Open access peer-reviewed chapter

AAV-Mediated Gene Therapy for CRB1-Hereditary Retinopathies

Written By

Celso Henrique Alves and Jan Wijnholds

Submitted: January 30th, 2018 Reviewed: June 5th, 2018 Published: November 5th, 2018

DOI: 10.5772/intechopen.79308

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Abstract

Variations in the Crumbs homolog-1 (CRB1) gene lead to autosomal recessive retinal dystrophies such as early-onset retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). No treatment is yet available for these patients. Adeno-associated virus (AAV) mediated gene therapy for hereditary retinal diseases holds great promise proven by the large number of active clinical trials. We here summarized the knowledge about the localization and function of CRB1 in the retina and the main pathological features resulting from loss of CRB1 function in humans and in rodents. This know-how is being applied to design and develop AAV gene therapy vectors for the treatment of CRB1-Hereditary retinopathies. Knowing which cell types express the CRB proteins, the possible redundancy of function between CRB1 and CRB2, and the AAV tropism in the human retina, will allow us to rationalize about the AAV capsid, promoter and route of administration that should be used in the AAV vector in order to efficiently and specifically deliver CRB1 or CRB2 into the human retina.

Keywords

  • crumbs homolog-1 (CRB1)
  • retinitis pigmentosa
  • Leber congenital amaurosis
  • gene therapy
  • adeno-associated virus (AAV)

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 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 RPE65gene 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 (CHMor REP-1) [5], achromatopsia (CNGA3) [6], wet age-related macular degeneration (AMD) (VEGFR1/FLTand 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 CRB1cDNA, 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 CRB1cDNA 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. CRB2cDNA 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].

Targeted diseaseAAV serotypePromoterGeneDelivery routeVolume injectedDosageClinicalTrials.gov IdentifierRef.
LCAAAV4RPE65hRPE65Subretinal400 or 800 μL1.22 × 1010 vg
4.8 × 1010 vg
NCT01496040[17]
AAV2CBAhRPE65Subretinal450 μL1.8 × 1011 vg
6 × 1011 vg
NCT00749957[18, 19]
AAV2hRPE65hRPE65Subretinalup to 1 mLup to 3 × 1012 vgNCT00643747[4, 20]
AAV2CBSBhRPE65Subretinal150–300 μL8.94 × 109
3.58 × 1010 vg
NCT00481546[21]
AAV2CBAhRPE65v2Subretinal150 μL1.5 × 1010 vg
4.8 × 1010 vg
1.5 × 1011 vg
NCT00999609[22]
ChoroideremiaAAV2CBAREP1Subretinal60–100 μL1010–1011 vgNCT01461213 NCT02407678 NCT02077361[5]
AAV2NRhCHMSubretinalNRNRNCT02341807NR
RP (RLBP1)AAV8sRLBP1 CPK850hRLBP1SubretinalNRNRNCT03374657[23]
RP (PDE6B)AAV5RKhPDE6BSubretinalNRNRNCT03328130[24]
RP (MERTK)AAV2VMD2hMERTKSubretinalNRNRNCT01482195[25]
X-linked RPNRNRRPGRSubretinalNRNRNCT03116113NR
AAV2tYFGRK1RPGRSubretinalNRNRNCT03316560[26, 27]
AchromatopsiaAAV8NRxhCNGA3SubretinalNR1 × 1010 vg
5 × 1010 vg
1 × 1011 vg
NCT02610582NR
AAV8hCARCNGB3SubretinalNRNRNCT03001310NR
AAV2tYFPR1.7CNGA3SubretinalNRNRNCT02935517[28]
AAV2tYFPR1.7CNGB3SubretinalNRNRNCT02599922[29]
X-linked retinoschisisAAV8scRS/IRBPshRSIntravitrealNRNRNCT02317887[30, 31]
AAV2tYFCBhRS1IntravitrealNRNRNCT02416622[32]
Leber hereditary optic neuropathy (LHON)scAAV2 (Y444,500,730F)CMV/CBAP1ND4v2Intravitreal200 μL5.00 × 109 vg
2.46 × 1010 vg
1.0 × 1011 vg
NCT02161380[8, 33]
AAV2CMVND4Intravitreal90 μL3 × 1010 vg
9 × x1010 vg
1.8 × 1011 vg
NCT02064569
NCT02652767
NCT02652780
NCT03293524
[34, 35]
Age-Related Macular Degeneration (AMD)AAV2CMVsFLT01Intravitreal100 μL2 × 108 vg
2 × 109 vg
6 × 109 vg
2 × 1010 vg
NCT01024998[36]
AAV8NRsoluble anti-VEGFSubretinalNR3 × 109 vg
1 × 1010 vg
6 × 1010 vg
NCT03066258NR

Table 1.

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

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.

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2. CRB1-Hereditary retinopathies

More than 240 different mutations in the CRB1gene 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 CRB1are 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 CRB1gene was initially characterized by RP with preservation of para-arteriolar 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 CRB1gene (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 CRB1mutations are about 1.5 times thicker than normal retinas, while retinas of patients with LCA due to mutations in other genes such as RPE65or GUCY2Dare 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, 43, 44].

No treatment is yet available for CRB1-associated retinal dystrophies. We achieved proof-of-concept for retinal CRB1gene therapy, using an AAV9-CMV-hCRB2vector 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 CRB1mutations [45]. This fact associated with the large spectrum of retinal dystrophies observed in patients with mutations in the CRB1gene [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, 48, 49, 50, 51, 52].

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, 54, 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 Crb1gene 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).

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.

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 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, 65, 66, 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:

  1. 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 Crb2specifically 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.

  2. early onset-RP: ablation of Crb2from retinal progenitor cells, and consequent loss of CRB2 in cone and rod photoreceptors and Müller glial cells [47, 49] or ablation of Crb2specifically 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 Crb1gene. Interestingly the retinal phenotype observed in this Crb1rat strain differs from the phenotype observed in the Crb1knockout mice. The Crb1rat 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 of age [64].

  3. 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 early loss of retinal function [44].

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 CRB1gene.

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3. Adeno-associated virus (AAV) biology

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 repgene encodes four proteins (Rep78, Rep68, Rep52 and Rep40), which are required for viral genome replication and packaging. While capgene 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 aapgene 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.

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 capgenes, 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 repand capgenes are supplied in trans on a helper plasmid along with helper genes from adenovirus (E4, E2aand 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 CRB1cDNAand 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 CRB1cDNAin 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 CRB2cDNAas 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

3.2.1. Subretinal injection

In pre-clinical studies performed in rodents ab externosubretinal 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.govdatabase, 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].

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 vivobetween 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 vivosetting. To obtain evidence-based data for clinical gene therapy studies, researchers optimize culture protocols for human retinal organotypic cultures [77, 78, 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 CRB1or CRB2into 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 vivoin 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 CRB1or CRB2specifically 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.

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4. Conclusion

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 RPE65gene. 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 CRB2gene augmentation therapy might be a promising medicine to prevent progression of retinitis pigmentosa in patients with mutations in the CRB1gene. In mice at mid-stage retinal disease CRB2gene augmentation therapy successfully improved retinal morphology with preservation of photoreceptor cells and retinal function, therefore providing good perspectives for the forthcoming clinical trial in patients with RP due to mutations in CRB1.

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Acknowledgments

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

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Conflict of interest

The LUMC is the holder of patent application PCT/NL2014/050549, which describes the potential clinical use of CRB2; JW is listed as inventor on this patent, and JW is an employee of the LUMC.

References

  1. 1. Glybera. Glybera, INN - alipogene tiparvovec [Internet]. 2015. Available from:http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Summary_for_the_public/human/002145/WC500135474.pdf[Accessed: 2018-03-21]
  2. 2. Hinderer C, Katz N, Buza EL, Dyer C, Goode T, Bell P, et al. Severe toxicity in nonhuman primates and piglets following high-dose intravenous administration of an AAV vector expressing human SMN. Human Gene Therapy. 2018;29(3):285-298
  3. 3. LUXTURNATM. FDA Approves Spark Therapeutics’ LUXTURNATM (voretigene neparvovec-rzyl), a One-time Gene Therapy for Patients with Confirmed Biallelic RPE65 Mutation-associated Retinal Dystrophy | Spark Therapeutics Inc. – IR Site [Internet]. 2018. Available from:http://ir.sparktx.com/news-releases/news-release-details/fda-approves-spark-therapeutics-luxturnatm-voretigene-neparvovec[Accessed: 2018-03-21]
  4. 4. Bainbridge JW, Smith AJ, Barker SS, Robbie S, Henderson R, Balaggan K, et al. Effect of gene therapy on visual function in Leber’s congenital Amaurosis. The New England Journal of Medicine. 2008;358(21):2231-2239
  5. 5. MacLaren RE, Groppe M, Barnard AR, Cottriall CL, Tolmachova T, Seymour L, et al. Retinal gene therapy in patients with choroideremia: Initial findings from a phase 1/2 clinical trial. Lancet. 2014;383(9923):1129-1137
  6. 6. Michalakis S, Mühlfriedel R, Tanimoto N, Krishnamoorthy V, Koch S, Fischer MD, et al. Restoration of cone vision in the CNGA3−/− mouse model of congenital complete lack of cone photoreceptor function. Molecular Therapy. 2010;18(12):2057-2063
  7. 7. Sanofi-Genzyme. Safety and Tolerability Study of AAV2- sFLT01 in Patients With Neovascular Age-Related Macular Degeneration (AMD). [Internet]. 2016. Available from:http://clinicaltrials.gov/ct2/show/NCT01024998[Accessed: 2018-03-21]
  8. 8. Feuer WJ, Schiffman JC, Davis JL, Porciatti V, Gonzalez P, Koilkonda RD, et al. Gene therapy for Leber hereditary optic neuropathy: Initial results. Ophthalmology. 2016;123(3):558-570
  9. 9. Ghazi NG, Abboud EB, Nowilaty SR, Alkuraya H, Alhommadi A, Cai H, et al. Treatment of retinitis pigmentosa due to MERTK mutations by ocular subretinal injection of adeno-associated virus gene vector: Results of a phase I trial. Human Genetics. 2016;135(3):327-343
  10. 10. Beltran WA, Cideciyan AV, Boye SE, Ye GJ, Iwabe S, Dufour VL, et al. Optimization of retinal gene therapy for X-linked retinitis Pigmentosa due to RPGR mutations. Molecular Therapy. 2017;25(8):1866-1880
  11. 11. Pang JJ, Boye SL, Kumar A, Dinculescu A, Deng W, Li J, et al. AAV-mediated gene therapy for retinal degeneration in the rd10 mouse containing a recessive PDEβ mutation. Investigative Ophthalmology and Visual Science. 2008;49(10):4278-4283
  12. 12. Choi VW, Bigelow CE, McGee TL, Gujar AN, Li H, Hanks SM, et al. AAV-mediated RLBP1 gene therapy improves the rate of dark adaptation in Rlbp1 knockout mice. Molecular Therapy - Methods & Clinical Development. 2015;2:15022
  13. 13. Ou J, Vijayasarathy C, Ziccardi L, Chen S, Zeng Y, Marangoni D, et al. Synaptic pathology and therapeutic repair in adult retinoschisis mouse by AAV-RS1 transfer. The Journal of Clinical Investigation. 2015;125(7):2891-2903
  14. 14. Janssen A, Min SH, Molday LL, Tanimoto N, Seeliger MW, Hauswirth WW, et al. Effect of late-stage therapy on disease progression in AAV-mediated rescue of photoreceptor cells in the retinoschisin-deficient mouse. Molecular Therapy. 2008;16(6):1010-1017
  15. 15. Pellissier LP, Hoek RM, Vos RM, Aartsen WM, Klimczak R, Hoyng S, et al. Specific tools for targeting and expression in Müller glial cells. Molecular Therapy - Methods & Clinical Development. 2014;1(2):14009
  16. 16. Pellissier LP, Quinn PM, Alves CH, et al. Gene therapy into photoreceptors and Müller glial cells restores retinal structure and function in CRB1 retinitis pigmentosa mouse models. Human Molecular Genetics. 2015;24(2):1-15
  17. 17. Le Meur G, Lebranchu P, Billaud F, Adjali O, Schmitt S, Bézieau S, et al. Safety and long-term efficacy of AAV4 gene therapy in patients with RPE65 Leber congenital Amaurosis. Molecular Therapy. 2018;26(1):256-268
  18. 18. Weleber RG, Pennesi ME, Wilson DJ, Kaushal S, Erker LR, Jensen L, et al. Results at 2 years after gene therapy for RPE65-deficient Leber congenital Amaurosis and severe early-childhood–onset retinal dystrophy. Ophthalmology. 2016;123(7):1606-1620
  19. 19. Acland GM, Aguirre GD, Bennett J, Aleman TS, Cideciyan AV, Bennicelli J, et al. Long-term restoration of rod and cone vision by single dose rAAV-mediated gene transfer to the retina in a canine model of childhood blindness. Molecular Therapy. 2005;12(6):1072-1082
  20. 20. Bainbridge JWB, Mehat MS, Sundaram V, Robbie SJ, Barker SE, Ripamonti C, et al. Long-term effect of gene therapy on Leber’s congenital Amaurosis. The New England Journal of Medicine. 2015;372(20):1887-1897
  21. 21. Cideciyan AV, Aleman TS, Boye SL, Schwartz SB, Kaushal S, Roman AJ, et al. Human gene therapy for RPE65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics. Proceedings of the National Academy of Sciences of the United States of America. 2008;105(39):15112-15117
  22. 22. Maguire AM, Simonelli F, Pierce EA, Pugh EN, Mingozzi F, Bennicelli J, et al. Safety and efficacy of gene transfer for Leber’s congenital amaurosis. The New England Journal of Medicine. 2008;358(21):2240-2248
  23. 23. Maclachlan TK, Milton MN, Turner O, Tukov F, Choi VW, Penraat J, et al. Nonclinical safety evaluation of scAAV8-RLBP1 for treatment of RLBP1 retinitis Pigmentosa. Molecular Therapy - Methods & Clinical Development. 2018;8(3):105-120
  24. 24. Pichard V, Provost N, Mendes-Madeira A, Libeau L, Hulin P, Tshilenge KT, et al. AAV-mediated gene therapy halts retinal degeneration in PDE6β-deficient dogs. Molecular Therapy. 2016;24(5):867-876
  25. 25. Parinot C, Nandrot EF. A comprehensive review of mutations in the MERTK proto-oncogene. Advances in Experimental Medicine and Biology. 2016;854:259-265
  26. 26. Huang WC, Wright AF, Roman AJ, Cideciyan AV, Manson FD, Gewaily DY, et al. RPGR-associated retinal degeneration in human X-linked RP and a murine model. Investigative Ophthalmology and Visual Science. 2012;53(9):5594-5608
  27. 27. Beltran WA, Cideciyan AV, Lewin AS, Iwabe S, Khanna H, Sumaroka A, et al. Gene therapy rescues photoreceptor blindness in dogs and paves the way for treating human X-linked retinitis pigmentosa. Proceedings of the National Academy of Sciences of the United States of America. 2012;109(6):2132-2137
  28. 28. Banin E, Gootwine E, Obolensky A, Ezra-Elia R, Ejzenberg A, Zelinger L, et al. Gene augmentation therapy restores retinal function and visual behavior in a sheep model of CNGA3 Achromatopsia. Molecular Therapy. 2015;23(9):1423-1433
  29. 29. Komáromy AM, Alexander JJ, Rowlan JS, Garcia MM, Chiodo VA, Kaya A, et al. Gene therapy rescues cone function in congenital achromatopsia. Human Molecular Genetics. 2010;19(13):2581-2593
  30. 30. Bush RA, Zeng Y, Colosi P, Kjellstrom S, Hiriyanna S, Vijayasarathy C, et al. Preclinical dose-escalation study of Intravitreal AAV-RS1 gene therapy in a mouse model of X-linked Retinoschisis: Dose-dependent expression and improved retinal structure and function. Human Gene Therapy. 2016;27(5):376-389
  31. 31. Marangoni D, Bush RA, Zeng Y, Wei LL, Ziccardi L, Vijayasarathy C, et al. Ocular and systemic safety of a recombinant AAV8 vector for X-linked retinoschisis gene therapy: GLP studies in rabbits and Rs1-KO mice. Molecular Therapy - Methods & Clinical Development. 2016;5:16011
  32. 32. Molday RS. Insights into the molecular properties of ABCA4 and its role in the visual cycle and Stargardt disease. Progress in Molecular Biology and Translational Science. 2015;134:415-431
  33. 33. Koilkonda RD, Chou TH, Porciatti V, Hauswirth WW, Guy J. Induction of rapid and highly efficient expression of the human ND4 complex I subunit in the mouse visual system by self-complementary adeno-associated virus. Archives of Ophthalmology. 2010;128(7):876-883
  34. 34. Vignal C, Uretsky S, Fitoussi S, Galy A, Blouin L, Girmens J-F, et al. Safety of rAAV2/2-ND4 gene therapy for Leber hereditary optic neuropathy. Ophthalmology. 2018;S0161-6420(17):33673-33674
  35. 35. Cwerman-Thibault H, Augustin S, Lechauve C, Ayache J, Ellouze S, Sahel JA, et al. Nuclear expression of mitochondrial ND4 leads to the protein assembling in complex I and prevents optic atrophy and visual loss. Molecular Therapy - Methods & Clinical Development. 2015;2:15003
  36. 36. Heier JS, Kherani S, Desai S, Dugel P, Kaushal S, Cheng SH, et al. Intravitreous injection of AAV2-sFLT01 in patients with advanced neovascular age-related macular degeneration: A phase 1, open-label trial. Lancet. 2017;390(10089):50-61
  37. 37. Talib M, van Schooneveld MJ, van Genderen MM, Wijnholds J, Florijn RJ, ten Brink JB, et al. Genotypic and phenotypic characteristics of CRB1-associated retinal dystrophies: A long-term follow-up study. Ophthalmology. 2017;124(6):884-895
  38. 38. den Hollander AI, Roepman R, Koenekoop RK, Cremers FPM. Leber congenital amaurosis: Genes, proteins and disease mechanisms. Progress in Retinal and Eye Research. 2008;27(4):391-419
  39. 39. Corton M, Tatu SD, Avila-Fernandez A, Vallespín E, Tapias I, Cantalapiedra D, et al. High frequency of CRB1 mutations as cause of early-onset retinal dystrophies in the Spanish population. Orphanet Journal of Rare Diseases. 2013;8:20
  40. 40. Lotery AJ, Malik A, Shami SA, Sindhi M, Chohan B, Maqbool C, et al. CRB1 mutations may result in retinitis pigmentosa without Para-arteriolar RPE preservation. Ophthalmic Genetics. 2001;22(3):163-169
  41. 41. Den Hollander AI, Davis J, Van Der Velde-Visser SD, Zonneveld MN, Pierrottet CO, Koenekoop RK, et al. CRB1 mutation spectrum in inherited retinal dystrophies. Human Mutation. 2004;24(5):355-369
  42. 42. Jacobson SG, Cideciyan AV, Aleman TS, Pianta MJ, Sumaroka A, Schwartz SB, et al. Crumbs homolog 1 (CRB1) mutations result in a thick human retina with abnormal lamination. Human Molecular Genetics. 2003;12(9):1073-1078
  43. 43. Aleman TS, Cideciyan AV, Aguirre GK, Huang WC, Mullins CL, Roman AJ, et al. Human CRB1-associated retinal degeneration: Comparison with the rd8 Crb1-mutant mouse model. Investigative Ophthalmology and Visual Science. 2011;52(9):6898-6910
  44. 44. Pellissier LP, Alves CH, Quinn PM, Vos RM, Tanimoto N, Lundvig DM, et al. Targeted ablation of Crb1 and Crb2 in retinal progenitor cells mimics Leber congenital Amaurosis. PLoS Genetics. 2013;9(12):e1003976
  45. 45. Bujakowska K, Audo I, Mohand-Saïd S, Lancelot M-E, Antonio A, Germain A, et al. CRB1 mutations in inherited retinal dystrophies. Human Mutation. 2012;33(2):306-315
  46. 46. Marquardt T, Gruss P. Generating neuronal diversity in the retina: One for nearly all. Trends in Neurosciences. 2002;25(1):32-38
  47. 47. Alves CH, Bossers K, Vos RM, Essing AH, Swagemakers S, Van Der Spek PJ, et al. Microarray and morphological analysis of early postnatal CRB2 mutant retinas on a pure C57BL/6J genetic background. PLoS One. 2013;8(12):1-17
  48. 48. Park B, Alves CH, Lundvig DM, Tanimoto N, Beck SC, Huber G, et al. PALS1 is essential for retinal pigment epithelium structure and neural retina stratification. The Journal of Neuroscience. 2011;31(47):17230-17241
  49. 49. Alves CH, Sanz A, Park B, Pellissier LP, Tanimoto N, Beck SC, et al. Loss of CRB2 in the mouse retina mimics human retinitis pigmentosa due to mutations in the CRB1 gene. Human Molecular Genetics. 2013;22(1):35-50
  50. 50. Alves CH, Pellissier LP, Vos RM, Garcia Garrido M, Sothilingam V, Seide C, et al. Targeted ablation of Crb2 in photoreceptor cells induces retinitis pigmentosa. Human Molecular Genetics. 2014;23(13):3384-3401
  51. 51. Dudok JJ, Sanz AS, Lundvig DMS, Sothilingam V, Garrido MG, Klooster J, et al. MPP3 regulates levels of PALS1 and adhesion between photoreceptors and Müller cells. Glia. 2013;61(10):1629-1644
  52. 52. van de Pavert SA, Kantardzhieva A, Malysheva A, Meuleman J, Versteeg I, Levelt C, et al. Crumbs homologue 1 is required for maintenance of photoreceptor cell polarization and adhesion during light exposure. Journal of Cell Science. 2004;117(18):4169-4177
  53. 53. Tepass U, Theres C, Knust E. Crumbs encodes an EGF-like protein expressed on apical membranes of Drosophila epithelial cells and required for organization of epithelia. Cell. 1990;61(5):787-799
  54. 54. Alves CH, Pellissier LP, Wijnholds J. The CRB1 and adherens junction complex proteins in retinal development and maintenance. Progress in Retinal and Eye Research. 2014;40:35-52
  55. 55. Quinn PM, Pellissier LP, Wijnholds J. The CRB1 complex: Following the trail of crumbs to a feasible gene therapy strategy. Frontiers in Neuroscience. 2017;11(3):175
  56. 56. Roh MH, Fan S, Liu CJ, Margolis B. The Crumbs3-Pals1 complex participates in the establishment of polarity in mammalian epithelial cells. Journal of Cell Science. 2003;116(14):2895-2906
  57. 57. Kantardzhieva A, Alexeeva S, Versteeg I, Wijnholds J. MPP3 is recruited to the MPP5 protein scaffold at the retinal outer limiting membrane. The FEBS Journal. 2006;273(6):1152-1165
  58. 58. Kantardzhieva A, Gosens I, Alexeeva S, Punte IM, Versteeg I, Krieger E, et al. MPP5 recruits MPP4 to the CRB1 complex in photoreceptors. Investigative Ophthalmology and Visual Science. 2005;46(6):2192-2201
  59. 59. Pellikka M, Tanentzapf G, Pinto M, Smith C, McGlade CJ, Ready DF, et al. Crumbs, the Drosophila homologue of human CRB1/RP12, is essential for photoreceptor morphogenesis. Nature. 2002;416(6877):143-149
  60. 60. van Rossum AG, Aartsen WM, Meuleman J, Klooster J, Malysheva A, Versteeg I, et al. Pals1/Mpp5 is required for correct localization of Crb1 at the subapical region in polarized Müller glia cells. Human Molecular Genetics. 2006;15(18):2659-2672
  61. 61. Den Hollander AI, Ghiani M, De Kok YJM, Wijnholds J, Ballabio A, Cremers FPM, et al. Isolation of Crb1, a mouse homologue of Drosophila crumbs, and analysis of its expression pattern in eye and brain. Mechanisms of Development. 2001;110(1-2):203-207
  62. 62. Herranz-Martín S, Jimeno D, Paniagua AE, Velasco A, Lara JM, Aijón J, et al. Immuno-cytochemical evidence of the localization of the crumbs homologue 3 protein (CRB3) in the developing and mature mouse retina. PLoS One. 2012;7(11):e50511
  63. 63. Quinn PM, Buck TM, Alves CH, Ohonin C, Chuva de Sousa Lopes SM, Mikkers HMM, et al. Recapitulation of the human fetal crumbs complex in human iPSCs-derived retinas and retinal pigment epithelium. Investigative Ophthalmology & Visual Science. 2017;58(8):3758
  64. 64. Zhao M, Andrieu-Soler C, Kowalczuk L, Paz Cortes M, Berdugo M, Dernigoghossian M, et al. A new CRB1 rat mutation links Muller glial cells to retinal telangiectasia. The Journal of Neuroscience. 2015;35(15):6093-6106
  65. 65. Aredo B, Zhang K, Chen X, Wang CX-Z, Li T, Ufret-Vincenty RLR, et al. Differences in the distribution, phenotype and gene expression of subretinal microglia/macrophages in C57BL/6N (Crb1rd8/rd8) versus C57BL6/J (Crb1wt/wt) mice. Journal of Neuro-inflammation. 2015;12(1):6
  66. 66. Mehalow AK, Kameya S, Smith RS, Hawes NL, Denegre JM, Young JA, et al. CRB1 is essential for external limiting membrane integrity and photoreceptor morphogenesis in the mammalian retina. Human Molecular Genetics. 2003;12(17):2179-2189
  67. 67. van, de Pavert SA, Meuleman J, Malysheva A, Aartsen WM, Versteeg I, Tonagel F, et al. A single amino acid substitution (Cys249Trp) in Crb1 causes retinal degeneration and deregulates expression of pituitary tumor transforming gene Pttg1. The Journal of Neuroscience. 2007;27(3):564-573
  68. 68. Naso MF, Tomkowicz B, Perry WL, Strohl WR. Adeno-associated virus (AAV) as a vector for gene therapy. BioDrugs. 2017;31(4):317-334
  69. 69. Maddalena A, Tornabene P, Tiberi P, Minopoli R, Manfredi A, Mutarelli M, et al. Triple vectors expand AAV transfer capacity in the retina. Molecular Therapy. 2017:524-541
  70. 70. Alves CH, Wijnholds J. AAV gene augmentation therapy for CRB1-associated retinitis pigmentosa. Methods in Molecular Biology. 1715;2018:135-151
  71. 71. Ochakovski GA, Bartz-Schmidt KU, Fischer MD. Retinal gene therapy: Surgical vector delivery in the translation to clinical trials. Frontiers in Neuroscience. 2017;11:174
  72. 72. Fischer MD, Hickey DG, Singh MS, MacLaren RE. Evaluation of an optimized injection system for retinal gene therapy in human patients. Human Gene Therapy Methods. 2016;27(4):150-158
  73. 73. Du L, Peng H, Wu Q, Zhu M, Luo D, Ke X, et al. Observation of total VEGF level in hyperglycemic mouse eyes after intravitreal injection of the novel anti-VEGF drug conbercept. Molecular Vision. 2015;21(7):185-193
  74. 74. Dureau P, Legat L, Neuner-Jehle M, Bonnel S, Pecqueur S, Abitbol M, et al. Quantitative analysis of subretinal injections in the rat. Graefe's Archive for Clinical and Experimental Ophthalmology. 2000;238:608-614
  75. 75. Chiu K, Chang RC-C, So K-F. Intravitreous injection for establishing ocular diseases model. Journal of Visualized Experiments. 2007;8:313
  76. 76. Klimczak RR, Koerber JT, Dalkara D, Flannery JG, Schaffer DV. A novel adeno-associated viral variant for efficient and selective intravitreal transduction of rat Müller cells. PLoS One. 2009;4(10):e7467
  77. 77. Buck TM, Pellissier LP, Vos RM, van Dijk EHC, Boon CJF, Wijnholds J. AAV serotype testing on cultured human donor retinal explants. Methods in Molecular Biology. 1715;2018:275-288
  78. 78. Orlans HO, Edwards TL, De Silva SR, Patrício MI, MacLaren RE. Human retinal explant culture for ex vivo validation of AAV gene therapy. Methods in Molecular Biology. 1715;2018:289-303
  79. 79. Wiley LA, Burnight ER, Kaalberg EE, Jiao C, Riker MJ, Halder JA, et al. Assessment of Adeno-associated virus serotype tropism in human retinal explants. Human Gene Therapy. 2018;29(4):424-436
  80. 80. Quinn PM, Buck TM, Ohonin C, Mikkers HMM, Wijnholds J. Production of iPS-derived human retinal organoids for use in transgene expression assays. Methods in Molecular Biology. 1715;2018:261-273
  81. 81. Hickey DG, Edwards TL, Barnard AR, Singh MS, De Silva SR, McClements ME, et al. Tropism of engineered and evolved recombinant AAV serotypes in the rd1 mouse and ex vivo primate retina. Gene Therapy. 2017;24(12):787-800
  82. 82. Vandenberghe LH, Bell P, Maguire AM, Xiao R, Hopkins TB, Grant R, et al. AAV9 targets cone photoreceptors in the nonhuman primate retina. PLoS One. 2013;8(1):e53463
  83. 83. Boye SE, Alexander JJ, Boye SL, Witherspoon CD, Sandefer KJ, Conlon TJ, et al. The human rhodopsin kinase promoter in an AAV5 vector confers rod- and cone-specific expression in the primate retina. Human Gene Therapy. 2012;23(10):1101-1115
  84. 84. Buck TM, Quinn PM, Alves CH, Van Dijk E, Ohonin C, Boon CJF, et al. Potency assay for AAV-gene vectors in human iPSCs-derived retinas and donor retinas. Investigative Ophthalmology & Visual Science. 2017;58(8):4093

Written By

Celso Henrique Alves and Jan Wijnholds

Submitted: January 30th, 2018 Reviewed: June 5th, 2018 Published: November 5th, 2018