Article Published in the Author Account of

Knut Stieger

Gene Therapy for Vision Loss — Recent Developments

Abstract: Retinal gene therapy mediated by adeno-associated virus (AAV) based gene transfer was recently proven to improve photoreceptor function in one form of inherited retinal blinding disorder associated with mutations in the RPE65 gene. Several clinical trials are currently ongoing, and more than 30 patients have been treated to date. Even though only a very limited number of patients will greatly benefit from this still experimental treatment protocol, the technique itself has been shown to be safe and will likely be used in other retinal disorders in the near future. A canine model for achromatopsia has been treated successfully as well as mouse models for different forms of Leber congenital amaurosis (LCA). For patients with autosomal dominant retinitis pigmentosa (adRP), a combined gene knockdown and gene addition therapy is being developed using RNA interference to block mRNA of the mutant allele. For those patients suffering from RP with unknown mutations, an AAV based transfer of bacterial forms of rhodopsin in the central retina might be an option to reactivate residual cones in the future.


Until recently, inherited retinal degenerative blinding disorders have been untreatable and the main task for the ophthalmologist was to accompany the patients on their long road to blindness and to advise them as to the risk for their offspring. This is presently changing with the advent of gene therapy as a therapeutic strategy aiming at slowing or even curing such degenerative disorders. The principle of gene therapy is based on the transfer of a therapeutic gene or part of a gene by use of viral or non-viral vectors administered to the eye. Generally, two different treatment strategies are followed, the specific or the non-specific approach. The term specific is used to highlight the need for the knowledge of the exact gene and the mutation responsible for the disease, while non-specific refers to the idea of trying to provide a protective environment that would prolong or even halt the process of photoreceptor degeneration rather than targeting the disease-causing mutation.

The possibility to generate transgenic animal models has paved the way for understanding the major pathologic pathways in retinal degenerative disorders, but the presence of naturally occurring animal models for many of these disorders has helped in developing successful treatment strategies that advance to the clinics or have already been used in patients. More than a dozen mouse models have been treated successfully by specific gene therapy approaches and many have shown tremendous improvement after non-specific therapy, some of them carrying naturally occurring mutations and other being transgenic in origin (Colella and Auricchio 2010; Smith et al., 2009; Stieger et al., 2010). However, the move to large animal models marked a critical turning point in research and accelerated gene-therapy’s pace to clinical application (Stieger et al., 2009). It was the successful treatment of dogs with mutations in the RPE65 gene that eventually opened the door for gene therapy applications to become a realistic treatment option in humans (Acland et al., 2001).

While in depth clinical characterization of the different pathologies in humans is a crucial prerequisite for effective treatment possibilities, it is the molecular basis of the disease that is most important in designing treatment strategies (Lorenz et al., 2010). Generally, retinal degenerative disorders have their origin either in the retinal pigment epithelium (RPE), in photoreceptor cells, or in bipolar cells. Depending on the target cell type, different essential issues in the design of the potential therapy have to be elucidated, such as the choice of the ideal gene vector, the optimal administration route, and the optimized gene expression cassette.

Of all currently available vector systems, viral or non-viral in origin, the adeno-associated virus (AAV) based vectors have emerged as the gold standard for retinal gene therapy (Surace and Auricchio, 2008). Vectors based on this virus enable long term and cell type specific transgene expression in RPE cells and/or photoreceptor cells following subretinal injection. The capacity to target certain cell types within the retina is based on the availability of different AAV serotypes, of which the serotype 2 is currently the most used in clinical applications, targeting RPE and photoreceptor cells in all species tested (Auricchio et al., 2001; Bennett et al., 1999). However, the serotypes 4, 5, and 8 are under intense investigation for clinical application, as they appear to be more efficient in transducing RPE cells specifically (serotype 4), RPE cells and photoreceptor cells (serotype 5), or RPE cells and all neuronal cells within the neuroretina (photoreceptor cells, inner nuclear layer cells, and ganglion cells) (serotype 8) (Allocca et al., 2007; Auricchio et al., 2001; Stieger et al., 2008; Weber et al., 2003). AAV vectors carrying a therapeutic gene can be produced following good manufacturing practice (GMP), which is a prerequisite for use in humans, and are listed as orphan drugs (Mitchell et al., 2010). After an international collaboration of the leading AAV producing laboratories, a standard batch of AAV2 vectors was released from the American Type Culture Collection (ATCC) that can be used to calibrate internal titer standards, allowing the production process of this still experimental drug to be even more rigid than ever (Lock et al., 2010).

Currently, different treatment strategies based on AAV mediated gene therapy approaches are in the pipeline for retinal blinding disorders. In this review, the authors will present the current status of clinical applications of gene therapy for patients with mutations in the RPE65 gene. Furthermore, interesting and promising studies for the treatment of other retinal blinding disorders are described that may soon advance to the clinics.

Clinical Trials for Patients with Mutations in the RPE65 Gene

The RPE specific gene RPE65 encodes for the isomerohydrolase that isomerizes bleached all-trans-retinal into photosensitive 11-cis-retinal (Jin et al., 2005; Moiseyev et al., 2005). If no 11-cis-retinal is produced due to loss of or impaired RPE65 function, the chromophore rhodopsin cannot be assembled, and the photoreceptors remain insensitive to light stimuli. Mutations in the RPE65 gene cause a severe phenotype, which is either called Leber congenital amaurosis (LCA) type 2 when symptoms are present at birth or within the first months of life, or early onset severe retinal dystrophy (EOSRD) when patients have significant vision during the first years of life (Lorenz et al., 2000; Lorenz et al., 2008). Mutations in the RPE65 gene account for 5-10% of all LCA cases. Even though the genetic origin of the pathology is located within the RPE cell, the clinical phenotype manifests because photoreceptors are not functioning and degenerate over time (Cideciyan, 2010).

The knowledge about the exact pathology enabled researchers to develop treatment strategies, which started in the large animal canine model of RPE65 deficiency a decade ago (Acland et al., 2001; Le Meur et al., 2007; Narfstrom et al., 2003) and culminated in the first clinical trials in humans in 2007. Initially, three trials started enrollment (i) at the Moorfields Eye Hospital/University College London, UK (phase I; NCT00643747) (Bainbridge et al., 2008), (ii) at the Children’s Hospital of Philadelphia (CHOP)/Second University of Naples, Italy (phase I/II; NCT00516477) (Maguire et al., 2008), and (iii) at the University of Pennsylvania/University of Florida/National Eye Institute (phase I; NCT00749957) (Cideciyan et al., 2008; Hauswirth et al., 2008). The vectors administered in all three trials were based on the AAV serotype 2, but contained different expression cassettes (MacLaren, 2009). A total of 9 patients (three per trial) were treated, and all received the therapeutic gene in the clinically worse eye because of safety considerations. Published clinical data demonstrated absence of major side effects after treatment with AAV-mediated gene therapy. Efficacy outcomes for the patients showed improvements in light sensitivity, ambulation through an obstacle course, and nystagmus frequency in some patients, but the results in other patients were less promising. All patients still had non-recordable electroretinography (ERG), which was in clear contrast to the results obtained in the treatment studies using the canine model of RPE65 deficiency. The reason for the absence of measurable ERG responses, even in those patients with the best recovery, remains unknown.

Since the first results were published, clinical data on more than 30 patients are now available, which revealed interesting and expected, but also some unexpected, data. As expected, the clinical benefit remained stable in all patients, and no severe adverse effects were observed (Cideciyan et al., 2009a; Simonelli et al., 2010). In general, younger patients responded better to the treatment, with greater improvements in light sensitivity compared with older patients (Maguire et al., 2009; Simonelli et al., 2010). It was interesting to note that one patient from the Philadelphia/Florida/NIH trial unexpected developed an extrafoveal fixation point under low light conditions, which was located within the injected area indicating a dynamic response of the retinal circuitry to the treatment (Cideciyan et al., 2009b). This may have implications as to the choice of the injection site.

Update of RPE65 Clinical Trials in 2010

Three years later, in 2010, eight trials referring to gene therapy and RPE65 mutations are open and listed on the website, including the above mentioned original trials. A phase I trial started in 2009 in Israel at the Hadassah Medical University (NCT 00821340) in collaboration with the University of Florida. Interestingly, researchers at the Hadassah Medical University identified a whole group of patients with North African descent carrying a population specific homozygous founder mutation in the RPE65 gene (Banin et al., 2010). Treatment of 10 patients all carrying the same mutations is anticipated and may reveal new information about the treatment efficiency, as this would be the first time to be able to compare treatment outcome in a relatively large group of genetically related patients. A phase I/II trial started in late 2008 at the Oregon Health and Science University/University of Massachusetts (NCT 00749957), but published results are not yet available.

Currently two trials are open, organized by scientists from CHOP and based on the original phase I/II trial. A phase III clinical trial, in collaboration with the University of Iowa (NCT00999609), will enroll a set of 12 patients and focus on the effectiveness of the treatment in young patients. A phase I trial (NCT01208389) is designed as a follow-up study to investigate the injection of the vector into the contralateral previously uninjected eye of those patients enrolled in the original phase I/II trial. This trial is based on observations in dogs and primates that repeated injections of AAV vectors in both eyes are safe and effective (Amado et al., 2010; Bennett et al., 1999).

The University of Nantes and other ophthalmological centers in France are currently recruiting and characterizing patients with RPE65 deficiency with the aim to prepare a gene therapy trial in the near future (NCT00422721). An alternative treatment protocol is investigated in a trial that started in 2009 at the Montreal Children’s Hospital (NCT01014052), which is based on the potential use of an orally available synthetic retinoid (QLT091001). Results of this trial are not yet published for patients with RPE65 mutations.

Due to the availability of different clinical trials for the treatment of RPE65 deficiency, patients now have the real option of receiving a treatment that at least halts the progression of the disease.

Recent Preclinical Developments in Specific Gene Therapy

There are numerous publications on successful specific gene therapy applications in small and large animal models. Here, we report only on the most recent or in our view most important preclinical developments.

Treatment of achromatopsia

Due to absent or severely impaired cone function, patients with achromatopsia suffer from color blindness, severely reduced visual acuity, photophobia, and nystagmus. The disease is caused by mutations in genes encoding cone ion channel subunits (cyclic nucleotide gated channel subunit alpha and beta 3 — CNGA3, CNGB3) (Kohl et al., 1998; Kohl et al., 2000) or cone specific proteins of the phototransduction cascade (alpha 2 subunit of transducin, GNAT2, and cone phosphodiesterase alpha subunit, PDE6C) (Kohl et al., 2002; Thiadens et al., 2009). Several animal models exist to date, and AAV mediated gene therapy successfully restored vision in most of them, indicating that this treatment paradigm might soon enter the clinical stage. A mouse model for mutations in the GNAT2 gene, the cone photoreceptor function loss 3 (cpfl3) mouse, was treated using an AAV serotype 5 vector and showed rescue of function for a period of at least 5 months (Alexander et al., 2007). In a similar study, the cnga3 knockout mouse was also successfully treated using a modified serotype 5 vector with treatment efficiency shown for up to 3 months (Michalakis et al., 2010). A canine model of achromatopsia associated with CNGB3 mutations was also successfully treated (Komaromy et al., 2010). Achromatopsia was found in dogs with a naturally occurring substitution in exon 6 (D262N) of the CNGB3 gene in German shorthaired pointer dogs and with a naturally occurring nonsense mutation in Alaskan malamute dogs (Sidjanin et al., 2002). Cone response was abolished at the age of 8 weeks in both models. Affected dogs were injected unilaterally into the subretinal space with an AAV serotype 5 vector. Four weeks after treatment, successful restoration of cone function was achieved in all 10 dogs, and the best outcome was observed in animals treated at a young age. Two animals were treated and monitored for over 14 months and no deterioration of the rescue effect was observed.

Efficient restoration of cone function and the absence of adverse events in all treated animal models open the way for preparing clinical trials for humans in the near future.

Successful treatment strategies in mouse models for Leber congenital amaurosis (LCA)

Recent focus in developing gene therapy strategies was given on mouse models with different forms of LCA caused by mutations in the GUCY2D gene (retinal guanylate cyclase 1, retGC1; LCA1), the AIPL1 gene (aryl hydrocarbon receptor-interacting protein-like 1; LCA4), and the RPGRIP1 gene (retinitis pigmentosa GTPase regulator interacting protein 1; LCA6).


The GUCY2D protein is one of the central enzymes within the phototransduction cascade in both types of photoreceptors, as it normally restores intracellular cGMP levels and therefore allows for the reopening of cGMP gated cation channels. Mutations within the gene cause LCA1 and represent one of the most severe forms of blindness in infants (Perrault et al., 1996). In an initial study, the subretinal injection of an AAV serotype 5 vector containing the murine retgc1 gene produced immunohistological evidence of transgene expression and interaction with other proteins in the cones (Haire et al., 2006). However, rescue of photoreceptor function as assessed by ERG was not observed. This has since changed with a recent study, in which an improved vector (again serotype 5) was injected into retgc1 knockout mice, and treatment efficacy was evaluated until 3 months post injection, with visual function, visual behavior, and morphology being rescued over the entire period (Boye et al., 2010).


The AIPL1 protein is involved in the biosynthesis and correct localization of phosphodiesterase in photoreceptors, and therefore indirectly involved in the correct mechanism of the phototransduction cascade (Sohocki et al., 2000). Three different mouse models with four different rates of photoreceptor degeneration have been used to study the effect of gene therapy: the aipl1 knockout mouse with a very rapid loss of photoreceptors; the hypomorphic aipl1h/h mouse showing a slow degeneration under low levels of illumination, and an accelerated retinal degeneration under increased light condition (Tan et al., 2009); and a crossbred mouse line aipl1hypo with a slightly faster onset of disease than the aiplh/h mouse under low light conditions (Sun et al., 2009). Subretinal injection of AAV serotype 2, 5, or 8 vectors carrying either the murine aipl1 or the human AIPL1 transgene was sufficient to preserve morphology and function in all mouse models tested (Sun et al., 2009; Tan et al., 2009). Especially, the injection of the AAV serotype 8 vector elicited preservation of morphology and function even in the light accelerated aipl1h/h mouse and in the aipl1 knockout mouse. Treatment benefit was detectable for 23 months (Sun et al., 2009).


The RPGRIP1 protein is involved in the ciliary transport mechanisms of photoreceptors and mutations in its encoding gene cause deregulation of protein trafficking across the connecting cilium and subsequently photoreceptor loss. In rpgrip1 knockout mice, the degeneration of photoreceptors and subsequently all retinal layers is rapid (Pawlyk et al., 2005). Initially, the subretinal injection of an AAV serotype 5 vector containing the murine rpgrip1 gene resulted only in a transient protection of photoreceptor morphology and function as assessed by histology and ERG (Pawlyk et al., 2005). Recently, an AAV serotype 8 vector with the human RPGRIP1 gene was administered subretinally into rpgrip1 knockout mice, and preservation of visual function and morphology was obtained over a period of at least 5 months (Pawlyk et al., 2010).

The present treatment strategies for all three forms of LCA were successful in different mouse models. However, in recent years it became evident that therapies that work well in mice may fail when it comes to their clinical application in humans. Unfortunately, large animal models do not exist for GUCY2D and AIPL1 deficiency, and the canine model of RPGRIP1 deficiency has not yet been treated by gene therapy. If no canine model is available for treatment strategies, the direct transfer from mouse models to human application is necessary but uncertain.

Combined gene knock-down and gene addition therapy for autosomal dominant retinitis pigmentosa

Autosomal dominant diseases are mainly caused by mutations that result either in a toxic gain of function or in a dominant negative effect of the encoded protein. Mutations within the rhodopsin gene account for the largest proportion of autosomal dominant retinitis pigmentosa (adRP) cases and affect the folding, stability, and intracellular processing of the protein. The presence of only one mutant allele is sufficient for the onset of retinal degeneration. To treat these kinds of disorders, gene addition therapy is not suitable, as the mutant allele, mRNA, or protein product must be silenced beforehand. Rather than targeting the specific allele, which would mean in the case of rhodopsin to target more than 100 different alleles, the treatment strategy recently evolved more towards a combined gene knock-down and gene addition therapy. Both endogenous rhodopsin alleles, regardless of whether mutated or not, are down-regulated by siRNA technology, while at the same time, a codon modified rhodopsin cDNA that is not sensitive to siRNA interference is added by AAV mediated gene transfer. However, two crucial points must be addressed for this strategy: (1) the necessity of complete downregulation of endogenous rhodopsin production and (2) at the same time a very effective gene addition by gene transfer.

(1) Almost complete downregulation of endogenous rhodopsin was achieved by expressing an siRNA specific for all forms of human rhodopsin following transfer by an AAV serotype 5 vector into a mouse model that carries the human rhodopsin gene on a mouse rhodopsin background (O’Reilly et al., 2007a) and in a mouse model containing a mutated human rhodopsin gene (Chadderton et al., 2009). In both models, endogenous mouse rhodopsin expression remained unaffected.

(2) Similar successful strategies were developed for expressing rhodopsin at sufficiently high levels within photoreceptor outer segments after knock-down for functional light sensitivity. As the protein makes up to 90% of the outer segment membrane proteins (Palczewski, 2006), it has been a challenge to achieve this goal until recently. After having isolated a functional codon modified rhodopsin gene that is not sensitive to siRNA interference (O’Reilly et al., 2007b), this cDNA was expressed following AAV serotype 5 vector mediated gene transfer on a rhodopsin knockout mouse and found to produce about 40% of endogenous rhodopsin, which seems to be sufficient for normal function of rod outer segments (Palfi et al., 2010).

A large animal model for autosomal dominant RP exists, which is the dog breed Mastiff with a mutation in the rhodopsin gene (Kijas et al., 2002). It remains to be seen, whether this combined strategy of gene knock-down and gene addition will be successful in this clinically more relevant model before the strategy advances to the clinics. Several questions remain to be answered including the safety issue of siRNA applications in vivo (Grimm, 2009) and the problem of whether even a small percentage of residual activity of mutant endogenous rhodopsin can induce toxic effects.

Non-specific Gene Therapy Approaches for Treating Retinitis Pigmentosa

Even though continued research into the genetic origin of retinal degenerative disorders reveals more and more mutations in yet unknown genes, only 50-70% of all patients can be presented with a correct genetic diagnosis to date. Furthermore, except from patients with mutations in the RPE65 gene, no actual treatment option exists for those patients with known mutations in other genes, as translation of successful treatment strategies from the mouse to humans is still ongoing (see above). Therefore, non-specific gene therapy approaches exist that have the goal to either protect photoreceptors from degeneration by providing high amounts of neurotrophic factors (CDNF, BDNF, EPO) (Buch et al., 2006; Rex et al., 2004; Sieving et al., 2006) or to actively inhibit the induction of apoptosis by blocking the activity of caspases through inhibitors of apoptosis (XIAP) (Leonard et al., 2007; Yao et al., 2010).

Yet another strategy is to render cells from the inner retina photosensitive through the introduction of microbial rhodopsin following gene transfer. It has been shown that photoreceptor degeneration precedes degeneration of neurons in the inner retina by several years in most diseases, and targeting the neurons from the inner retina is possible until late stages of the disease. Expression of the bacterial protein channelrhodopsin-2 would generate a light-gated cation channel within the cellular membrane that would allow for depolarization events upon light stimuli (Nagel et al., 2003). It has been shown that targeting ganglion cells and bipolar cells have rendered them light sensitive and resulted in the restoration of basic vision-driven behavior in rodents (Lagali et al., 2008; Tomita et al., 2009; Tomita et al., 2010). Very recently, channelrhodopsin-2 was expressed even in ganglion cells of the marmorset retina, a well known nonhuman primate model (Ivanova et al., 2010).

However, targeting second or third order neurons in the retina would preclude the therapy from profiting from basic retinal circuitries, as they function correctly only when stimulated from first order neurons. Interestingly, cone bodies remain present within the retina longer than rod bodies in at least some forms of retinal degenerative disorders. This observation was taken further by a recent study that identified cone cell bodies in two mouse models of retinal degeneration long after the function of these cells was below sensitivity threshold. These light-insensitive cones were reactivated by the introduction of a bacterial form of rhodopsin, halorhodopsin, through AAV mediated gene transfer (Busskamp et al., 2010). The use of AAV serotype 7 and 8 vectors allowed for the rapid and strong expression of different forms of halorhodopsin in cones, which resulted in a measurable ERG, and normal spike depolarization pattern in ganglion cells, indicating reactivation of cone retinal circuitry. In addition, cortical circuits were also reactivated and visually guided behaviors were observed in treated animals. Human patients with retinal degenerative disorders who still possess residual cone bodies within the retina may benefit from this therapeutic strategy in the near future.


Clinical gene therapy trials for one form of inherited retinal blinding disorder have been started and revealed crucial information about the safety and efficacy of such treatment forms. Even though only a very small number of patients will directly benefit from this particular treatment, the general technique of AAV mediated transfer of a cDNA into retinal cells has been shown to work well, and will be used in other forms of retinal blinding disorder in the near future.

The question of which retinal disorder will be the next to be treated by gene therapy is not an easy one to answer, as several protocols are in the pipeline. The successful treatment of dogs with achromatopsia makes this disorder likely to be the next in line for a clinical application. On the other hand, as the safe administration of AAV vectors has been demonstrated in the RPE65 trials, researchers may feel confident to advance with the results of mouse models directly into the clinics and treat other forms of LCA soon.

The combined gene knock-down and gene addition strategy that has been proposed as a treatment option for autosomal dominant RP is still several steps away from any clinical application as a number of questions still remain unanswered. The presence of a large animal model may provide the platform for studying this treatment paradigm in a model system close to humans.

Finally, reactivating residual cones through halorhodopsin expression in retinas of patients regardless of the underlying disorder may provide a technique to help those patients where the disease-causing mutations are still unknown or where specific treatment options are still far from becoming a reality.


The authors report no conflicts of interest.

(Corresponding author: Knut Stieger, D.V.M. Ph.D., Department of Ophthalmology, Justus-Liebig-University Giessen, Friedrichstr. 18, 35385 Giessen, Germany.)


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[Discovery Medicine; ISSN: 1539-6509; Discov Med 10(54):425-433, November 2010.]

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