at USB are negotiated and administered by the institution (USB) which receives them on its proper accounts. Individual investigators who participate in the sponsored project(s) are not directly compensated by the sponsor but may receive support from the institution for their project(s). Hendrik P.N. Scholl is an editor-in-chief of Ophthalmic Research. Daniel C. Chung is an employee of SparingVision, a consultant for Spark Therapeutics Inc., and an advisory board member for Novartis. Claudio Spera, Daniel Viriato, and Judit Banhazi are all employees of Novartis Pharma AG.

The sponsorship of this study and article processing charges were funded by Novartis Pharma AG, Basel, Switzerland.

Bart P. Leroy, M. Dominik Fischer, John G. Flannery, Robert E. MacLaren, Deniz Dalkara, Hendrik P.N. Scholl, Daniel C. Chung, Claudio Spera, Daniel Viriato, and Judit Banhazi made substantial contributions to the conception and design of the work and interpretation of data and participated in drafting and revising it critically for important intellectual content. All the authors have read and approved the final version to be published and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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