**1. Introduction**

A new generation of medicines emerged in 2012 with the first ever European market authorization of Glybera (alipogene tiparvovec), an adeno-associated virus (AAV) gene therapy medicine for the treatment of a rare inherited autosomal recessive lipid disorder, lipoprotein lipase deficiency. Five years later the company did not seek for renewal of the marketing authorization for Glybera due to patient's lack of demand [1]. Despite the marketing failure

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


of Glybera, the use of AAV gene therapy in the eye is very attractive since the marketing prospects look better for the small amounts of AAV medicine to be transferred into the retinal tissue or retinal pigment epithelium. The eye is well accessible for surgery and allows direct observation, *in vivo*, of the retinal tissue in microscopic detail. Moreover, the eye is considered an immune-privileged tissue. Therefore, the risks of an immune response against the virus and/or the transgene itself are reduced. The local application in the "compartmentalized" eye of low amounts of AAV drug will minimize side effects expected if systemically applied at high doses [2]. But most importantly, potential drug efficacy for retinal orphan diseases can be efficiently proven thanks to a plethora of non-invasive retinal investigation techniques.

**Table 1.** Summary of the clinical trials for retinopathies using AAV as delivery system registered on ClinicalTrials.gov

CBA: chicken β-actin promoter (CBA); CBSB: Hybrid modified short cytomegalovirus (CMV) enhancer and chicken β-actin promoter (CBA); GRK1: G protein-coupled receptor kinase; hCAR: human cone arrestin; NR: not reported; PR1.7: 1.7-kb L-opsin promoter; REF: References; RK: Rhodopsin kinase; scRS/IRBP: Retinoschisin/interphotoreceptor retinoid

**route**

AAV2 CMV *sFLT01* Intravitreal 100 μL 2 × 108

*anti-VEGF*

**Volume injected**

Subretinal NR 3 × 109

**Dosage ClinicalTrials.**

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

vg

 vg 1 × 1010 vg 6 × 1010 vg

2 × 109 vg

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

6 × 10<sup>9</sup> vg 2 × 1010 vg **gov Identifier**

NCT01024998 [36]

NCT03066258 NR

**Ref.**

**AAV serotype Promoter Gene Delivery** 

AAV8 NR *soluble* 

binding protein; VMD2: Vitelliform macular dystrophy-2.

**Targeted disease**

database.

Age-Related Macular Degeneration (AMD)

At the end of 2017, Luxturna (voretigene neparvovec-rzyl) became the first FDA-approved AAV gene therapy medicine for patients with hereditary retinal disease caused by biallelic *RPE65* gene mutations [3, 4]. The market approvals of the first gene therapy medicines in Europa and in the USA paved the road to similar programs, reflected on the large number of clinical trials registered on the ClinicalTrials.gov website using AAVs as a delivery strategy to treat hereditary retinal diseases such as choroideremia (*CHM* or *REP-1*) [5], achromatopsia (*CNGA3*) [6], wet age-related macular degeneration (AMD) (*VEGFR1*/*FLT* and a gene encoding soluble anti-VEGF protein) [7], Leber hereditary optic neuropathy (LHON) (*ND4*) [8], autosomal recessive retinitis pigmentosa (arRP) (*MERTK*) [9], X-linked RP (*RPGR*) [10], RP (*PDE6B*) [11] and (*RLBP1*) [12] and X-linked Retinoschisis (*RS1*) [13, 14] (**Table 1**). Developing an AAV gene therapy to treat patients with mutations in the Crumbs homolog-1 (*CRB1*) gene was particularly challenging due to its large cDNA (4.2 kb) which approached the packaging limit of the AAV genome (~4.7–4.9 kb). Thus, to build an AAV vector that allowed efficient packaging of the human *CRB1* cDNA, the use of a short promoter (<350 bp) and a short synthetic polyadenylation sequence was required to efficiently express the CRB1 protein *in vivo*. Codon optimization of the *CRB1* cDNA was used to achieve sufficient levels of expression [15]. A second strategy that implied the replacement of CRB1 by its structural and functional family member CRB2 was used to overcome the size limitation and potential toxicity due to expression of CRB1. *CRB2* cDNA was only


**Targeted disease**

X-linked retinoschisis

Leber hereditary optic neuropathy (LHON)

**AAV serotype Promoter Gene Delivery** 

LCA AAV4 RPE65 *hRPE65* Subretinal 400 or

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

Choroideremia AAV2 CBA *REP1* Subretinal 60–100

CPK850

Achromatopsia AAV8 NRx *hCNGA3* Subretinal NR 1 × 1010 vg

RP (*RLBP1*) AAV8 sRLBP1

AAV8 scRS/

scAAV2 (Y444,500,730F) IRBP

CMV/ CBA

**route**

AAV2 CBA *hRPE65* Subretinal 450 μL 1.8 × 1011 vg

AAV2 CBA *hRPE65v2* Subretinal 150 μL 1.5 × 1010 vg

RP (*PDE6B*) AAV5 RK *hPDE6B* Subretinal NR NR NCT03328130 [24] RP (*MERTK*) AAV2 VMD2 *hMERTK* Subretinal NR NR NCT01482195 [25] X-linked RP NR NR *RPGR* Subretinal NR NR NCT03116113 NR

AAV2 hRPE65 *hRPE65* Subretinal up to

AAV2 CBSB *hRPE65* Subretinal 150–

**Volume injected**

800 μL

1 mL

300 μL

μL

*hRLBP1* Subretinal NR NR NCT03374657 [23]

AAV2 NR *hCHM* Subretinal NR NR NCT02341807 NR

AAV2tYF GRK1 *RPGR* Subretinal NR NR NCT03316560 [26,

AAV8 hCAR *CNGB3* Subretinal NR NR NCT03001310 NR AAV2tYF PR1.7 *CNGA3* Subretinal NR NR NCT02935517 [28] AAV2tYF PR1.7 *CNGB3* Subretinal NR NR NCT02599922 [29]

AAV2tYF CB *hRS1* Intravitreal NR NR NCT02416622 [32]

AAV2 CMV *ND4* Intravitreal 90 μL 3 × 1010 vg

*P1ND4v2* Intravitreal 200 μL 5.00 × 10<sup>9</sup>

**Dosage ClinicalTrials.**

1.22 × 1010 vg

4.8 × 1010 vg

6 × 1011 vg

8.94 × 109 3.58 × 10<sup>10</sup> vg

4.8 × 1010 vg 1.5 × 1011 vg

5 × 1010 vg 1 × 1011 vg

*shRS* Intravitreal NR NR NCT02317887 [30,

vg 2.46 × 10<sup>10</sup> vg

1.0 × 1011 vg

9 × x1010 vg 1.8 × 1011 vg

1010–1011 vg NCT01461213

up to 3 × 1012 vg **gov Identifier**

NCT01496040 [17]

NCT00749957 [18,

NCT00643747 [4,

NCT00481546 [21]

NCT00999609 [22]

NCT02407678 NCT02077361

**Ref.**

19]

20]

[5]

27]

31]

33]

[34, 35]

NCT02610582 NR

NCT02161380 [8,

NCT02064569 NCT02652767 NCT02652780 NCT03293524 CBA: chicken β-actin promoter (CBA); CBSB: Hybrid modified short cytomegalovirus (CMV) enhancer and chicken β-actin promoter (CBA); GRK1: G protein-coupled receptor kinase; hCAR: human cone arrestin; NR: not reported; PR1.7: 1.7-kb L-opsin promoter; REF: References; RK: Rhodopsin kinase; scRS/IRBP: Retinoschisin/interphotoreceptor retinoid binding protein; VMD2: Vitelliform macular dystrophy-2.

**Table 1.** Summary of the clinical trials for retinopathies using AAV as delivery system registered on ClinicalTrials.gov database.

of Glybera, the use of AAV gene therapy in the eye is very attractive since the marketing prospects look better for the small amounts of AAV medicine to be transferred into the retinal tissue or retinal pigment epithelium. The eye is well accessible for surgery and allows direct observation, *in vivo*, of the retinal tissue in microscopic detail. Moreover, the eye is considered an immune-privileged tissue. Therefore, the risks of an immune response against the virus and/or the transgene itself are reduced. The local application in the "compartmentalized" eye of low amounts of AAV drug will minimize side effects expected if systemically applied at high doses [2]. But most importantly, potential drug efficacy for retinal orphan diseases can be efficiently proven thanks to a plethora of non-invasive retinal investigation techniques.

At the end of 2017, Luxturna (voretigene neparvovec-rzyl) became the first FDA-approved AAV gene therapy medicine for patients with hereditary retinal disease caused by biallelic *RPE65* gene mutations [3, 4]. The market approvals of the first gene therapy medicines in Europa and in the USA paved the road to similar programs, reflected on the large number of clinical trials registered on the ClinicalTrials.gov website using AAVs as a delivery strategy to treat hereditary retinal diseases such as choroideremia (*CHM* or *REP-1*) [5], achromatopsia (*CNGA3*) [6], wet age-related macular degeneration (AMD) (*VEGFR1*/*FLT* and a gene encoding soluble anti-VEGF protein) [7], Leber hereditary optic neuropathy (LHON) (*ND4*) [8], autosomal recessive retinitis pigmentosa (arRP) (*MERTK*) [9], X-linked RP (*RPGR*) [10], RP (*PDE6B*) [11] and (*RLBP1*) [12] and X-linked Retinoschisis (*RS1*) [13, 14] (**Table 1**). Developing an AAV gene therapy to treat patients with mutations in the Crumbs homolog-1 (*CRB1*) gene was particularly challenging due to its large cDNA (4.2 kb) which approached the packaging limit of the AAV genome (~4.7–4.9 kb). Thus, to build an AAV vector that allowed efficient packaging of the human *CRB1* cDNA, the use of a short promoter (<350 bp) and a short synthetic polyadenylation sequence was required to efficiently express the CRB1 protein *in vivo*. Codon optimization of the *CRB1* cDNA was used to achieve sufficient levels of expression [15]. A second strategy that implied the replacement of CRB1 by its structural and functional family member CRB2 was used to overcome the size limitation and potential toxicity due to expression of CRB1. *CRB2* cDNA was only 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 stabilized the transcript [16].

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 pho-

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

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

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

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

from the subapical region [52, 60] (**Figure 1**).

toreceptors, bipolar cells, late-born amacrine cells and Müller glial cells [47–52].
