**3. Adeno-associated virus (AAV) biology**

at a distance from the subapical region. However, when expressed in both photoreceptors and Müller glial cells, human CRB2 localization was restricted to the subapical region, which suggested that expression of CRB2 in both cells types might be required for proper protein

**Figure 1.** Model depicting the localization of CRB1 and CRB2 proteins in the human retina at 2 days post-mortem. CRB proteins are present at the subapical region above the adherens junctions between Müller glial cells, between photoreceptor and Müller glial cells and between photoreceptor cells. CRB1 is located in both Müller glial cells and cone and rod photoreceptor cells at the subapical region. CRB2 is located in Müller glial cells at the subapical region, and in

Animal models able to recapitulate features of the *CRB1*-retinopathies are of value to understand the molecular mechanism behind retinopathies and to test new AAV gene therapy vectors. Over the recent years several rodent models were described in the literature. The retinal phenotypes observed in these animals mimic the wide spectrum of clinical features as described in *CRB1*-patients, including early and late onset RP, LCA and telangiectasia [44, 49, 50, 52, 64–67]. The onset and severity of the phenotype observed in these animal models seem closely associated with the total levels of the CRB proteins in the different cell compartments.

**a.** late onset-RP: homozygous knockout *Crb1* [52], hemizygous knockin *Crb1C249W/−* [67] and homozygous naturally occurring mutant *Crb1rd8* [66] mice showed, at foci, loss of integrity of the outer limiting membrane, with protrusions of rows of photoreceptor nuclei into the inner- and outer segments layer and ingression of photoreceptor nuclei into the photoreceptor synaptic layer. Microglial cell infiltration and upregulation of glial fibrillary acidic protein (GFAP) were observed at the foci of photoreceptor dysplasia. Conditional ablation of *Crb2* specifically in Müller glial cells resulted in disruptions at the outer limiting

localization and function [16].

**2.2. Animal models for** *CRB1***-retinopathies**

The available models can be grouped into three major categories:

photoreceptors at vesicles in the inner segments at a distance from the subapical region.

124 In Vivo and Ex Vivo Gene Therapy for Inherited and Non-Inherited Disorders

Adeno-associated virus belongs to the parvovirus family, but is placed in the genus Dependovirus since it is dependent on co-infection with other viruses, mainly adenoviruses, in order to replicate. AAV is a small, non-enveloped single-stranded DNA virus. The genome of the AAV is approximately 4.7 kb and has three open reading frames to express the *rep* (Replication), *cap* (Capsid) and assembly activating protein (*aap*) (Assembly) genes, flanked by two 145 nucleotide-long inverted terminal repeats (ITRs). The ITRs self-assemble into hairpin structures required for genome replication, integration and encapsidation. The *rep* gene encodes four proteins (Rep78, Rep68, Rep52 and Rep40), which are required for viral genome replication and packaging. While *cap* gene transcripts gives rise to the viral capsid proteins, virion protein 1 (VP1), VP2 and VP3, with molecular weights of 87, 72 and 62 kDa, respectively. These capsid proteins assemble into an icosahedral symmetry protein shell of 60 subunits, in a molar ratio of 1:1:10 (VP1:VP2:VP3). The *aap* gene encodes the assembly-activating protein (AAP) that is thought to have a scaffolding function for capsid assembly [68]. Wild-type AAV integrates into the human host genome at a specific site, AAVS1 on chromosome 19.

important parameter to take into consideration in the testing of gene therapy vectors, together with the selection of the AAV capsid and promoter since all these parameters have effects on

AAV-Mediated Gene Therapy for *CRB1*-Hereditary Retinopathies

http://dx.doi.org/10.5772/intechopen.79308

In pre-clinical studies performed in rodents *ab externo* subretinal injections are commonly performed [16, 70]. This method uses a small needle (34 gauge) to penetrate (*ab externo*) the sclera at the limbus and under direct observation the needle can be guided through the retina to create a subretinal space between the retinal pigment epithelium and the outer limiting membrane. Normally, a volume of 1 μL is injected to form an injection fluid bleb that transiently detaches a large portion of neural retina from the RPE in one single injection. Incorrect surgery might cause cataract due to damage to the lens. It is also common to have a large

Subretinal injections in human can be performed using the "single-step" or the "two-step" approach [71]. With the "single-step" approach the fluid, containing the gene therapy vector, is directly delivered into the subretinal space without previous retinal detachment [22].

The "two-step" approach consists of first the generation of a bleb in the subretinal space by injection of a balanced salt solution (BSS), followed by injection of the therapeutic agent using a controlled flow rate [4, 5, 72]. The second approach offers several advantages like the possibility to better assess the direction of bleb spread as well as to minimize vector loss by misguided injection [71]. The subretinal surgery and injection is a specialized technique and can in principle be executed by surgeons operating an ophthalmic surgery robot to obtain most reproducible results. According to information collected from the different clinical trials registered in the *Clinicaltrials.gov* database, a volume ranging from 60 to 1000 μL can be

Subretinal injections seem the logical choice when RPE or photoreceptors are the target cells, since these cells will be in direct contact with the fluid containing the AAV particles. However, degenerating retinas at an advanced stage are often quite thin, with disruptions at the outer limiting membrane, loss of inner/outer segments and/or photoreceptor cells, neovascularization and infiltration of microglial cells. All these features might lead to a reduction in the potential subretinal space between the neural retina and the RPE, or to leaking of the AAV vector to the choroid vasculature system and influence the AAV tropism. The retinal detachment caused during the subretinal injection might potentially also either aggravate or allevi-

Intravitreal injection implies direct delivery into the space in the back of the eye called the vitreous cavity, which is filled with a jelly-like fluid called the vitreous humor gel. Intravitreal injections are generally limited to volumes of up to 2 μL in mice [15, 16, 73], while in rats the

volume of backflow of injected AAV particles when the injection needle is retracted.

the tropism of the vectors.

**3.2. Route of delivery**

*3.2.1. Subretinal injection*

injected via this route (**Table 1**).

ate the processes of retinal degeneration.

*3.2.2. Intravitreal injection*

In gene therapy a recombinant AAV (rAAV) and not the wild-type AAV are used. In rAAV the viral genome required for viral replication, the rep and *cap* genes, and the element required for site-specific integration are deleted. A sequence containing a promoter, a 5′-untranslated region, the cDNA of a transgene of interest, and a 3′-untranslated region containing a polyadenylation site are then inserted in between the AAV vector containing the two ITRs. To produce AAV particles from the AAV gene therapy plasmid in a human cell line, the *rep* and *cap* genes are supplied in trans on a helper plasmid along with helper genes from adenovirus (*E4*, *E2a* and *VA*) necessary for replication.

The major advantages of the use of rAAVs are the safety profile, low immunogenicity, lack of toxicity and the property that the rAAV genomes do not integrate into the host genome. The rAAV capsid enters the cells by receptor-mediated endocytosis, the rAAV genomes are processed into nuclear episomal structures and are maintained extrachromosomally. Dependent on the gene therapy vector used, and the life span of the targeted cell, the rAAV genomes can express a transgene for more than 10 years. AAV vectors also have the ability to transduce non-dividing cells, including non-dividing retinal neurons like photoreceptors. One major disadvantage of the rAAV technology is the size limitation of the total DNA that can be efficiently packaged in the AAV vector (4.7–4.9 kb) which makes it difficult to design AAV-mediated gene therapy for larger genes (≥ 4 kb). The development of dual and triple AAV vectors with a maximum transfer capacity of around 9 and 14 kb, respectively, might in the future overcome in part this limitation [69].

The generation of a gene therapy vector able to deliver CRB1 is particularly challenging due to its large size of cDNA (4.2 kb). To assemble the gene therapy vector, the 4.2 kb *CRB1 cDNA* and the two ITR sequences (0.29 kb) need to be added which make up to 4.49 kb. Therefore, only 0.2–0.41 kb space is left for the promoter and polyadenylation sequences. Although challenging it was shown that it is possible to efficiently package human *CRB1 cDNA* in AAV vectors and to express CRB1 protein *in vivo* [15, 16]. Another strategy to overcome the size limitation is to use the 3.85 kb *CRB2 cDNA* as replacement [16].

#### **3.1. Gene delivery in the retina using AAVs**

The eye offers a set of unique features for the application of gene therapy vectors. The eye is a small, compartmentalized, immunoprivileged, paired organ and easily accessible using minimally invasive techniques. There are also high resolution functional and structural diagnostics, such as, optical coherence tomography, scanning laser ophthalmoscopy and electroretinography, as well as psychophysical tests such as microperimetry, kinetic perimetry, visual acuity testing, and multi-luminance mobility test (MLMT) in the ophthalmology field that allow to examine the eye/retina structure and to test as well retinal function and vision. Gene therapy vectors for retinal disease can be delivered mainly by two routes: subretinally into the "subretinal space" between the neural retina and the RPE, or intravitreally, into the vitreous body, both approaches are described below. The administration route is an important parameter to take into consideration in the testing of gene therapy vectors, together with the selection of the AAV capsid and promoter since all these parameters have effects on the tropism of the vectors.

#### **3.2. Route of delivery**

a molar ratio of 1:1:10 (VP1:VP2:VP3). The *aap* gene encodes the assembly-activating protein (AAP) that is thought to have a scaffolding function for capsid assembly [68]. Wild-type AAV

In gene therapy a recombinant AAV (rAAV) and not the wild-type AAV are used. In rAAV the viral genome required for viral replication, the rep and *cap* genes, and the element required for site-specific integration are deleted. A sequence containing a promoter, a 5′-untranslated region, the cDNA of a transgene of interest, and a 3′-untranslated region containing a polyadenylation site are then inserted in between the AAV vector containing the two ITRs. To produce AAV particles from the AAV gene therapy plasmid in a human cell line, the *rep* and *cap* genes are supplied in trans on a helper plasmid along with helper genes from adenovirus

The major advantages of the use of rAAVs are the safety profile, low immunogenicity, lack of toxicity and the property that the rAAV genomes do not integrate into the host genome. The rAAV capsid enters the cells by receptor-mediated endocytosis, the rAAV genomes are processed into nuclear episomal structures and are maintained extrachromosomally. Dependent on the gene therapy vector used, and the life span of the targeted cell, the rAAV genomes can express a transgene for more than 10 years. AAV vectors also have the ability to transduce non-dividing cells, including non-dividing retinal neurons like photoreceptors. One major disadvantage of the rAAV technology is the size limitation of the total DNA that can be efficiently packaged in the AAV vector (4.7–4.9 kb) which makes it difficult to design AAV-mediated gene therapy for larger genes (≥ 4 kb). The development of dual and triple AAV vectors with a maximum transfer capacity of around 9 and 14 kb, respectively, might in

The generation of a gene therapy vector able to deliver CRB1 is particularly challenging due to its large size of cDNA (4.2 kb). To assemble the gene therapy vector, the 4.2 kb *CRB1 cDNA* and the two ITR sequences (0.29 kb) need to be added which make up to 4.49 kb. Therefore, only 0.2–0.41 kb space is left for the promoter and polyadenylation sequences. Although challenging it was shown that it is possible to efficiently package human *CRB1 cDNA* in AAV vectors and to express CRB1 protein *in vivo* [15, 16]. Another strategy to overcome the size

The eye offers a set of unique features for the application of gene therapy vectors. The eye is a small, compartmentalized, immunoprivileged, paired organ and easily accessible using minimally invasive techniques. There are also high resolution functional and structural diagnostics, such as, optical coherence tomography, scanning laser ophthalmoscopy and electroretinography, as well as psychophysical tests such as microperimetry, kinetic perimetry, visual acuity testing, and multi-luminance mobility test (MLMT) in the ophthalmology field that allow to examine the eye/retina structure and to test as well retinal function and vision. Gene therapy vectors for retinal disease can be delivered mainly by two routes: subretinally into the "subretinal space" between the neural retina and the RPE, or intravitreally, into the vitreous body, both approaches are described below. The administration route is an

integrates into the human host genome at a specific site, AAVS1 on chromosome 19.

(*E4*, *E2a* and *VA*) necessary for replication.

126 In Vivo and Ex Vivo Gene Therapy for Inherited and Non-Inherited Disorders

the future overcome in part this limitation [69].

**3.1. Gene delivery in the retina using AAVs**

limitation is to use the 3.85 kb *CRB2 cDNA* as replacement [16].

#### *3.2.1. Subretinal injection*

In pre-clinical studies performed in rodents *ab externo* subretinal injections are commonly performed [16, 70]. This method uses a small needle (34 gauge) to penetrate (*ab externo*) the sclera at the limbus and under direct observation the needle can be guided through the retina to create a subretinal space between the retinal pigment epithelium and the outer limiting membrane. Normally, a volume of 1 μL is injected to form an injection fluid bleb that transiently detaches a large portion of neural retina from the RPE in one single injection. Incorrect surgery might cause cataract due to damage to the lens. It is also common to have a large volume of backflow of injected AAV particles when the injection needle is retracted.

Subretinal injections in human can be performed using the "single-step" or the "two-step" approach [71]. With the "single-step" approach the fluid, containing the gene therapy vector, is directly delivered into the subretinal space without previous retinal detachment [22].

The "two-step" approach consists of first the generation of a bleb in the subretinal space by injection of a balanced salt solution (BSS), followed by injection of the therapeutic agent using a controlled flow rate [4, 5, 72]. The second approach offers several advantages like the possibility to better assess the direction of bleb spread as well as to minimize vector loss by misguided injection [71]. The subretinal surgery and injection is a specialized technique and can in principle be executed by surgeons operating an ophthalmic surgery robot to obtain most reproducible results. According to information collected from the different clinical trials registered in the *Clinicaltrials.gov* database, a volume ranging from 60 to 1000 μL can be injected via this route (**Table 1**).

Subretinal injections seem the logical choice when RPE or photoreceptors are the target cells, since these cells will be in direct contact with the fluid containing the AAV particles. However, degenerating retinas at an advanced stage are often quite thin, with disruptions at the outer limiting membrane, loss of inner/outer segments and/or photoreceptor cells, neovascularization and infiltration of microglial cells. All these features might lead to a reduction in the potential subretinal space between the neural retina and the RPE, or to leaking of the AAV vector to the choroid vasculature system and influence the AAV tropism. The retinal detachment caused during the subretinal injection might potentially also either aggravate or alleviate the processes of retinal degeneration.

#### *3.2.2. Intravitreal injection*

Intravitreal injection implies direct delivery into the space in the back of the eye called the vitreous cavity, which is filled with a jelly-like fluid called the vitreous humor gel. Intravitreal injections are generally limited to volumes of up to 2 μL in mice [15, 16, 73], while in rats the volumes are limited to 3–5 μL [74]. The main surgical complications observed are cataract formation due to lens-induced damage and retinal perforation [75].

cultures. This study suggested that serotypes AAV4, AAV5 and AAV6 were particularly efficient at transducing photoreceptor cells, whereas serotype AAV8 displayed consistently low transduction of these cells [79, 81]. Actually several AAV serotypes and ubiquitous promoters or cell specific promoters are being used in clinical trials (**Table 1**), the results from these studies will provide us with important clues about the best promoters and capsids to use in

AAV-Mediated Gene Therapy for *CRB1*-Hereditary Retinopathies

http://dx.doi.org/10.5772/intechopen.79308

In order to deliver *CRB1* or *CRB2* into rod and cone photoreceptor and Müller glial cells in the human retina an AAV capsid able to infect all the three cell types needs to be used in combination with a promoter active in the same cells. Studies performed in mice suggested that a combination of AAV9 and a CMV promoter might be a possibility but further studies are required to test its suitability for human retinal cells [16]. Subretinal injection of expression vectors packaged into serotypes AAV5 or AAV9 infect photoreceptors *in vivo* in macaques [82, 83]. Tropism studies in human retinal explants reported that AAV5 would be more efficacious than AAV9 [84]. Another strategy would be the use of one vector to deliver *CRB1* or *CRB2* specifically in Müller glial cells and a second vector to deliver specifically in photoreceptors. Besides regulatory and financial issues, the main technical issues here resides with the lack of a short promoter (≤ 300 bp) specific for Müller cells, and the lack of an AAV serotype that in human

retina efficiently infects Müller glial cells upon intravitreal or subretinal injection.

trial in patients with RP due to mutations in *CRB1*.

**Acknowledgements**

LUMC.

In recent years the scientific progress in the field of gene therapy for inherited retinal dystrophies culminated in the first ever approved AAV gene therapy medicine to treat LCA patients carrying mutations in the *RPE65* gene. The number of engineered AAV capsid variants and new promoters to drive expression in the different retinal cell types is raising at great speed allowing the design of more specific and more efficient viral vectors. Likewise, the number of clinical trials using AAV gene therapy is increasing at a similar rhythm, the data collected from these studies will be very useful for the development of similar therapies. Pre-clinical studies performed in mice demonstrated that AAV-mediated *CRB2* gene augmentation therapy might be a promising medicine to prevent progression of retinitis pigmentosa in patients with mutations in the *CRB1* gene. In mice at mid-stage retinal disease *CRB2* gene augmentation therapy successfully improved retinal morphology with preservation of photoreceptor cells and retinal function, therefore providing good perspectives for the forthcoming clinical

This work was supported by ZonMw project nr 43200004; FFB project nr TA-GT-0715-0665-

the human retina.

**4. Conclusion**

In humans intravitreal injections are generally performed under local anesthesia [71], by inserting a 30 gauge needle through the sclera at the pars plana region, 3.5–4 mm posterior to the limbus between vertical and horizontal muscles with limited reflux [71, 73]. In clinical trials volumes between 90 and 200 μL have been injected via this route (**Table 1**).

Intravitreal administration of AAV gene therapy might look tempting since it is an easier procedure with less potential surgical complications compared to the subretinal injection, especially when treating thinned degenerative retinas. However, administration of AAV intravitreally has its own caveats namely the difficulty of AAV capsids to cross the thick inner limiting membrane in the human retina and the current lack of AAV serotypes capable of transducing efficiently the human photoreceptors or RPE cells. Another obstacle is the potential AAV transduction and subsequent expression in other eye tissues, as for example, the ciliary body especially when using a ubiquitous promoter.

Pre-clinical studies in mice and rats showed that Müller glial cells can efficiently be infected after intravitreal administration of AAV2/6 or AAV2/shH10Y445F [15, 76], therefore these AAV capsids might be used to deliver CRB1 or CRB2 into Müller cells. AAV serotype shH10Y445F is however known to transduce efficiently the ciliary body epithelium when applied intravitreally [16].

#### **3.3. AAV capsids and cell type specific promoters**

The existence of 11 natural AAV serotypes and derivatives that differ in their tropism, and the different types of cells they infect, makes AAV a very useful system to infect the various cell types of the retina. The cell specificity of the AAV vector can be further increased by using cell type specific promoters, for example RPE65 or VMD2 to drive expression in retinal pigment epithelium. Or by using e.g. the rhodopsin (RH), G protein-coupled receptor kinase 1 (GRK1), 1.7-kb L-opsin promoter (PR1.7) or cone arrestin (hCAR) promoter to drive expression in rod and/or cone photoreceptors. Or using e.g. the RLBP1, GFAP or NR2E1 promoter to drive expression in Müller cells [17, 23, 24, 28].

Several pre-clinical studies showed the tropism and/or potency of the different capsids and promoter (cell specific or ubiquitous) in infecting retinal cell types such as RPE, photoreceptors and Müller glial cells. However, AAV tropism might differ *in vivo* between rodent species, dogs, non-human primates and human. AAV tropism is dependent of the route of administration, the stage of retinal development and severity of retinal dystrophy. Therefore, is quite difficult to extrapolate the data from pre-clinical studies performed in rodents directly to the human *in vivo* setting. To obtain evidence-based data for clinical gene therapy studies, researchers optimize culture protocols for human retinal organotypic cultures [77–79] or human iPSC-derived retinas to study the AAV tropism [80]. Recently, the capacity of different AAV serotypes to infect and express in human retinal cells was studied in organotypic cultures. This study suggested that serotypes AAV4, AAV5 and AAV6 were particularly efficient at transducing photoreceptor cells, whereas serotype AAV8 displayed consistently low transduction of these cells [79, 81]. Actually several AAV serotypes and ubiquitous promoters or cell specific promoters are being used in clinical trials (**Table 1**), the results from these studies will provide us with important clues about the best promoters and capsids to use in the human retina.

In order to deliver *CRB1* or *CRB2* into rod and cone photoreceptor and Müller glial cells in the human retina an AAV capsid able to infect all the three cell types needs to be used in combination with a promoter active in the same cells. Studies performed in mice suggested that a combination of AAV9 and a CMV promoter might be a possibility but further studies are required to test its suitability for human retinal cells [16]. Subretinal injection of expression vectors packaged into serotypes AAV5 or AAV9 infect photoreceptors *in vivo* in macaques [82, 83]. Tropism studies in human retinal explants reported that AAV5 would be more efficacious than AAV9 [84]. Another strategy would be the use of one vector to deliver *CRB1* or *CRB2* specifically in Müller glial cells and a second vector to deliver specifically in photoreceptors. Besides regulatory and financial issues, the main technical issues here resides with the lack of a short promoter (≤ 300 bp) specific for Müller cells, and the lack of an AAV serotype that in human retina efficiently infects Müller glial cells upon intravitreal or subretinal injection.
