**2.** *CRB1***-Hereditary retinopathies**

More than 240 different mutations in the *CRB1* gene have been described so far (http:// www.LOVD.nl/CRB1). These gene variations are associated with a wide variety of retinal dystrophies, including autosomal recessive retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), cone-rod dystrophy, isolated macular dystrophy and foveal retinoschisis [37]. Furthermore, mutations in *CRB1* are responsible for 7–17% of all the LCA cases and for approximately 3–9% of all cases of RP [38, 39]. Retinitis pigmentosa type 12 (RP12) due to mutations in the *CRB1* gene was initially characterized by RP with preservation of paraarteriolar retinal pigment epithelium (PPRPE), progressive visual field loss starting from the first decades of life, and early macular involvement. Later on it became clear that RP12 commonly presents early-onset retinitis pigmentosa, hyperopia and optic disc drusen, with or without PPRPE [37, 40, 41]. Leber congenital amaurosis type 8, due to mutation in the *CRB1* gene (LCA8), is a severe form of retinal dystrophy characterized by roving eye movements or nystagmus, nonrecordable or severely reduced cone and rod electroretinography amplitudes and severe loss of vision within the first years of life. Retinas of LCA8 patients with *CRB1* mutations are about 1.5 times thicker than normal retinas, while retinas of patients with LCA due to mutations in other genes such as *RPE65* or *GUCY2D* are thinner [42]. In addition, LCA8 retinas showed abnormal retinal architecture suggesting that loss of CRB1 function might interrupt the naturally occurring process of proliferation, apoptosis and cell migration during retinal development [42–44].

No treatment is yet available for *CRB1*-associated retinal dystrophies. We achieved proofof-concept for retinal *CRB1* gene therapy, using an AAV9-CMV-*hCRB2* vector in two mouse models. A first model lacked CRB1 and had reduced levels of CRB2 in Müller glial cells and photoreceptors, and a second model lacked CRB2 from Müller glial cells and photoreceptors [16]. These two pre-clinical studies opened the perspective for therapeutic trials for human *CRB1*-associated dystrophies.

Intriguingly, there is no clear genotype–phenotype correlation for *CRB1* mutations [45]. This fact associated with the large spectrum of retinal dystrophies observed in patients with mutations in the *CRB1* gene [37], reinforced the need to study in detail the clinical features and natural disease progression of *CRB1*-associated retinal dystrophies before moving towards a clinical trial. This knowledge is required to establish patient eligibility criteria and clinical outcomes for the forthcoming clinical trial.

#### **2.1. The** *CRB1***-complex in the retina**

In the developing mouse retina, the retinal neuroepithelium is composed of multipotent retinal progenitor cells that differentiate in a time-dependent manner, giving rise to six major types of neuronal and one type of glial cells. The first cell type to be generated from the progenitors are the ganglion cells, followed in overlapping sequential phases by horizontal cells, cone photoreceptors, amacrine cells, rod photoreceptors, bipolar cells and the Müller glial cells. The seven retinal cell types organize or "laminate" in three orderly distinct nuclear layers divided by two plexiform layers [46]. The CRB complex plays a crucial role during retinogenesis by the establishment of polarity, adhesion, retinal lamination and restricting proliferation and apoptosis of progenitors and the number of late born cells such as rod photoreceptors, bipolar cells, late-born amacrine cells and Müller glial cells [47–52].

3.85 kb in size and gave more flexibility to design the AAV gene therapy vector in terms of promoter sequence size, polyadenylation sequence and other optimized sequences that

More than 240 different mutations in the *CRB1* gene have been described so far (http:// www.LOVD.nl/CRB1). These gene variations are associated with a wide variety of retinal dystrophies, including autosomal recessive retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), cone-rod dystrophy, isolated macular dystrophy and foveal retinoschisis [37]. Furthermore, mutations in *CRB1* are responsible for 7–17% of all the LCA cases and for approximately 3–9% of all cases of RP [38, 39]. Retinitis pigmentosa type 12 (RP12) due to mutations in the *CRB1* gene was initially characterized by RP with preservation of paraarteriolar retinal pigment epithelium (PPRPE), progressive visual field loss starting from the first decades of life, and early macular involvement. Later on it became clear that RP12 commonly presents early-onset retinitis pigmentosa, hyperopia and optic disc drusen, with or without PPRPE [37, 40, 41]. Leber congenital amaurosis type 8, due to mutation in the *CRB1* gene (LCA8), is a severe form of retinal dystrophy characterized by roving eye movements or nystagmus, nonrecordable or severely reduced cone and rod electroretinography amplitudes and severe loss of vision within the first years of life. Retinas of LCA8 patients with *CRB1* mutations are about 1.5 times thicker than normal retinas, while retinas of patients with LCA due to mutations in other genes such as *RPE65* or *GUCY2D* are thinner [42]. In addition, LCA8 retinas showed abnormal retinal architecture suggesting that loss of CRB1 function might interrupt the naturally occurring process of proliferation, apoptosis and cell migration during

No treatment is yet available for *CRB1*-associated retinal dystrophies. We achieved proofof-concept for retinal *CRB1* gene therapy, using an AAV9-CMV-*hCRB2* vector in two mouse models. A first model lacked CRB1 and had reduced levels of CRB2 in Müller glial cells and photoreceptors, and a second model lacked CRB2 from Müller glial cells and photoreceptors [16]. These two pre-clinical studies opened the perspective for therapeutic trials for human

Intriguingly, there is no clear genotype–phenotype correlation for *CRB1* mutations [45]. This fact associated with the large spectrum of retinal dystrophies observed in patients with mutations in the *CRB1* gene [37], reinforced the need to study in detail the clinical features and natural disease progression of *CRB1*-associated retinal dystrophies before moving towards a clinical trial. This knowledge is required to establish patient eligibility criteria and clinical

In the developing mouse retina, the retinal neuroepithelium is composed of multipotent retinal progenitor cells that differentiate in a time-dependent manner, giving rise to six major types of neuronal and one type of glial cells. The first cell type to be generated from the

stabilized the transcript [16].

retinal development [42–44].

*CRB1*-associated dystrophies.

outcomes for the forthcoming clinical trial.

**2.1. The** *CRB1***-complex in the retina**

**2.** *CRB1***-Hereditary retinopathies**

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

The CRB family in mammals consists of three members CRB1, CRB2 and CRB3. Both the CRB1 and CRB2 have a large extracellular domain with epidermal growth factor-like and laminin-A globular domains, a single transmembrane domain and a short intracellular C-terminal domain. The C-terminal domain of 37 amino acids has a single FERM-protein-binding motif juxtaposed to the transmembrane domain and a single C-terminal PDZ protein-binding motif [53–55]. While CRB3, the third family member, contains the transmembrane and C-terminal domain but is very short in length since it lacks the large extracellular domain. The C-terminal PDZ motifs of CRB proteins bind to the PDZ domain of PALS1 (also called MPP5). PALS1 binds via its N-terminal L27 domain to the L27 domain of the multiple PDZ proteins PATJ and MUPP1 [56]. The multi-adapter protein PALS1 recruits MPP3 and MPP4 to the subapical protein complex at the so called subapical region adjacent to adherens junctions at the outer limiting membrane [57, 58]. Loss of the CRB1, CRB2, PALS1, or MPP3 but not MPP4 resulted in disruption of adhesion between photoreceptors and Müller glial cells. In summary, the core of the retinal CRB-complex is composed of CRB1, CRB2, PALS1, PATJ, MUPP1, and MPP3 [52, 59].

In the embryonic mouse retina, CRB1, CRB2, PALS1, PATJ and MUPP1 are expressed at the subapical region adjacent to the adherens junctions of the retinal progenitor cells [49]. In the adult mouse retina, CRB2 is present at the subapical region in photoreceptors and Müller glial cells. The mouse *Crb1* gene transcript is expressed in photoreceptors and Müller glial cells but expression of the CRB1 protein is limited to the subapical region of Müller glial cells [60, 61]. CRB3 has a broader expression pattern being located at the subapical region in both photoreceptors and Müller glial cells [52, 60], at the photoreceptor inner segments and photoreceptor synaptic terminals and at sub-populations of amacrine and bipolar cells in the inner plexiform layer [62]. The expression patterns of CRB1 and CRB2 observed in the mouse retina do in part match with the ones observed in the human retina. In the first trimester human fetal retina, CRB2 but not CRB1 is expressed at the subapical region. While in the second trimester CRB1, CRB2 and PALS1 localize at the subapical region. A similar expression pattern is observed in early (differentiation day 28) versus late (differentiation day 160) human induced pluripotent stem cells (iPSCs)-derived retinas [63]. Immunoelectron microscopic protein localization studies performed on adult human retinas, collected at two to 3 days post-mortem, showed CRB1 and CRB2 localization at the subapical region of Müller glial cells as found in the mouse retina. Human CRB1 localized also at the subapical region in photoreceptor cells, whereas human CRB2 localized at vesicles in the photoreceptor inner segments some distance away from the subapical region [52, 60] (**Figure 1**).

Interestingly, the overexpression of human CRB2 protein specifically in mouse photoreceptors that lacked endogenous mouse CRB2 in photoreceptors and Müller glial cells, caused aberrant localization of human CRB2 predominantly at vesicles in photoreceptor inner segments

membrane and ectopic photoreceptor nuclei in the inner- and outer segment layer [50]. The morphological abnormalities observed in all these models do not lead to a decrease in

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

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

**b.** early onset-RP: ablation of *Crb2* from retinal progenitor cells, and consequent loss of CRB2 in cone and rod photoreceptors and Müller glial cells [47, 49] or ablation of *Crb2* specifically in immature photoreceptors [50] leads to disruptions at the outer limiting membrane during late-stage embryonic development resulting in abnormalities in retinal lamination, severe retinal degeneration and early loss of retinal function. More recently, a naturally occurring substrain of Brown Norway rats (BN-J) was described as a model for retinal telangiectasia due to homozygous variations in the *Crb1* gene. Interestingly the retinal phenotype observed in this *Crb1* rat strain differs from the phenotype observed in the *Crb1* knockout mice. The *Crb1* rat displays retinal dysplasia at early postnatal days, leading to early-onset disruption of photoreceptor synapses and subsequent loss of retinal function at 1 month of age and near to complete photoreceptor cell death at 6 months

**c.** LCA: mouse retinas with loss of CRB1 and CRB2 proteins from retinal progenitor cells showed lack of a proper retinal lamination with loss of a photoreceptor synaptic layer, intermingling of photoreceptor nuclei with the nuclei of inner nuclear layer cells, and

The lack of a genotype–phenotype correlation in humans might correlate with the different retinal phenotypes as observed in mice with lowered levels of CRB1 and/or CRB2 in retinal progenitors, photoreceptors and Müller glial cells. Cumulative data suggest that not only the levels of CRB1 are important for the pathogenesis observed in humans but also the total levels of CRB1 and CRB2 proteins. Or that the levels of functional CRB2 variants in retinal progenitors, photoreceptors or Müller glial cells might play a role in determining the severity of the

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

electrical retinal function.

of age [64].

early loss of retinal function [44].

retinal dystrophy caused by mutations in the *CRB1* gene.

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

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

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 localization and function [16].

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

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. The available models can be grouped into three major categories:

**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 membrane and ectopic photoreceptor nuclei in the inner- and outer segment layer [50]. The morphological abnormalities observed in all these models do not lead to a decrease in electrical retinal function.


The lack of a genotype–phenotype correlation in humans might correlate with the different retinal phenotypes as observed in mice with lowered levels of CRB1 and/or CRB2 in retinal progenitors, photoreceptors and Müller glial cells. Cumulative data suggest that not only the levels of CRB1 are important for the pathogenesis observed in humans but also the total levels of CRB1 and CRB2 proteins. Or that the levels of functional CRB2 variants in retinal progenitors, photoreceptors or Müller glial cells might play a role in determining the severity of the retinal dystrophy caused by mutations in the *CRB1* gene.
