Adeno-Associated Virus Mediated Gene Therapy for Retinal Degenerative Diseases

  • Knut Stieger
  • Therese Cronin
  • Jean BennettEmail author
  • Fabienne Rolling
Part of the Methods in Molecular Biology book series (MIMB, volume 807)


Retinal gene therapy holds great promise for the treatment of inherited and noninherited blinding diseases such as retinitis pigmentosa and age-related macular degeneration. The most widely used vectors for ocular gene delivery are based on adeno-associated virus (AAV) because it mediates long-term transgene expression in a variety of retinal cell types and elicits minimal immune responses. Inherited retinal diseases are nonlethal and have a wide level of genetic heterogeneity. Many of the genes have now been identified and their function elucidated, providing a major step towards the development of gene-based treatments. Extensive preclinical evaluation of gene transfer strategies in small and large animal models is key to the development of successful gene-based therapies for the retina. These preclinical studies have already allowed the field to reach the point where gene therapy to treat inherited blindness has been brought to clinical trial.

In this chapter, we focus on AAV-mediated specific gene therapy for inherited retinal degenerative diseases, describing the disease targets, the preclinical studies in animal models and the recent success of the LCA-RPE65 clinical trials.

Key words

Inherited retinal diseases Retina Gene therapy AAV AAV serotypes Animal models RPE65 clinical trial 

1 Introduction

Several human clinical trials are ongoing or in preparation using recombinant Adeno-Associated Virus (rAAV), derived from a nonpathogenic virus, as a vehicle to treat or cure disease. rAAV has proven to be effective for treatments of a variety of eye disorders in preclinical studies (reviewed by refs. 1, 2, 3) partly because its ability to transduce nondividing cells has made it especially suitable for the retina. Indeed retinal tissue has distinct advantages for rAAV-mediated therapy: the tissue can be easily imaged and researchers have a contralateral eye as the ideal internal control for an in vivo experiment. The diverse populations of retinal cells are arranged in discrete layers and their connections are so well mapped that investigators can determine the specific tropism of the injected virus (see Fig. 1 for retinal anatomy). Apart from these practical advantages, a less well understood but potentially critical benefit to retinal gene delivery is the relative naivety of the tissue to the innate and adaptive immune system. Corneal transplantation already takes advantage of the eye’s “immune privileged” status. Re-administration studies in mice, dogs, and in nonhuman primates have verified the lack of inflammatory response after delivery and repeated delivery of AAV to the retina (4, 5, 6). Thus there are many contributing factors that have motivated investigators to make the retina an early candidate tissue for human gene therapy. Recent successes in this field have shown it to be a judicious choice. In fact AAV’s promise in the retina may give renewed validity to gene therapy in general, as it has allowed lessons learned from early clinical trials to ultimately be put into practice.
Fig. 1.

Schematic and histological overview on the most prominent retinal layers. The different neuronal cell types are connected by different forms of synapses, thus allowing the converted electrical impulses from the photoreceptors to reach the axons of the ganglion cells, which will transport the information into the brain.

Retinal diseases are non-lethal and do not in general have a significant adverse effect on fertility or reproduction within a population. This accounts for the wide level of genetic heterogeneity, which presents the pathologies with an equally broad range of clinical guises; in brief, retinal degeneration (RD) as a disease group is a challenge to categorize. Nonetheless, this mutational complexity has attracted the attention of molecular and clinical geneticists worldwide. Oftentimes their research efforts are supported by highly motivated patient groups. In addition to tireless fund raising, these groups provide information on family genealogy, enabling projects that map linked genes and reveal the identity of many causal mutations. Emerging alongside this gene discovery effort has been the advances in transgenic animal modelling, primarily using mice, frogs and zebrafish. This has helped investigators to elucidate the molecular pathology that leads from a mutant gene to such conditions as choroidemia (7), Stargardt’s disease (8), retinitis pigmentosa (9, 10) and X-linked juvenile retinoschisis (11). The molecular genetics behind these diseases will be discussed in greater depth in Subheading 2. In many cases it is the naturally occurring animal models that have been more relevant in discerning the physiopathological bases of the degeneration. While the Royal College of Surgeons (RCS) rat is the first known animal model of inherited RD, the rd1 mouse is the best characterised, having been studied for well over 80 years (12). Screens for spontaneous mutations in zebrafish have helped in the understanding of retinal development (13) and have been used to test therapeutic agents (14). However, timing therapeutic rescue in the animal models can be complicated, with the pathology being either too severe where it arises early in development, or too mild where it arises late in the lifespan of the animal.

The move to large animal testing marked a critical turning point in the research and accelerated the pace of discovery to clinical application. This was largely due to the many differences in retinal anatomy across species and the reality that direct comparisons cannot be made between a mouse and a man. While dogs do not have the highly pigmented macular region near the center of the retina found in primates, they do have a cone-enriched portion of the area (area centralis). Because of the size, surgical approaches similar to those that would be used in a human can be used (15). Further, canine models of inherited RDs have been very informative with respect to AAV therapies. The rescue of the disease in one such model, the Briard dog, established the therapeutic window available for the successful treatment of a form of retinal degeneration, Leber’s congenital amaurosis (LCA), by gene augmentation therapy (16, 17, 18, 19, 20, 21). This therapeutic approach has taken advantage of the long-term stability of transgene expression mediated by AAV. Where the dog model has been important in treatment design, the nonhuman primate has been important for preclinical testing of toxicity and immune response to both AAV and the therapeutic transgene it delivers (6, 22).

Furthermore, the work in large animal models has allowed investigators to optimise technical aspects of the injection procedure, retinal function testing, and imaging used to evaluate efficacy. All this has brought the field to the point where gene therapy to treat inherited blindness has been brought to clinical trial (23, 24, 25); a point that would not yet have been reached but for the animals with naturally occurring and engineered retinal phenotypes. Although challenges lie ahead, it is only a matter of time before proof-of-concept of gene augmentation therapy for other forms of retinal degeneration brings other blinding diseases to clinical trial.

2 Disease Targets

The retinal diseases can be divided into two groups, the hereditary degenerative disorders, and the neovascular disorders. While the first group of diseases is solely due to mutations in certain genes, the second group has its origins in a more complex mix of genetic and environmental causes.

In the following chapter, the authors will present the hereditary retinal disorders, which have been targeted for gene therapy using AAV vectors (Table 1).
Table 1

Retinal degenerative diseases that have been targeted by AAV-mediated gene therapy



Inheritance pattern

Target genes for gene therapy

Location of mutated gene

Rod-cone dystrophy


ar, ad, X

Rho, PDE6ß, ABCA4, RPE65, LRAT, RDS/Peripherin, MERTK, IMPDH1


Cone-rod dystrophy


ar, ad, X





ar, (ad)



Juvenile maculopathy (M. Stargardt)





Stationary cone dystrophy (achromatopsia)





Juvenile retinoschisis





Ocular albinism





Oculocutaneous albinism

Not known


(OCA1) tyrosinase


ar autosomal recessive, ad autosomal dominant, X X-linked, RPE retinal pigmented epithelium, PR photoreceptor, LCA Leber congenital amaurosis, EOSRD early onset severe retinal dystrophy

The hereditary degenerative disorders of the retina are primarily divided into three groups, depending on which type of photoreceptor manifests the initial insult, rods, or cones. These three groups include rod-cone dystrophies (Retinitis pigmentosa, RP), cone-rod dystrophies and cone dystrophies (maculopathies). In addition, the wide range of clinical symptoms in these diseases has resulted in the definition of two subgroups, in which the most severe forms of the degenerative disorders are included, LCA and early onset severe retinal dystrophy (EOSRD). However, more hereditary retinal diseases exist that have been subjected to the development of retinal gene therapy strategies. They include the stationary cone dysfunction disorder achromatopsia, retinoschisis, which is due to the separation of retinal layers and subsequent loss of retinal circuits, and albinism.

The visual symptoms in retinal degenerative disorders indicate the gradual loss of the two photoreceptor types: rods, which are present in the periphery and mediate achromatic vision under poor lightning conditions; and cones, which are concentrated in the central area (macula) and are important for color vision and fine visual acuity in daylight.

The different forms of hereditary retinal disorders will be shortly presented with prevalence, clinical symptoms and genetic situation with the aim to provide the reader with some background for the better understanding of the therapeutic applications.

2.1 Retintis Pigmentosa (Rod-Cone Dystrophies)

Retintis pigmentosa (RP) has a worldwide prevalence of 1 in 4,000 people, leading to more than one million total affected individuals worldwide (26).

RP is a heterogenous disorder. The onset of the disease can vary significantly: some patients may lose vision very early in life while others remain asymptomatic until midadulthood. Patients develop difficulties with dark adaptation and night blindness in adolescence and loss of mid-peripheral visual field in early adulthood. In a more advanced stage of the disease, patients have only a very limited visual field and will eventually also lose the central vision.

Most cases of RP are monogenic, but the disease itself is very heterogenous. To date, about 200 loci have been associated with retinal dystrophies, and about 45 genes have been identified and characterized causing RP ( Interestingly, these 45 genes collectively account for only a little over half of all patients. The mode of inheritance can be divided into three groups, autosomal recessive (50–60%), autosomal dominant (30–40%), and X-linked (5–15%). Other forms of inheritance exist for RP such as uniparental isodisomy or non-Mendelian inheritance (mitochondrial or digenic), but these occur only very rarely. Most genes cause only a small proportion of cases, but some exceptions are known; the RHO gene causes about 25% of autosomal dominant RP cases, the USH2A gene causes 20% of autosomal recessive cases, and RPGR causes 70% of X-linked cases. Mutations in these three genes account for about 30% of all RP cases (26).

2.2 Cone-Rod Dystrophies

The prevalence of this type of photoreceptor degeneration is 1 in 40,000 people worldwide (27).

Because of the initial insult in cones, patients with cone-rod dystrophy rapidly lose central vision and develop abnormalities of color vision associated with a variable degree of nystagmus and photophobia. The clinical course of the disease is often more severe when compared to patients with rod-cone dystrophy, as the central visual field loss severely hampers the vision early in these patients. Over time, the loss of cones and associated rod system abnormalities will lead to impaired peripheral vision as well (28). As with the rod-cone dystrophies, inheritance pattern of autosomal recessive, autosomal dominant and X-linked have been reported. The major genes responsible for autosomal recessive cone-rod dystrophies are ABCA4 (29) (see below), which is also called Stargardt disease, the ion channel subunit CNGA3 (30) that can also be responsible for complete achromatopsia, and the ciliary transport protein RPGRIP-1 (31). The most important genes responsible for the onset of autosomal dominant cone-rod dystrophy are CRX (32) that is involved in the embryogenesis of photoreceptors, GUCY2D (33) that is involved in the Calcium homeostasis of the photoreceptors, peripherin/RDS (28) being an important structural protein in the outer segments and AIPL1 (34), which assists in the assembly of PDE in the photoreceptor membrane. Finally, mutations in the gene RPGR have been reported to be responsible in some rare cases of X-linked cone-rod dystrophy (35).


LCA is the most severe form of all hereditary retinal dystrophies and is responsible for congenital blindness. Its prevalence is estimated at about 1:80,000 (36). If the symptoms start in early childhood and legal blindness is diagnosed before the age of 20, the disease should be called as EOSRD (37, 38).

Clinical signs include severe impairments of visual function from birth or with onset in the first months of life (for LCA), or during the first years of life (for EOSRD). In infants, these signs include the inability to visually fix on objects, nystagmus, a diminished photomotor reflex, and the presence of the oculo-digital Franceschetti sign. The funduscopic image of such patients is normal and the electroretinogram (ERG) nonrecordable (36).

In most cases the inheritance pattern is autosomal recessive, although some autosomal dominant mutations are known. To date, 15 genes have been identified as being involved in the onset of the disease: IMPDH1, AIPL1, CRB1, CEP290, CRX, GUCY2D, LRAT, RD3, RDH12, MERTK, RPGRIP1, TULP1, SPATA5, RPE65 and LCA5 ( (39). Only about 50% of all cases are associated with mutations in these genes, indicating that many genes responsible for the onset remain undiscovered.

2.4 Juvenile Maculopathy/Stargardt Disease

The most common form of juvenile maculopathy is called Stargardt disease and has a prevalence of 1 in 10,000 people in the world.

Onset of symptoms in patients with Stargardt disease starts in the second decade and will deteriorate progressively (40). Typical clinical signs are central scotoma, the bulls eye formed macula in the fundus autofluorescence image and yellow dots dispersed over the entire retina in the fundus examination (fundus flavimaculatus) (41). The gene responsible for Stargardt disease is the ABCA4 gene, the gene product of which is involved in the clearance of all trans-retinol from the photoreceptor cells. If ABCA4 function is impaired, the chromophore is accumulating in the RPE as lipofuscin and hampering the functional correlation of RPE cells and photoreceptors.

Some mutations are also associated with milder forms of the disease and later onset of symptoms, and are therefore often classified as AMD. The inheritance pattern of Stargardt disease is autosomal recessive (42).

2.5 Stationary Cone Dystrophies/Achromatopsia

For gene therapy studies, the most relevant stationary cone dystrophy is complete achromatopsia, in which function of cones is completely lost. The prevalence of the disease is unknown but is estimated at about 1:30,000 (43).

Patients suffer from reduced visual acuity, photophobia and dayblindness. Residual ERG recordings are measurable in very few patients. Mutations in three different genes are known to cause achromatopsia, CNGB3 (cyclic nucleotide gated channel β-3), CNGA3 (cyclic nucleotide gated channel α-3) and GNAT2 (guanine nucleotide α transducin) (44, 45, 46). All three gene products are involved in the phototransduction cascade, with CNGA3 and CNGB3 being subunits of the cone-specific cGMP-gated cation channel, and GNAT2 being the subunit of the cone-specific G-protein transducin.

The inheritance pattern for all three genes is autosomal recessive.

2.6 Juvenile Retinoschisis

Juvenile retinoschisis is the leading cause of juvenile macular degeneration in males and leads to a schisis (splitting) of retinal layers (47). The prevalence is estimated to be between 1:15,000 and 1:30,000.

Clinical symptoms differ greatly among patients and no correlation with underlying mutations has been made. Visual acuity varies between 20/20 and 20/600. The cartwheel like structure of folds radiating out from the fovea is the characteristic sign of juvenile retinoschisis (47). This phenomenon can become less distinct with age. Generally, large bullous lesions that can be observed by OCT imaging in infancy disappear and become invisible in older patients. Visual function can remain stable until midadulthood but can also deteriorate rapidly when complications such as hemorrhage or retinal detachments occur. Affected individuals have a relatively normal a-wave in the ERG, while the b-wave is completely abolished (negative ERG), indicating deficient responses from the inner retina (48). Female carriers of the mutation remain normally asymptomatic.

The underlying cause of the disease is a mutation in the retinoschisis gene RS1, which encodes the retinoshisin gene (49). The inheritance pattern is X-linked.

2.7 Albinism

The onset of albinism with ocular involvement has to be divided into two groups, (1) the pure ocular albinism (OA) and (2) the oculo-cutaneous albinism (OCA). All forms share the typical lack of abnormal production of the pigment melanin, which causes an increased light sensibility in organs that are exposed to light, such as the skin and the eye. There are at least ten forms of OA and four forms of OCA known due to different genes that can be mutated. Patients with OA develop pathologies primarily in the eye, while patients with OCA have also skin and hair abnormalities to a varying degree.

X-linked ocular albinism type 1 (OA1) is the most common form of ocular albinism and is caused by mutations in the oa1 gene. The prevalence of this disease is reported to be 1 in 60,000 (50). Patients have reduced and abnormal ERG recordings, severely reduced visual acuity and photophobia. The iris is in part translucent, the ocular fundus is hypopigmented and the macula shows foveal hypoplasia. The crossing of optic nerve fibers in the optic chiasm is reduced, which leads to the typical results of ipsilateral visually evoked potentials after unilateral light stimuli in these patients (albino-VEP). The protein encoded by OA1 is responsible for the correct organization of RPE melanosoms, and mutations lead to the occurrence of large pigment granules, the macromelanosoms. Rare forms of ocular albinism that are not X-linked have been reported.

OCA can be caused by mutations in different genes, the most severe form is OCA1 due to mutations in the tyrosinase gene (51). The protein tyrosinase is involved in early steps of the generation of melanin granula in epithelial cells such as the RPE in the eye. The clinical symptoms are similar to OA1 but the inheritance pattern of the disease is autosomal recessive (52). Clinically, the disease can be divided into OCA1-A with absence of any tyrosinase activity and OCA1-B with reduced tyrosinase activity.

3 Delivery Methods

3.1 Injection Method

Because of the blood/retina barrier, it is not possible to inject AAV intravenously and then obtain transduction of the retina. Instead, the rAAV must be delivered to an area in contact with the target cells (the intravitreal space or the subretinal space) so that those cells can be transduced (see Fig. 2). The serotypes of AAV most widely evaluated do not diffuse across the layers of the retina.
Fig. 2.

Anatomical features of the eye and the positions, where intravitreal and subretinal deliveries are performed. For intravitreal injection, the needle is introduced into the vitreous. For subretinal injection, the needle is advanced through the vitreous and positioned in the retina between the photoreceptors and the RPE.

3.1.1 Intravitreal

Intravitreal injection exposes cells lining both the inner surface of the retina and the anterior segment. Thus, depending on the rAAV capsid serotype and the presence of appropriate receptors (see below), retinal ganglion cells and Muller cells can be transduced and in the anterior segment, trabecular meshwork, Schlemm’s canal, corneal endothelial cells, lens, iris and ciliary body epithelial cells can be transduced (53, 54, 55). Although transduction of anterior segment structures is not desirable in retinal gene augmentation or gene knockdown strategies, it may be useful in the future for delivery of neurotrophic factors (not discussed further in this review). Retinal ganglion cell transduction, which can be achieved through intravitreal delivery can deliver transgene product to those areas of the brain where ganglion cells synapse (lateral geniculate nucleus, superior colliculus, etc.) (54, 55, 56).

3.1.2 Subretinal

Subretinal injection exposes cells lining both the outer surface of the retina and the retinal pigment epithelium (RPE). Thus, retinal photoreceptors, Muller cells and RPE cells can be transduced after subretinal injection (53, 57, 58, 59). Because of the different anatomical features of the mouse eye versus the large animal eye, different subretinal injection techniques must be used in the different species. The lens of the mouse eye occupies nearly the entire vitreous cavity. Thus it is difficult to deliver the AAV from an anterior approach in mice due to risk of damage to the lens with a resultant cataract. In contrast, subretinal injections in dogs, monkeys, and men can be carried out under direct visualization through an operating microscope (6, 15, 19, 22, 23, 24, 25).

3.2 Tropism of rAAV Vectors

Different AAV serotypes display tissue preference and specific cellular tropism in a variety of organs. The retina contains within its layers many different cell types, some of them unique to the eye. Therefore, cellular tropism of each serotype needs to be tested in the retina of every animal model before starting any gene therapy protocol (Table 2). Over the last decade, serotypes AAV2/1, 2/2, 2/3, 2/4, 2/5, 2/6, 2/8, and 2/9 and 2/rhu10 have been tested for cellular tropism and long-term expression in the retina of rodents, dogs and nonhuman primates. In addition, AAV serotypes isolated from porcine tissue as well as newly engineered AAV capsid variants have been tested for optimized cellular transduction efficacy.
Table 2

Cellular tropism of rAAV serotypes following subretinal injection

rAAV serotype



























































RPE retinal pigmented epithelium, PR photoreceptors, MC Muller cells, INL inner nuclear layer, GC ganglion cells, n.d. not determined

3.2.1 Cellular Tropism Following Intravitreal Injection

Generally, the transduction efficiency of AAV vectors of any serotype following intravitreal delivery is much lower when compared to subretinal injections. However, the best studied AAV serotype, serotype 2, transduces retinal cells after intravitreal injection, mainly retinal Ganglion cells. Efficacy of transduction and level of transgene expression remain low in all species (54, 56, 60, 61, 62). Nonetheless, clinical trials based on the intravitreal injection of AAV2 vectors to express anti-VEGF molecules are currently in progress (63, 64). The intravitreal injection of AAV2/2 vectors expressing the transgene under GFAP promoter resulted in detectable transgene expression in Muller cells (60). Due to minor capsid changes, the AAV variant SSH10, closely related to AAV serotype 6, is capable of transducing Muller cells in the retina following intravitreal injection in mice (65).

One of the reasons for the limited capacity of AAV vectors to transduce retinal cells after intravitreal injection is the presence of the inner limiting membrane (ILM) at the junction of neuroretina and vitreous. Following the digestion of the ILM using a nonspecific protease, AAV serotypes 2/1, 2/2, 2/5, 2/8, and 2/9 transduced retinal cells to a variable degree, with the AAV5 serotype being the most efficient (66).

Interestingly, even though it has been shown several times that the AAV8 serotype is not able to efficiently transduce retinal cells following intravitreal injection, one study by Park and colleagues reported on the successful expression of the retinoschisin (rs1) gene in photoreceptor and other retinal cells of rs1 knockout mice after intravitreal delivery of the vector (67). However, one potential explanation for this discrepancy would be the altered retinal morphology in rs1−/− mice, in which the normal cellular junctions and barriers of the ILM may be less restrictive to the viral passage into the neuroretina.

3.2.2 Cellular Tropism Following Subretinal Injection

Following subretinal injection in the mouse retina, AAV2/2 vector transduces RPE and photoreceptors (53, 57, 68). Similar cellular specificity but more efficient transduction is obtained using the AAV2/5 serotype (69). AAV2/1 serotype transduces only the RPE in the mouse, with rapid onset of transgene expression (3–4 days) (53). AAV2/3 serotype does not transduce retinal cells; rAAV2/6 seems to weakly transduce RPE cells (70). The more recently developed AAV serotypes AAV7, 8, and 9 were all shown to very efficiently transduce photoreceptors and RPE in mice (55, 58, 71). In addition, AAV serotype 9 also transduces Mueller cells (58) and cells that have synaptic connections in the outer plexiform layer (72). The AAV variant SSH13, developed through minor capsid changes from AAV serotype 6, transduces Mueller cells in the retina following subretinal injection (73). The porcine AAV serotype AAV2/po1 was as efficient in transducing mouse photoreceptors and RPE cells as AAV2/5 following subretinal injection (74).

Subretinal injection of serotypes AAV2/1, 2/2, 2/3, 2/4, and 2/5 in rats resulted in a hierarchy in the level of transgene expression, with AAV2/4 and AAV2/5 being the most efficient (75). The more recently utilized serotype AAV8 transduces very efficiently cells in all retinal layers, including RPE, photoreceptors, cells of the inner nuclear layer (INL) and ganglion cells (76). Interestingly, this serotype transduces cells that are located inside and outside of the injected area, a characteristic that is unique to AAV8, as it has not yet been reported for other serotypes (76). A way to enhance RPE transduction with AAV serotype 2 after subretinal injection is the co-delivery of ultrasound targeted microbubbles, which would temporarily destroy small areas of the cellular membrane to increase the uptake of the vector (77).

Brainbridge and colleagues observed transgene expression in RPE and photoreceptor cells following subretinal injection of AAV2/2 vector in dogs (78). Comparative studies using AAV2/2 and AAV2/5 serotypes showed that transduction efficacy of photoreceptors by AAV2/5 was superior to that of AAV2/2 in rats, dogs and nonhuman primates (59). The same study showed, that AAV2/4 serotype allowed exclusive and stable transduction of RPE cells in all three animal models tested. Subretinal injection of a complete AAV5/5 showed higher transduction efficacy than AAV2/2 in RPE and photoreceptors of mice and nonhuman primates (79). An interesting observation was that self complementary (sc)AAV vectors of the serotype 5 were capable of transducing RPE and photoreceptor cells at a faster rate with higher level of transgene expression when compared to single-stranded (ss)AAV vectors (55, 80).

Similar to the results in rats following subretinal injection, the serotype AAV8 transduces all cells of the retina inside and outside of the injected area in dogs (76). This finding may have important consequences for gene therapy trials, where widespread expression of the transgene in the retina is the objective.

3.3 Specific Versus Nonspecific Promoter

Combining a cell-type specific promoter and a cell-type specific vector would ensure the most specific and efficient transgene regulation in one cell type and at the same time limit widespread distribution of vector and/or transgene products within the host organism.

In vivo gene transfer studies used mostly strong and ubiquitous viral promoters for driving reporter or therapeutic transgene expression, such as the immediate early cytomegalovirus (CMV) promoter (20, 53, 57) or a chimeric chicken beta-actin (CBA)/CMV enhancer promoter (16). Gene regulation studies revealed a number of ocular specific promoters capable of driving transgene expression in one cell type: the human RPE65 specific promoter (81) and the VMD2 specific promoter (82) in the RPE, the mouse GFAP specific promoter in Muller cells (83), the human rhodopsin kinase specific promoter in both types of photoreceptors, rods and cones (84), and different opsin promoters specific for rod or cone photoreceptor subtypes: mouse (85), bovine (86) and human rod opsin specific promoter (87), human blue opsin specific promoter (88) and the human red and green opsin specific promoter (89, 90, 91)

3.4 Safety and Biodistribution in the Retina

Evaluation of safety of gene transfer in the eye of large animal models is highly relevant for the development of clinical trials. As direct visualization of GFP expression in the retina can easily be carried out by fluorescence fundus photography, vectors encoding this reporter gene have been used to study the capacity of AAV for long term expression in the retina of rodents and large animal models such as dogs and primates. Subretinal injection of AAV2/2.CMV.gfp in dogs resulted in efficient and stable transgene expression for at least 18 months (78). Using the same vector in the primate retina led to efficient transduction of RPE cells and rod photoreceptors for more than 12 months (6). Another study, using the complete AAV5/5 vector, showed similar cellular tropism in the primate retina and persistent transgene expression for 10 months, the duration of the study (79).

To evaluate the short-term morbidity of subretinal injections and the safety of long-term rAAV-mediated transgene expression, Le Meur and colleagues injected the clinically interesting serotypes AAV2/2, AAV2/4 and AAV2/5 unilaterally in 14 beagle dogs and 9 nonhuman primates. They found that subretinal injection of rAAV vectors in dogs and primates is a safe procedure with no perioperative complications and a high rate of successful retinal gene transfer (95%). As assessed by angiography and electroretinography, retinal anatomy and function remained unchanged, with persistent transgene expression up to now 6 years (92) (Rolling, unpublished data).

A primary requirement for safe and successful ocular gene therapy using rAAV is an accurate evaluation of vector distribution after subretinal or intravitreal delivery. Not only are multiple cell types in the retina exposed to the vector, and some scattering of virus to the systemic circulation is possible, but also the eye is closely connected to the brain via the optic nerve and the presence of vector DNA in the brain needs to be examined. Weber and colleagues documented vector shedding after subretinal delivery of rAAVs in dogs and nonhuman primates (59). Vector DNA could be detected in the serum of injected animals as early as 15 min after AAV administration and for up to 25 days in some individuals. Viral genome was detected in nasal and lacrimal fluid after 15 min and for up to 4 days. The extent of vector shedding is likely affected by the dose delivered.

Regarding the distribution of vector DNA in the optic nerve and brain, investigators have to distinguish between the presence of transgene product and the actual expression of the transgene in the brain. Early studies showed that after intravitreal injection of AAV2/2.CMV.gfp in mice and dogs, levels of GFP protein persists for 6 months in the brain and optic nerve. However, no vector DNA could be detected outside of the retina (54). Other studies showed the presence of lacZ and GUSB as transgene products in the optic nerve or the brain of mice and guinea pigs, respectively (56, 93). Again, vector DNA could not be detected in the optic nerve or brain in both studies. Griffey and colleagues showed improved enzyme activity along the visual pathway in the brain after intravitreal delivery of an AAV2/2 vector encoding for the missing enzyme in a mouse model of infantile neuronal ceroid lipofuscinosis (94). These data suggest that AAV2/2 mediated intravitreal gene transfer results in the transduction of retinal ganglion cells and transgene products are anterogradely transported along the visual pathway into the brain. However, these data do not show any anterograde transport of vector DNA into the brain via the optic nerve.

In contrast, Provost and colleagues studied the vector DNA distribution in rats, dogs and nonhuman primates after subretinal or intravitreal injection of AAV2/2, 2/4 and 2/5 vectors (95). They detected vector DNA occasionally in peripheral blood mononuclear cells at early time points after injection in all animals. Specifically, vector DNA sequences were present in the optic nerve of dogs and primates that received subretinal injections of AAV2/4 or AAV2/5; intravitreal injection of rAAV2/2 in dogs resulted in the detection of vector DNA along the visual pathway in the brain – the optic nerve, optic chiasm, optic tract, lateral geniculate nucleus and visual cortex. These results suggested for the first time the anterograde transport of rAAV2/2 from the retina to central visual structures.

Jacobson and colleagues studied vector DNA distribution in dogs and primates after subretinal or intravitreal injection of AAV2/2 vector at different time points post injection (22, 96). Vector DNA could not be detected in a dose escalation study of subretinally injected AAV2/2 in dogs (96). In contrast, vector DNA could be temporarily detected in the optic nerve and brain of primates injected subretinally with the AAV2/2 vector at 1 week post injection (22). At 3 months post injection, only one primate had a detectable number of copies in the left geniculate nucleus.

In all cases, even though vector DNA was detected in the brain, no transgene expression was observed in brain structures after subretinal injection. This observation may be due to spreading of nonfunctional vector that does not allow transgene expression, or lack of expression due to particular issues related to the transgene cassette, or because transgene expression was below the detection level. However, a recent study by Stieger et al. reported the presence of vector DNA, GFP mRNA and GFP protein in neurons in the left lateral geniculate nucleus (contralateral to the injected eye) after subretinal delivery in dogs (76). In addition, vector DNA was found in many parts of the brain, but chiefly on the contralateral side. These results demonstrated for the first time that subretinal injection of AAV2/8 vector leads to gene transfer into distal parts of the brain. Because axons of the retinal ganglion cells project to the contralateral geniculate nucleus, the data also suggest trans-synaptic transport as the mode of delivery to these neurons; however, due to limitations in the experimental design, this could not be proved.

An unexpected observation was made by the group of Rolling and colleagues when they studied the ultrastructure of the retina in dogs and primates several years after subretinal injections of AAV vectors. They observed persisting particles of the AAV serotypes 2, 4, and 5 in the outer plexiform layer (OPL) and in other retinal layers of dogs and primates up to 6 years following successful gene transfer into the retina (97). This observation opens the concept of in vivo intracellular clearance of vector particles as a potentially critical factor for the design of gene therapy clinical trials, especially in terms of immunological safety.

3.5 Preclinical Studies for Inherited Degenerative Diseases

This chapter focuses on specific gene therapy, which includes gene addition or gene silencing to treat autosomal recessive and autosomal dominant inherited degenerative diseases, respectively (Table 3). Nonspecific gene therapy strategy, which involves expression of neurotrophic factors to promote cell survival in retinal degenerations is not discussed.
Table 3

Preclinical studies in animal models of retinal degeneration

Gene therapy


Targeted gene

Animal model

Human disease


Gene addition

Targeting RPE



Oa 1



RPE65 −/− mouse, RPE65−/− Briard dog

LRAT−/− mouse

Oa1−/− mouse

OCA mouse

RCS rat



Ocular albinism

Oculocutaneous albinism


(16, 17, 18, 19, 20, 21, 104, 105, 106, 107)






Targeting PR








Peripherin 2


rd1 mouse, rd10 mouse

GNAT2cpfl3 mouse

CNGB3 dog

RetGC1−/− mouse

Aipl1−/− mouse, Aipl1h/h mouse

abca4−/− mouse


Prph2 rd2/rd2 mouse

RS1h−/Y mouse






Morbus Stargardt



Juvenile retinoschisis

(114, 116)




(121, 122)



(127, 128, 129, 130)

(67, 132, 133, 134, 135, 136)

Gene silencing






RHO +/− mouse, P23H rat

RHO +/− mouse, RHO-M mouse, RHO347−/− Rho −/−mouse, P23H rat

AAVmut IMPDH1 mouse




(140, 141, 142, 143)

(144, 145, 146, 147, 149, 150)


RPE retinal pigmented epithelium, PR photoreceptor, LCA Leber congenital amaurosis, EOSRD early onset severe retinal dystrophy, adRP autosomal dominant retinitis pigmentosa, arRP autosomal recessive retinitis pigmentosa

3.5.1 Treatment of Autosomal Recessive Diseases

The strategy to treat autosomal recessive diseases, in which the mutated gene product is not existent or does not cause toxic effects, is to provide the cell with a correct allele of the mutated gene by gene addition technology. As it has been mentioned above, choice of vector and promoter for the expression cassette will depend on the targeted cell that is in most of the cases either the RPE or the photoreceptors. Therefore, a general differentiation of the gene therapy approaches has been established into those targeting diseases originating in the RPE and those targeting diseases originating in photoreceptors.

In general, it turned out that diseases originating in the RPE seem to be more amenable to gene therapy treatment than diseases from the second group. This is in part due to the single layer ­characteristic of the RPE, which can be more efficiently targeted by AAV vectors. Another reason is the impact of corrected and functional RPE cells on the adjacent photoreceptors, because up to 40 of these specialized and very sensitive neurons depend on nutritional as well as metabolic support from one RPE cell. On the other hand, diseases originating in the photoreceptor cells often result in rapid induction of apoptosis limiting the extent of therapeutic intervention. The complex expression pattern of some of the genes in photoreceptors (i.e. RPGR, RPGRIP) and the involvement in complex enzymatic cascades (i.e. phototransduction cascade) is likely to require further technologic improvements, such as the ability to confer physiologic transgene expression and the production of cell specific isoforms.

Concerning the RPE cell layer, gene augmentation protocols targeting five different genes have been reported: the all-trans retinylester isomerohydrolase RPE65, the lecithin retinal acyltransferase (LRAT), the mer tyrosine kinase (Mertk) and the genes OA1 and tyrosinase (Tyr). While the last two genes are involved in the onset of albinism, mutations in LRAT, MERTK and RPE65 cause LCA or EOSRD.

Two of the genes targeted in photoreceptors are involved in the visual cycle, the LCA genes RPE65 and the LRAT.

The RPE65 gene encodes for an RPE specific 65-kDa protein that has been identified as the isomerohydrolase in the visual cycles, which restores the rhodopsin ligand 11-cis retinal (98, 99). Mutations in this gene are responsible for about 6% of LCA cases. In addition to a genetically engineered rep65 knockout mouse model (100), two naturally occurring models of LCA caused by mutations in rpe65 exist: the homozygous Swedish Briard dog (101, 102) and the rd12 mouse (103). Similar to humans, these animals do not develop sufficient amounts of the photopigment rhodopsin, they have poor vision and diminished light and dark adapted ERG responses. Especially the Briard dog model represents a valuable model for the development of a gene replacement therapy for RPE65 deficient patients, as the phenotype of the retinal degeneration is very similar to the one in humans. In these animals, a 4-bp deletion leads to a premature stop codon and the absence of functional protein in the retina (101, 102).

In mice, Lai and colleagues reported successful restoration of vision in rpe65 knockout animals following rAAV2/2 vector administration that carried the mouse rpe65 gene under the control of the CMV promoter (104). Pang and colleagues reported the successful restoration of vision in rd12 mice using a rAAV2/5 vector carrying the human RPE65 gene driven by the CBA promoter (105). To verify functional vision in these animals, the morris water maze test was modified to test rod function under dim light, and indeed, treated animals behaved like wildtype mice, while untreated control mice failed this test. Dejneka et al. showed that delivery of human RPE65 through rAAV2/1 resulted in rescue in adult rpe65 knockout mice (106). The authors of the last mentioned study showed in addition that in utero administration of rAAV2/1 vector carrying the human RPE65 gene driven by a CMV promoter could restore visual function in the KO mouse (106). However, there are limitations to this approach that make in utero gene therapy in humans rather unlikely.

In dogs, two groups demonstrated that a single subretinal injection of a rAAV2/2 vector carrying the canine rpe65 gene under the control of a strong CBA promoter was sufficient to restore visual function in affected dogs, as assessed by ERG, immunochemistry and behavioral testing (16, 20). In an attempt to improve the expression cassette and the vector, the first group injected several different constructs using AAV2/1, 2/2 or 2/5 capsids, the non-specific CBA promoter or the tissue-specific RPE65 promoter to drive either the canine or the human rpe65 gene and reported long-term restoration of visual function for up to 3 years (17). Finally, improvements to the promoter were done in order to increase specificity of the method (18). The second group studied the long-term outcome of their AAV2/2.CMV.crpe65 injected animals and reported a gradual loss of visual function as measured by ERG responses over 3 years, the reason for which is not clear (107). In addition, Le Meur and colleagues have demonstrated the capacity of a rAAV2/4 vector carrying the human rpe65 gene under control of the human RPE65 promoter to restore vision in the Briard dog model (19). As mentioned above, rAAV2/4 vectors transduce exclusively the RPE and therefore, the use of this serotype increases the specificity and safety of the therapy. In addition, the tissue-specific RPE65 promoter also increases specificity by allowing transgene expression only in RPE cells. In two safety studies on AAV2/2 mediated retinal gene transfer, Jacobson and colleagues and Bennicelli and colleagues reported preclinical safety data on a total of 21 rpe65 −/−dogs and 17 normal nonhuman primates subretinally injected with a rAAV2/2 vector carrying the human rpe65 gene driven by the CMV/β-actin hybrid promoter (18, 22, 96).

LRAT is an enzyme located in the RPE, which converts all-trans-retinol into all-trans-retinyl ester, a key step in the restoration of 11-cis retinal during the retinoid cycle. In lrat −/− mice, no 11-cis retinal is produced and visual function is severely impaired at 2 months of age, as measured by scotopic and photopic ERG (108). Subretinal injection of a rAAV2/1 vector carrying the mouse lrat gene under the control of the human VMD2 promoter restored ERG waves to 50% of wildtype mice (109). Interestingly, oral administration of 9-cis-retinyl succinate and 9-cis-retinyl acetate, which bypass the LRAT function in the retinoid cycle, similarly restored ERG responses to 50% of wildtype mice (109).

Two genes targeted in the RPE are involved in melanin synthesis and mutations in these genes are responsible for OA1 or OCA1.

A mouse model of ocular albinism type 1 has been generated, which mimics the human phenotype (110). Subretinal injection of a rAAV2/1 vector carrying the murine oa1 gene driven by a CMV promoter in 8 months old mice resulted in an increased number and normal density of melanosomes in the RPE (111). More importantly, ERG recordings in dark adapted oa1 −/− mice were improved, regardless of the age at which the mice were treated (1 or 8 month of age).

A spontaneous null mouse of OCA1 (Tyr c-2j) that has several retinal function anomalies due to photoreceptor loss beginning at 7 month of age was used to study the effect of AAV-mediated gene therapy in this disease (112). An AAV2/1 vector driving the human tyrosinase gene under the control of the CMV promoter was injected subretinally into 1-month old mice. Animals had detectable levels of melanin at 2 months post injection, indicating that the pathway for the production of the pigment is functional. Similarly, electrophysiological examination revealed increased light sensitivity and improved photoreceptor function.

In the RCS rat, phagocytosis of photoreceptor outer segments by RPE cells is impaired due to a mutation in the receptor tyrosine kinase gene, mertk. This leads to the accumulation of outer segment debris in the subretinal space and subsequently to progressive loss of photoreceptors by apoptosis. Subretinal injection of rAAV2/2 vector carrying the mouse mertk gene driven by either a strong CMV promoter or the cell-specific RPE65 promoter led to prolonged photoreceptor survival for as long as 9 weeks compared to untreated control eyes (113).

Concerning photoreceptor cells, nine different genes have been targeted by AAV-mediated gene therapy, encoding the phosphodiesterase 6 beta subunit (PDE6B), alpha transducin subunit (GNAT2), the cyclic nucleotide gated channel subunit beta (CNGB3), the retinal guanylate cyclase 1 (RetGC1, GUCY2D), the aryl hydrocarbon receptor-interacting protein like 1 (AIPL1), the ATP binding cassette transporter (ABCA4), the Retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1), peripherin and retinoschisin (Rsh1).

The rd1 mouse contains a mutation in the ß subunit of the phosphodiesterase (pde6b) gene, which results in a rapid onset of photoreceptor degeneration. At postnatal day 21, only one row of photoreceptors remains in the retina. This mouse model was the first to be used in a gene replacement protocol using AAV vectors. In 1997, Jomary and colleagues injected intravitreally a rAAV vector containing the PDE6β gene under the control of a truncated opsin promoter into 8 days old mice (114). At 21 days, treated eyes showed increased numbers of photoreceptor rows when compared to noninjected control eyes. Unfortunately, functional recovery was not evaluated in vivo, but only in vitro. Treated retinas showed a twofold increase in sensitivity to light. This study provided for the first time evidence that AAV-mediated gene augmentation therapy may rescue photoreceptors morphologically and functionally.

A second mouse (rd10) strain that has a mutation in exon 13 of the pde6b gene shows a slightly less rapid degeneration. Loss of photoreceptors starts at 2 weeks of age and highest rates of apoptosis are observed at 5 weeks (115). Subretinal injection of an AAV2/5 vector carrying the mouse pde6b gene under the control of the ubiquitous CBA into 2-week-old mice resulted in prolonged survival of photoreceptors until p35 (3 weeks post injection) and substantial scotopic ERG recordings (116). However, long-term examination was not reported.

Gnat2 cpfl3 homozygous mice have a mutation in the murine gnat2 gene homolog cpfl3 (cone photoreceptor function loss 3), and exhibit cone dysfunctioning and progressive loss of cone α-transducin immunolabeling (117). This mouse model of achromatopsia was recently used to study an AAV-mediated gene therapy protocol targeting cones (89). Subretinal injection of a rAAV2/5 vector containing the murine gnat2 gene under the control of a red/green opsin promoter in 3–4 weeks old gnat2 cpfl3 mice resulted in rescue of cone function and visual acuity for at least 5 months.

Achromatopsia has been associated in dogs with a substitution in exon 6 (D262N) of the CNGB3 gene in German shorthaired pointer dogs and with a nonsense mutation in Alaskan malamute dogs (118). In both canine models of achromatopsia, cone response was abolished at the age of 8 weeks. Affected dogs were injected unilaterally into the subretinal space with an AAV5 vector containing the canine CNGB3 gene under the control of four different promoters (PRO.5, 3LCR-PRO.5, PR2.1, and the human cone arrestin promoter). Four weeks after treatment, successful restoration of cone function was achieved in ten dogs with either CNGB3 null or missense mutation. The best outcome was observed when animals were treated at a young age. While positive results were seen with all four promoters, the most robust restoration of cone function was obtained with the PR2.1 promoter. Two dogs treated with AAV5.PR2.1.CNGB3 were monitored for over 14 months and no deterioration of the rescue effect was observed (91, 119).

In retgc1 knockout mice, the light-driven translocation of cone arrestin is compromized, which leads to loss of function and photoreceptor cell death within the first 6 month of life. Subretinal injection of a rAAV2/5 vector containing the retgc1 gene driven by either the cell specific mouse opsin promoter or the strong CBA promoter lead to restoration of light-driven translocation of cone arrestin, but not rescue of function of cones as assessed by ERG (120). Failure to restore function of cones was potentially due to toxic side effects caused by retgc1 overexpression in these cells.

The AIPL1 is involved in the biosynthesis and correct localisation of PDE in photoreceptors and mutations in this gene lead to the rapid onset of LCA (34). 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 (all gone by 3 weeks of age); the hypomorphic Aipl1h/h mouse under low levels of illumination showing a slow degeneration (degeneration starts at 12 weeks and half of all photoreceptors are gone by 6 months of age) and the same hypomorphic Aipl1h/h mouse under increased light condition showing an accelerated retinal degeneration by two- to threefold when compared to the low illuminance condition (121). Finally, a crossbred mouse line called Aipl1hypo that was produced by mating the Aipl1−/− and AIpl1h/h mice and has a slightly faster onset of disease as the original Aiplh/h mouse under low lightning conditions (122). Subretinal injection of an AAV2/2 vector carrying the murine Aipl1 transgene under the control of the CMV promoter was sufficient to preserve morphology and function in the Aipl1h/h mouse under low lightning conditions, and a similarly constructed AAV2/8 vector was even capable to preserve morphology and function in the light accelerated Aipl1h/h mouse and in the Aipl1−/− knockout mouse (121). In a related approach, AAV2/5 and AAV2/8 vectors driving the human Aipl1 gene under the control of the rhodopsin kinase (Rk) promoter were injected subretinally into the Aipl1hpo mouse model at 5 months of age and photoreceptors were still detectable after 23 months (122). In addition, the AAV2/8 vector was also able to preserve morphology and function in the Aipl1−/− when injected at p9 to p13 (122).

An ABCA4−/− knockout mouse model based on pigmented (123) or albino (124) mice, which resembles the phenotype of Stargardt disease has been used for the study of AAV-mediated gene therapy. Subretinal injection of an AAV2/5 vector driving the ABCA4 gene under the control of the CMV promoter into this mouse model resulted in properly localized ABCA4 protein in the photoreceptor layer, normal RPE cell appearance and decreased RPE thickness (125). Functionally, the recovery time from light desensitization was significantly improved.

Retinitis pigmentosa GTPase regulator (RPGR) is a protein connected to the photoreceptor cilium by RPGR interacting protein 1 (RPGRIP1). If RPGRIP1 is nonfunctional due to mutation in its encoding gene, RPGR is delocalized, causing deregulation of protein trafficking across the connecting cilium and subsequently photoreceptor loss. In RPGRIP-1 knockout mice, the degeneration of photoreceptors and subsequently all retinal layers is rapid (126). The subretinal injection of a rAAV2/2 vector containing the rpgrip1 gene driven by a mouse opsin promoter resulted in the transient morphological and functional protection of photoreceptors as assessed by histology and ERG (126).

PrphRd2/Rd2 mice, formerly known as rds (retinal degeneration slow) mice carry a homozygous null mutation in the Prph2 gene, encoding the structural protein peripherin2, which is essential for outer-segment formation. Due to the failure to develop photoreceptor discs and outer segments, photoreceptor cells undergo progressive apoptosis. ERGs are diminished early after birth and decline to undetectable levels within 2 month. Gene replacement has been tested using an AAV2/2 vector carrying the wildtype Prph2 gene under the control of a rhodopsin promoter. Subretinal injection of this vector led to improvement of photoreceptor structure and function as shown by electron microscopy and ERG (127, 128). Sarra et al. demonstrated that the potential of morphological improvement depends upon the age at which animals are treated and overexpression of the peripherin 2 protein might be harmful to photoreceptor cells (129). Another study by Schlichtenbrede et al. showed the improvement of higher visual functions after gene replacement therapy in Prph Rd2/Rd2 mice based on recordings from visually responsive neurons in the superior colliculus (130).

The mouse ortholog of the retinoschisin gene is known as rs1h (131). Two knockout mouse models of RS have been generated, both of which exhibit to a certain degree a similar phenotype when compared to the human disease (11, 132). Each mouse model was then used to study the efficacy of gene therapy protocols. The first group of studies used an AAV2/2 vector containing the murine rs1h gene under the control of a CMV promoter. In 13 weeks old rs1h −/Y mice (132), subretinal injection of this vector leads to the detection of retinoschisin in all retinal cell layers and the restoration of the normal b-wave amplitude (132). However, normal retinal structure could not be restored. Following the initial results, the authors presented the long-term results of the retinal changes following AAV-mediated gene transfer in this mouse model (133). A correlation between functional and morphological changes for up to 14 months post injection was demonstrated. Furthermore, in a different study, the authors demonstrated mislocalization and reduced levels of pre- and postsynaptic markers in photoreceptors in mice (PSD95, mGluR6, synaptophysin) at 4 weeks, which explains the reduced b-wave in ERG recordings and therefore the block in transmitting information from the photoreceptors to ganglion cells and subsequently to higher visual regions in the brain (134). Apparently, the Retinoschisin expression helps at least in part in the preservation of normal synaptic structures and gene addition therapy using the AAV2/2.CMV.rs1 vector partially restored synaptic integrity in these animals. Very recently, the intravitreal injection of an AAV2/8 vector containing the rs1 gene (including a truncated first intron) under the control of a retinoschisin promoter resulted in detectable retinoschisin expression in rs1 knockout mice (67). This successful gene transfer resulted in improved ERG recordings up to 15 weeks post injection. The second group of studies employed an AAV2/5 vector carrying the human rs-1 gene driven by a mouse opsin promoter, which was subretinally injected into 2 weeks old rs1h −/− mice (135). Using this approach, retinochisin was detected in all cell layers identical to that seen in wild type animals, and normal visual function was restored as observed by the increased b-wave and lasted for at least 1 year. In addition, structural integrity of the retina was improved and photoreceptors were preserved for the duration of the study. The authors explored in a second study the feasibility of subretinal injections of the vector at later time points during the disease (136). They demonstrated that a treatment with the AAV2/5.mopsin.Rs1 vector at later time points (up until 7 months of age) can result in detectable retinoschisin expression, even though morphological or functional rescue remains marginal if treatment is performed later than 2 months of age.

3.5.2 Treatment of Autosomal Dominant Diseases

Autosomal dominant diseases are caused by mutations, which result in a toxic “gain of function” effect of the encoded protein. Mutations of the rhodopsin gene account for the largest proportion of autosomal dominant retinitis pigmentosa (adRP) and affect the folding, stability and intracellular processing of the protein. The presence of only one allele is sufficient for the onset of retinal degeneration. One particular rhodopsin mutation, P23H, is responsible for 12% of all adRP cases in the US (137) and transgenic mice and rats containing this mutation have been developed to study the disease and evaluate potential treatment strategies. Mutations in another gene, the inosine 5′-monophosphate dehydrogenase 1 (IMPDH1) cause the adRP form that is called RP10 (138).

To treat these kinds of disorders, gene augmentation therapy is not suitable, as the mutant allele, mRNA or protein product must be silenced beforehand. To down-regulate the production of toxic proteins in a cell, two strategies have been developed involving either ribozymes or siRNA.

Ribozymes are RNA enzymes capable of cleaving a complementary mRNA sequence and according to their form in the cytoplasm they are called either hammerhead or hairpin ribozymes (139). Naturally occurring ribozymes have been screened to identify molecules, that are capable of cleaving specifically the mutant rhodopsin mRNA and allow the expression of the normal allele. Subretinal injection of a rAAV2/2 containing the allele specific ribozyme (hammerhead or hairpin) driven by an opsin promoter in P23H rats at postnatal day P15 led to preservation of photoreceptors for at least 3 months (140). The ribozymes were able to decrease the mRNA level of the mutant allele by 10–15%. LaVail and colleagues demonstrated that the same treatment strategy slowed photoreceptor degeneration for up to 8 months when animals were treated at either P15 or later (P30 or P45) (141). In addition, compared with control eyes, the treatment resulted in higher ERG amplitudes up to 6 months after injection.

As more than 200 mutations in the rhodopsin gene have been found to cause adRP, allele specific ribozymes would have to be found for each mutation. An alternative approach is to develop allele independent ribozymes, that digest all endogenous rhodopsin mRNA, mutant or wild type. At the same time, a ribozyme-resistant rhodopsin gene would be introduced into photoreceptors by gene transfer.

One such ribozyme, Rz397, was introduced into an rAAV2/2 vector driven by a mouse opsin promoter, and subretinally injected into rhodopsin knockout hemizygous (rho +/−) mice at P6 and P60 (142). The ribozyme was capable of decreasing the amount of rhoposin by 80% in these animals. In a second experiment, the same group developed a ribozyme, Rz525, which is capable of cleaving all dog, mouse, and human, but not rat rhodopsin (143). Subretinal injection of a rAAV2/5 vector containing this ribozyme driven by a mouse opsin promoter into P23H hemizygous rats carrying one rat and one mouse rhodopsin allele resulted in 46% reduction of mouse mRNA but no change in rat mRNA. By diminishing the amount of mouse rhodopsin in these rats, toxic effects were less important and photoreceptor morphology could partially be preserved.

Small interfering RNAs (siRNAs) are double stranded RNA molecules of 20–25 bp length which are capable of cleaving complementary mRNA. These molecules are processed from small hairpin (sh) RNA driven by an RNA polymerase III promoter like U6 or H1, or by a polymerase II promoter like CMV.

Subretinal injection of a rAAV2/5 vector carrying an shRNA specifically targeting the mouse P23H rhodopsin gene under the control of a U6 promoter were injected subretinally into P23H transgenic rats (144). The processed and allele specific siRNA diminished the amount of mouse mutant rhodopsin in these animals but was not sufficient to prevent or block photoreceptor degeneration.

Another study used allele independent siRNA molecules capable of degrading mutant and wild type rhodopsin (145). Subretinal injection of a rAAV2/5 vector carrying a shRNA complementary to mouse rhodopsin driven by a H1 promoter were injected subretinally into heterozygous rhodopsin knockout mice at P16. Endogenous rhodopsin protein could be decrease by 60%, mRNA levels by 30%. Retinal function was diminished due to the reduction of rhodopsin content.

Similarly, the allele independent strategy of nonspecific suppression of all Rhodopsin alleles and replacement with a codon modified allele is the basis of the gene therapy strategy developed by the group of Farrar and colleagues. They developed different strategies to overcome the major hurdles of such an approach, which is the necessity of complete downregulation of endogenous Rho and at the same time a very effective replacement by gene transfer.

To overcome the first hurdle, functional siRNAs were developed, screened for efficiency in vitro and tested in vivo using an AAV2/5 vector (AAV2/5.shBB.EGFP) in mouse model that expresses the human Rhodopsin gene on a mouse Rhodopsin background (NHR+/− Rho −/−) (146). The siRNA expression downregulated the human Rhodopsin mRNA level for up to 90%. In addition, the same vector was shown to be efficient in downregulating the human Rhodopsin allele containing the P347S mutation that was crossbred on a normal mouse line (RHO347+/− Rho +/+) (147). As the siRNA is specific for the human Rhodopsin gene, suppression of the mouse Rhodopsin in this mouse model was not observed and retinal function and morphology remained normal following the administration of the vector.

A more challenging point is the second hurdle, which is the sufficient replacement of the Rhodopsin protein, as it makes up to 90% of the outer segment membrane proteins (148). Furthermore, the replaced Rhodopsin gene has to be unspecific for the siRNA mediated downregulation, which can be achieved by modifying the DNA sequence at crucial points (codon modification). In order to study the effect of a codon modified human Rhodopsin gene, a mouse model was generated that expresses such an allele on a mouse rhodopsin background (Rho-M) (149). It was shown that the codon modified gene was equivalent to the endogenous Rhodposin gene in producing sufficient protein with the correct function in the retina of these animals. In order to test the capacity of an rAAV-mediated gene transfer of the codon modified Rhodopsin gene into the retina of Rho −/− mice, eight different constructs containing the transgene, different promoters and the siRNA cassette were incorporated into the AAV2/5 serotype (150). Subretinal injections, performed as early as p0, resulted in transgene expression of up to 40% of normal levels and the formation of outer segments and significant rod-derived ERG recordings in treated eyes. To summarize the treatment strategies for adRP caused by mutations in the Rhodopsin gene, the combined strategy of downregulation and replacement seems to be a practical and reliable way to circumvent the extensive allelic heterogeneity in the human population.

Downregulating the mutant gene by siRNA was also used as treatment strategy for adRP due to mutations in the IMPDH1 gene (151). AAV-mediated expression of the mutant human or mouse IMPDH1 gene in the retinas of wild type mice was performed and retinal degeneration with complete loss of the ONL was observed in both models 4 weeks after the injection (151). In order to treat the pathologic condition, an AAV2/5 vector was constructed containing the IMPDH1 specific siRNA and was co-injected with an AAV2/5 vector expressing the mutant IMPDH1 gene in to adult wild type mice. After 4 weeks, the morphology of the retina was conserved in co-injected mice compared to mice injected with the mutant IMPDH1 gene alone, indicating that the down regulation of all endogenous genes, including the toxic allele would be beneficial for patients suffering from this form of retinal degeneration (also known as RP10).

3.6 Human Clinical Trials

This chapter focuses on design and strategy of the first gene therapy human clinical trials for inherited retinal degeneration. These trials were proposed after proof-of-concept of gene augmentation therapy had been demonstrated in dog and mouse models of LCA due to RPE65 mutations.

3.6.1 Design

Three separate groups near simultaneously initiated open label, dose escalation, unilateral subretinal gene transfer studies in a small number of individuals with LCA due to RPE65 mutations (Table 4). In all studies, baseline studies of retinal/visual function were carried out and then, after surgery, the studies were repeated as a function of time after injection. All three groups used rAAV2/2 vectors; two of them used the CBA promoter and one used a RPE-cell specific (RPE65) promoter. The doses were 1.5E10 (3 subjects), 4.8E10 (6 subjects) and 1.5E11 vector genomes (vg) (3 subjects), (Children’s Hospital of Philadelphia, CHOP), up to 1E11 vg in three subjects (dose was dependant on volume; University College London, UCL) and 5.9E10 vg in three subjects (University of Florida-University of Pennsylvania, UFl). The volumes of injection ranged from 150 μl to 1.0 ml (UCL). Subjects in the UCL and UFl studies were young adults while those in the CHOP study ranged in age from 8 years old to 44 years old (23, 24, 25).
Table 4

Published human clinical trials for patients with mutations in the RPE65 gene


NCT number



No. of patients



UCL (23)


phase I

AAV2/2.hRPE65p.hRPE65 (hRPE65 promoter)




CHOP (25, 153, 156)


phase I/II

AAV2-hRPE65v2 (CBA promoter)




Ufl (24, 154, 155, 157)


phase I

AAV2-CBSB-hRPE65 (CBA promoter with shortened CMV enhancer)




UCL University College London, CHOP Children’s Hospital of Philadelphia, UFl University of Florida, NCT registry number, CBA chicken beta actin, CMV cytomegalovirus

Because of the many variables that differed between the three studies (regulatory sequences, method of preparation and purification of the vector, dose, volume, disease-causing mutations, age of subject, location of injection, outcome measures, etc.), it is not possible to compare the data directly from one study to another. However, several important conclusions can be drawn: (1) The procedure was safe. There were no harmful inflammatory responses and immune responses were benign. The procedure was well tolerated by the 18 subjects (total number reported in these three studies). There were no severe adverse events and the few adverse events were mild; (2) The majority of the subjects benefited from the procedure. All 12 of the subjects in the CHOP study showed both improvements in retinal and visual function as assessed by both subjective and objective measures. The level of improvement was most striking in the youngest subjects, as might have been expected based on results from animal studies (152). All four of the children (8–11 years old) and one of the adults (26 years old) in the CHOP study gained the ability to ambulate independently using the injected eye (153). All three of the subjects in the UFL study and one of the subjects in the UCL study showed improvements in subjective measures of visual function (23, 24). The subject in the UCL study who showed improvements in microperimetry and dark-adapted perimetry also became able to navigate independently; and (3) The safety profile persisted and the improvements in retinal/visual function that were reported soon after injection were maintained in at least in two of the three trials. Cideciyan reported that the improvements in visual sensitivity were maintained between 3 and 12 months (154). In addition, this group reported that one of the three patients was able to foveate using the injected portion of her retina (155). The excellent safety and efficacy profile of the CHOP trial was also maintained through the 2-years timepoint (153, 156). All three of the subjects followed the longest continued to show significant improvements in visual acuity and pupillary light reflexes, and the ability to ambulate independently was maintained in the one individual in whom that had been documented at the previous timepoints (156).

3.6.2 General Considerations

The success of the LCA-RPE65 clinical trials to date provides a great incentive to evaluate gene augmentation approaches for other retinal degenerative diseases in future clinical trials. The data from the LCA-RPE65 studies will be useful in preparing for these other targets. The safety data to date using rAAV2/2 in a dose range of 1.5E10–1.5E11 vg will be helpful in designing these follow-up studies. However, additional preclinical safety studies will need to be carried out using hybrid rAAVs with capsids of other serotypes. Photoreceptors are the primary disease-causing cells in the majority of hereditary retinal degenerations, and more efficient transduction can be achieved in this cell type using other rAAV vectors than AAV2/2 (see above). The dosing strategy will present more of a challenge for these situations because there will not be efficacy if too low a dose is given but too high of a dose could lead to toxicity.

Preclinical data have demonstrated the stability of AAV-mediated retinal gene transfer and, so far, this has been verified at least through a 1–2 years time period in the human clinical trials (153, 154, 156). At present, it is not known how long rAAV-mediated transgene expression will persist in the human retina. However, there are two facts that lend hope to the idea that it will persist long term: (1) Retinal cells are post-mitotic at birth and so even though the transgene cassette is maintained episomally in retinal cells, it is unlikely to be diluted due to cell division; and (2) Given the long follow-up in dogs (6 and 11 years, Rolling and Bennett, personal communication) and the stable expression of transgenes after subretinal injection in these animals, rAAV-mediated transgene expression should persist in other species. Therefore, as long as the gene transfer slows down the degenerative component of the disease, rAAV-mediated transgene expression is expected to be stable.

A real challenge will be an economic one – to develop and test a gene-based treatment for each one of the hundreds of different genes involved in retinal degenerative disease. Preclinical studies can generally be funded through traditional mechanisms, such as government-issued grants and private foundations. Successful proof-of-concept studies then lead to candidates for a clinical trial. There are very few funding sources, however, that will cover the costs of initiating and completing a clinical trial. Following demonstration of proof-of-concept, additional studies aimed at optimizing a vector (selecting the appropriate regulatory sequence, carrying out dose-ranging studies, completing and submitting regulatory documents, etc.) can cost $250,000. The cost of generating a clinical-grade vector is currently about $500,000. Preclinical safety studies will likely require testing in nonhuman primates, since eyes of those animals are most similar anatomically to those of humans (Primates are the only animals with a macula). Those safety studies can easily cost $250,000. One must be absolutely sure that the rAAV vector is optimal prior to generating the clinical vector, because if there is a change thereafter, the entire process starts anew.

Next, clinical trials themselves are costly. The cost of a phase 1 clinical trial involving 12 subjects seen at one site was about $1 million. If there is a second Center, bills are incurred not only for the clinical trial itself but also to monitor regulatory compliance. Thus, it can easily cost $2 million to carry out a phase 1 clinical trial. Assuming safety and efficacy are identified in the phase 1 study, the subsequent studies (phase 2/3 trials) are even more costly.

For all retinal degenerative diseases, the best chance of success (recovery of vision or of prevention of further deterioration of vision), will be to intervene early in the disease process. Once the photoreceptor cells have died, there is nothing left to treat. In many forms of RP, it is possible that rescue could be achieved in teens or young adults. In choroideremia, it may be desirable to target teens and in Stargardt disease, it may be desirable to target pre-teens. For LCA, the optimal results may be obtained in treating infants as vision deficits are present in that disease early in life. In LCA, not only would it be most desirable to treat the retina before cells are lost to degenerative processes but also because there is a loss in plasticity of the connections between the retina and brain after about age 3. For example, in LCA-CEP290 and LCA-AIPL1, vision is severely abnormal even in infancy and so it would be sensible to treat as early as possible. In contrast, individuals with LCA-RPE65 generally have formed (but poor) vision before age 3 years old and thus the retinal/CNS circuitry is established. Therefore, individuals in their teens, twenties and even as old as 44 years old can benefit from delivery of the wildtype RPE65 cDNA (23, 24, 153).

One question that will need to be addressed, however, is, whether it is safe to re-administer the vector to the contralateral eye at a later timepoint. Although re-administration in animals appears safe, the safety of re-administration in humans is unknown. In young children, a unilateral injection could result in a favoring of one eye versus the other and amblyopia, a condition whereby the brain is incapable of recognizing signal from an eye. If this were to happen because one eye was treated and was seeing better than the other, or if one eye was patched due to a complication from surgery, it would prevent benefit of future surgery on the amblyopic eye.

The ages of inclusion for each clinical trial will be determined through an evaluation of risk:benefit ratios and through consideration of principles of clinical research involving vulnerable (pediatric) subjects. In the USA in the clinical trial run at CHOP, enrollment of pediatric subjects was approved even though there was thought to be slightly greater than minimal risk, classified as code 45 CFR 46.405 in the USA. This code states that research can be carried out which involves greater than minimal risk on vulnerable subjects but there must be a prospect of direct benefit to the individual. In the CHOP study, this code forced the team to use a dose that had been shown to rescue vision in >90% of dogs (instead of a lower, potentially ineffective dose). Finally, the lower age limit may be selected by determining whether a child is able to give assent. It is widely felt that children 8 years old and above are capable of understanding what they are agreeing to as long as the language is appropriate (

3.6.3 Outcome

One of the most important details of clinical trial design is selection of the most appropriate outcome measures. The tests should be sensitive, quantitative, and reproducible. Some retinal function/visual function tests are subjective in nature (i.e., the subject tells the examiner the response and the results can be influenced by the subject’s levels of wakefulness, cooperation, etc.). It is thus desirable to include objective tests as well. That way, one can be sure that the subject is not simply showing a placebo or learning effect or is too tired to comply. It is important that the tests/test preparation do not overburden the subject. This is particularly a concern in children with short attention spans. Finally, it is desirable to carry out sufficient testing and to repeat the testing as warranted in order to be able to obtain sufficient data for robust statistical analyses.

3.7 Comparison of the Results of rAAV-Mediated Gene Therapy in Human Trials and Animal Models

Interestingly, the data obtained in animal models of LCA-RPE65 were largely predictive of the results in the human clinical trials. First, subretinal delivery was safe in both animals and humans, the vector remained predominantly in the eye (i.e., there was minimal “shedding”) and the delivery resulted in stable improvements in vision relating to the area/portion of retina treated. A single unilateral subretinal injection of 1.5E10 (or higher) vg in all of the Briard dogs younger than 2 years old resulted in increased light sensitivity, improvements in the full field ERG and also the ability of the animals to navigate. In dogs, the first suggestion that there was efficacy was an improvement in pupillary light reflexes noted at 2 weeks after injection. PLRs and ERGS were also readily recordable in Rpe65−/− mice treated subretinally up to 4 months of age (“young adult mice”), but were severely reduced in aged mice. An improvement in PLRs was noted soon after injection in human subjects – the earliest timepoint being 8 days (153). A benefit with the humans was that they could also describe their sense if increased brightness in the injected eye at early (and late) timepoints.

A full field ERG has not been recorded to date in any of the 18 individuals age 8 years old or older enrolled to date in any of the three trials. However, multifocal mf ERGs were recorded from two young subjects in the CHOP study (153). Further, all of the youngest subjects (and two older ones – see above) gained the ability to navigate independently in the clinical trials (25, 153, 156). The ability to ambulate reflects a combination of different visual abilities: light sensitivity, visual acuity and visual fields. Visual acuity and visual fields could not be measured in the dogs, but improved in the majority of the humans enrolled in the CHOP trial (153). Like the dogs (17), both rod and cone-mediated responses were recovered in humans tested in the UFl trial (24).

What was not predictive based on the animal experiments was that while there were permanent pigmentary changes in the dog retinas indicating the borders of the original retinal detachments (17, 18), no such changes were detectable in the injected human retinas (25, 153, 156). It may be that injection-induced changes in the underlying reflective tapetum in dogs contribute to the ophthalmoscopically detectable changes in this species. The other finding that was not predictive based on the animal experiments was that there was improvement in retinal/visual function in a human as old as 44 years old. This individual did not recover normal vision, but did show improved PLRs, and improvements in subjective measures of retinal/visual function. Bilateral PLRS were not measured in the older dogs that were evaluated and so a direct comparison across species cannot be made. However, there has been no clear-cut demonstration of improvement in the older (>2 years old) dogs that have been tested.

4 Conclusion

There has been great progress over the past decade in characterizing and generating animal models for retinal degeneration, developing and testing strategies for ameliorating the disease process and moving the promising proof-of-concept data into clinical trials. Because of these strides, there is great promise for future applications of AAV-mediated gene therapy for blindness. There are a number of technical and economic challenges that must be tackled to be able to successfully ameliorate these inherited conditions. In addition, there are still a number of safety concerns, including issues relating to immune response, stability, and long-term effects of high levels of transgene expression. However, meticulous preclinical safety and efficacy studies combined with a cautious, stepwise translational approach based on a careful evaluation of risk: benefit ratios will lead the way to the ultimate goal of being able to use rAAV to ameliorate or possibly even cure forms of inherited blindness.


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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Knut Stieger
    • 1
  • Therese Cronin
    • 2
  • Jean Bennett
    • 3
    Email author
  • Fabienne Rolling
    • 4
  1. 1.Department of OphthalmologyJustus-Liebig-University GiessenGissenGermany
  2. 2.Scheie Eye InstituteUniversity of PennsylvaniaPhiladelphiaUSA
  3. 3.Department of Ophthalmology, F.M. Kirby Center for Molecular Ophthalmology, Scheie Eye InstituteUniversity of PennsylvaniaPhiladelphiaUSA
  4. 4.Laboratoire de Thérapie GéniqueINSERM UMR649, Université de NantesNantesFrance

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