Accepted Manuscript Treatment of ocular disorders by gene therapy M. Ángeles Solinís, Ana del Pozo-Rodríguez, Paola S. Apaolaza, Alicia Rodríguez-Gascón PII: DOI: Reference:

S0939-6411(14)00377-4 http://dx.doi.org/10.1016/j.ejpb.2014.12.022 EJPB 11787

To appear in:

European Journal of Pharmaceutics and Biopharmaceutics

Received Date: Accepted Date:

19 September 2014 15 December 2014

Please cite this article as: M. Ángeles Solinís, A. del Pozo-Rodríguez, P.S. Apaolaza, A. Rodríguez-Gascón, Treatment of ocular disorders by gene therapy, European Journal of Pharmaceutics and Biopharmaceutics (2014), doi: http://dx.doi.org/10.1016/j.ejpb.2014.12.022

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Title: Treatment of ocular disorders by gene therapy

Authors: M. Ángeles Solinís1, Ana del Pozo-Rodríguez1, Paola S. Apaolaza1, Alicia RodríguezGascón1,*

1

Pharmacokinetic, Nanotechnology and Gene Therapy Group (PharmaNanoGene). Faculty of

Pharmacy. Centro de investigación Lascaray ikergunea. University of the Basque Country UPV/EHU. Paseo de la Universidad, 7. 01006. Vitoria-Gasteiz (Spain)

*

Author for correspondence: Alicia Rodríguez Gascón

Pharmacy and Pharmaceutical Technology Laboratory, Pharmacy Faculty, University of the Basque Country (UPV-EHU), Paseo de la Universidad 7, 01006 Vitoria-Gasteiz. Tel.:+34 945 01 30 94. Fax. +34 945 01 30 40. e-mail: [email protected]

1

Abstract

Gene therapy to treat ocular disorders is still starting, and current therapies are primarily experimental, with most human clinical trials still in research state, although beginning to show encouraging results. Currently 33 clinical trials have been approved, are in progress, or have been completed. The most promising results have been obtained in clinical trials of ocular gene therapy for Leber Congenital Amaurosis, which have prompted the study of several ocular diseases that are good candidates to be treated with gene therapy: glaucoma, age-related macular degeneration, retinitis pigmentosa, or choroideremia. The success of gene therapy relies on the efficient delivery of the genetic material to target cells, achieving optimum long-term gene expression. Although viral vectors have been widely used, their potential risk associated mainly with immunogenicity and mutagenesis has promoted the design of non-viral vectors. In this review, the main administration routes and the most studied delivery systems, viral and non-viral, for ocular gene therapy are presented. The primary ocular diseases candidates to be treated with gene therapy have been also reviewed, including the genetic basis and the most relevant preclinical and clinical studies.

Keywords: ocular disorder, gene therapy, viral vector, non-viral vector, clinical trial

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Highlights We examine the promises and challenges of applying gene therapy to ocular diseases We revise preclinical and clinical studies in which ocular gene therapy was applied The eye presents advantages for gene therapy: accessibility and ease evaluation Viral vectors are the most common gene delivery systems for ocular diseases Gene replacement therapy is the most widely used in clinical approach Introduction

Gene therapy, which involves intracellular delivery of genetic material either to block a dysfunctional gene or to deliver a gene as a therapeutic, has huge potential for treating diseases with a genetic component. In spite of the promising strategy that gene therapy supposes for several diseases, its potential risk still makes necessary studies to extend safeness and effectiveness concerns. Actually, clinical application of gene therapy is currently limited to serious diseases that have no cure. The eye possesses important advantages for gene therapy: a well-defined anatomy, it is relatively immune privileged, the accessibility, it is easily examined and, in the same subject one eye can be used as the experimental target and the other one as a control. The progresses in gene therapy hold considerable promise for the management of ophthalmic conditions, and ocular gene therapy has been extensively explored in recent years as a therapeutic avenue to target diseases of the cornea, retina and retinal pigment epithelium (RPE). The success of gene therapy relies on the efficient delivery of the genetic material to target cells, achieving optimum long-term gene expression. In this review we focus on the main methods for gene delivery and on the application of gene therapy in ocular diseases.

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1.

Gene delivery to the eye: methods of administration

To treat ocular diseases with gene therapy three main parameters have to be perfectly selected: the administration route, the delivery system and, the use of specific promoter elements. 2.1. Ocular administration routes Ocular gene delivery can be performed through different routes including topical instillation, periocular routes, intracameral injection, intravitreal injection, subretinal injection or suprachoroidal injection (Figure 1). Each administration route shows advantages and disadvantages, and the selection should be based upon the targeted cells and the characteristics of the vector used [1, 2].

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Figure 1. Scheme of the different ocular administration routes. NFL: Nerve Fiber Layer, GCL: Ganglion Cells Layer, IPL: Inner Plexiform Layer, INL: Inner Nuclear Layer, OPL: Outer Plexiform layer, ONL: Outer Nuclear Layer, ONL: Outer Nuclear Layer, PRL: Photoreceptor Layer, RPE: Retinal Pigment Epithelia.

Topical instillation Topical administration is the easiest non-invasive method of drug delivery in the eye. However, ocular bioavailability of instilled molecules is very poor [3], especially in the case of large molecules such as nucleic acids. On the one hand, an important fraction of the low drug dose that can be administered topically is drained from the ocular surface [4] and, even absorbed into the systemic circulation through conjunctival and nasal vessels [5]. On the other hand, penetration of the cornea and conjunctival epithelia to reach the posterior chamber is limited by the size of nucleic acids [3]. Therefore, the topical route is usually limited to the treatment of diseases related to the anterior segment of the eye. 5

Periocular routes The term periocular route includes the administration of drugs via peribulbar, retrobulbar, posterior juxtascleral, sub-tenon and subconjunctival injections [6], being this last one the most studied for delivery of nucleic acids. Suconjunctival injection is a little invasive method that allows large administration volumes and can be repeated as necessary [3]. Through this route, drugs can penetrate to the anterior and posterior segments of the eye, but potential complications may appear due to systemic absorption [3]. Disposition of particles and molecules subconjunctivally injected depends on the particle size: larger particles (> 200 nm) are retained for long time in the subconjunctival space and are more appropriate for sustained delivery to the anterior chamber [7]. Nucleic acids, due to their large molecular weight are also located in the injection site without major penetration into other ocular tissues or systemic circulation [8].

Intracameral injection Delivery of nucleic acids into the anterior chamber induces transduction of anterior eye segment tissues, although the rapid turnover of aqueous humor and the short contact time with ocular tissues may result in low efficacy [3]. This kind of injection has produced stable protein expression in corneal endothelial cells and trabecular meshwork by using pseudotyped equine infectious anaemia virus (EIAV) as vector [9]. Intracameral administration has been also used to control intraocular pressure (IOP) by gene therapy [10]. Intravitreal injection

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Drug delivery by intravitreal injection has been extensively studied as a way to access to vitreous and retina structures. Administration into intravitreal humour is relatively easy and high doses are possible, although adverse events such as retinal detachment, endophthalmitis [11] and increase of IOP [12] may occur. Several works about the distribution of nucleic acids after intravitreal injection have been carried out. Shen et al. [13] administered a short interfering RNA (siRNA) against vascular endothelial growth factor (VEGF) in mice. siRNA was detected in several ocular tissues, including ganglion cells and photoreceptors. In another study, the distribution of a siRNA against VEGF-A in rabbit eyes was similar [14]. Subretinal injection Injection of the vectors into the subretinal space allows the contact of the nucleic acids with photoreceptors, outer retinal layers and RPE cells. Therefore, this route is useful for the treatment of retinal degenerations caused by gene mutations in photoreceptors or RPE. However, like intravitreal injection, subretinal administration is an invasive method and there is a risk of ocular damage, i.e. lesions in RPE, hemorrhages, retinal tears, sub- or pre-retinal fibrosis, and retinal detachment [3]. Subretinal administration has been used in clinical trials to treat Leber Congenital Amaurois Type 2 (LCA2) with very promising results [15,16]. Suprachoroidal injection Suprachoroidal administration, below the sclera and above choroid, has been also explored for drug delivery to the posterior segment of the eye [17]. This route of administration does not interfere with optical pathways and improves diffusional access to the choroid, but macromolecules are cleared rapidly and a sustained release formulation is necessary for longer duration [18]. Touchard et al. [19] have developed a transfection method called suprachoroidal electrotransfer, combining the administration 7

of a non-viral plasmid DNA into the suprachoroidal space with the application of an electrical field. After administration in rat eyes, choroidal cells, RPE, and the outer segment of photoreceptors, were efficiently transduced for at least 1 month. In a previous work [20], the administration of a viral vector in the suprachoroidal space of rabbit eyes demonstrated robust transfection in all treated eyes at the level of the choroid, RPE, photoreceptors and, retinal ganglion cells 6 weeks after treatment. 2.2. Delivery systems The ideal gene delivery system should be taken up efficiently and broadly in the target tissue after administration by a less-invasive method, without ectopic expression. In chronic disease treatments gene expression should start fast, be high enough to induce phenotypic improvement and keep on long time. Additionally, the vector should be well tolerated and should not cause adverse effects, such as inflammation, immune response or integrational toxicity [21]. The research of an effective gene therapy for ocular diseases has conducted to the development of several delivery systems. In the case of interference RNA (RNAi), clinical trials targeting ocular diseases use direct injections of naked siRNA [22], but in order to improve the efficacy and reduce the number of injections, a sustained release of siRNAs for extended periods of time could be very useful [23]. Among gene delivery systems, viral vectors have been widely used, but their potential risk associated with immunogenicity [24], mutagenesis [25] and, even persistence in the brain [26], have promoted the design of non-viral vectors as alternative. Non-viral vectors are less immunogenic, do not induce major ocular inflammatory responses, are safe and ease to produce in large scales, although their main limitation still remains the lower transfection efficacy [3]. Viral vectors 8

Up to date viral vectors continue being the gene delivery system of choice for ocular diseases [27]. Several viral vectors have been applied to deliver genes to ocular tissues, including adenovirus, adeno-associated virus, retrovirus and lentivirus. The selection of the most appropriate viral vector will depend on the target cell and the duration of the response (long- or short-term response). Adenoviral vectors Adenoviruses (Ads) are double-stranded DNA vectors able to effectively transfect both dividing and non-dividing cells. These vectors show various advantages that make them interesting for gene therapy: they do not integrate their nucleic acid into their host genomes, thus avoiding the risk of mutagenesis, are ease of amplification and purification and, they can incorporate large genes [1]. However, Ads usually induce immune responses [28] and the duration of gene expression is short [29]. Therefore, Ads have potential use in scenarios where transient gene expression is necessary, such as over-expression of proteins in corneal epithelium of patients with diabetes mellitus, but their application for inherited gene defects is discouraged [30,31]. Nevertheless, Ads have demonstrated transfection efficacy both in anterior [10,32-35] and posterior eye segment [36-39]. The most commonly used Ads for gene delivery are serotypes 2 and 5, which have suffered recombinant modifications to reduce immunogenicity and viral replication capacity [28]. Recombinant Ads (r-Ads) carrying interleukin(IL)-10 [32] or nerve growth factor [33] genes have demonstrated ability to improve allograft survival in corneal transplants in rats. rAds are also able to transfect trabecular meshwork, which is interesting to treat glaucoma and steroid-induced IOP [10,34,35]. Regarding retinaassociated diseases, rAds have been designed as vectors for genes involved in retinitis pigmentosa[36], age-related macular degeneration[37,38], or diabetic retinopathies[39]. 9

Recently, Ads carrying three different genes (Ascl1, Brn3b and Ngn2) have been used to reprogram ex vivo mouse fibroblasts to retinal ganglion-like cells [40]. Adeno-associate viral vectors Adeno-associate viruses (AAVs) are nonpathogenic single-stranded DNA vectors able to transduce slow or non-dividing cells and provide long-term transgene expression, which can persist up to 6 years after a single dose [41]. Among the numerous AAV serotypes described, AAV2 is the most commonly used for gene delivery [1], but transduction efficacy, specificity and onset of transgene expression depends not only on the serotype but also on the capsid. Capsid proteins may be exchanged among various AAV serotypes, leading to recombinant AAVs (AAVn1/n2) in which the first number of the vectors denotes serotype and the second number corresponds to the capsid [42]. In the retina, subretinal administration of AAV2 vectors resulted in transduction of RPE and photoreceptor cells [43,44], whereas intravitreal injection leads to ganglion cells transduction [45,46]. The experience with AAVs has demonstrated that AAV2/5 and AAV2/8 are the two most efficient serotypes for photoreceptor targeting [47]. However, AAVs transduce predominantly rods, and gene transfer to both cones and rods is essential for gene therapy of inherited retinal degenerations such as LCA1. Recently, Manfredi et al. [48] have shown that the chimeric AAV2/8 transduces both pig cones and rods, which supports the use of AAV2/8 for gene therapy of retinal diseases. AAV vectors have been also extensively documented as effective delivery systems to treat Xlinked retinoschisis (XLRS) in a mice model (Rs1h knockout mice) [49-53]. Results showed retinal structure and function repair, joint with a slower degeneration of the retina. AAVs are also useful to transfect corneal cells. In primary cultures of human corneal fibroblasts exposed to different AAV infectious particles, AAV2/6 displayed 30-50-fold 10

higher transduction efficiency in comparison with AAV2/8 or AAV2/9 [54]. However, when the same viral vectors were topically applied onto mouse cornea in vivo and human cornea ex vivo [55], the order of transduction efficiency was AAV2/9 > AAV2/8 > AAV2/6. Importantly, AAVs resulted safe, and cell death or inflammation was not detected. Tyrosine-mutant AAVs (substitution of tyrosine by phenylalanine in the capside) enhanced nuclear transport and increased transgene expression [56]. This new generation of AAVs requires lower doses for transduction, which leads to a decreased immune response. These vectors have been shown to provide long-term rescue of a variety of photoreceptor dystrophies in several animal species [57-59]. Lentiviral vectors Lentiviruses are single stranded RNA vectors that possess distinct properties that make them particularly suitable for gene delivery in ophthalmic diseases: high and stable expression, ability to infect dividing and non-dividing cells, as well as primary cells, and a lack of associated intraocular inflammation [60]. Because they are integrating vectors, highly deleted [61], self-inactivating [62] and non-integrating [63] lentiviral vectors have been developed as safer alternatives; in fact, clinically serious adverse events have not been reported with this type of vectors [64]. Lentiviral vectors have been shown to transduce both anterior and posterior segment of the eye. Parker et al. [65] used a human immunodeficiency virus (HIV)-based vector and obtained stable transgene expression in over 80–90% of corneal endothelial cells of mice, sheep and humans in vitro. In addition, when corneas were transduced ex vivo and transplanted as allografts, sustained expression of donor corneal endothelium in the transplanted ovine cornea in vivo was detected. Lentiviral vectors are also able to transduce retinal cells [66-68], and have been studied as vectors for different genes 11

related to retinal dystrophies such as rod photoreceptor cGMP phosphodiesterase beta subunit (PDEbeta) [69], RPE65 [66] or photoreceptor-specific adenosine triphosphate (ATP)-binding cassette transporter (ABCA4) protein [67,68]. Binley et al. [70] have designed a lentiviral vector that expresses the angiostatic proteins endostatin and angiostatin, to be delivered via a subretinal injection for the treatment of the wet form of age-related macular degeneration (RetinoStat®). Studies carried out in macaques and rabbits showed that the formulation was well tolerated, localized, and capable of induce persistent expression after subretinal delivery. Lentiviruses have been recently reported as vectors for RNAi with potential utility for the treatment of diabetic retinopathy, retinopathy of prematurity and other ocular neovascular diseases [71]. Retroviral vectors Retroviral vectors are also RNA viruses with oncogenetic capacity, which implies a risk of major adverse effects. The use of retroviruses for gene therapy has been drastically reduced due to the development of T-cell leukemia in 4 patients enrolled in a clinical trial for X-linked severe combined immunodeficiency treatment with this kind of vectors [72] and, the death of one of those individuals [73]. Non-viral vectors In spite of the significant success with viral vectors, non-viral approaches are being extensively explored, since their controlled synthesis offers the possibility of improving the design to reach more efficient results [74]. Among non-viral strategies, physical methods (iontophoresis, electroporation, gene gun, nucleofection) have achieved considerable progress, but gene expression efficiency is still a limitation [21,74,75], and chemical non-viral vectors have gained more attention. A typical non-viral vector contains a cationic compound that bounds electrostatically the genetic material and form a stable complex. The nucleic acid is so protected, and the 12

resulting cationic vector also facilitates the attachment to cell surface and the subsequent endocytosis [76]. The nature of the cationic components generally is lipidic or polymeric. More detailed description of non-viral vectors has been recently published [77]. Lipid-based vectors Cationic lipids are positive charge amphiphilic molecules formed by a cationic head moiety and a lipid hydrophobic group. These lipids may form complexes (so-called lipoplexes) with negatively charged nucleic acids [78]. Among lipid-based systems, liposomes and solid lipid nanoparticles (SLNs) are the preferred to deliver nucleic acids. Liposomes are spherical vesicles composed of an aqueous compartment surrounded by a phospholipid bilayer of natural or synthetic origin, with sizes that can range from 20 nm to a few microns. Due to their resemblance to biological membranes, liposomes show higher biocompatibility than polymeric vehicles, which contribute to be better delivery systems [79]. Technological factors, such as the lipid to nucleic acid ratio or total lipid concentration in the final complex are determinant for efficient gene delivery. Liposomal encapsulation of nucleic acids has been shown an effective method to transfect corneal cells, inner retinal layer and RPE [80]. Topical instillation of liposomes carrying the β-galactosidase gene led to transfection of retinal ganglion cells. Injection into the anterior chamber delivered the gene to the basal layer of the corneal epithelium, the ciliary epithelium, the stroma of the ciliary body and iris, and retinal ganglion cells. Injection into the vitreous or subretinal space resulted in the expression of the gene in the ciliary epithelium, the stroma of the ciliary body and iris, retinal ganglion cells, and RPE cells. In order to improve the efficacy and site specificity, significant effort has been dedicated to modify the composition and chemical structure of liposomes [81]. 13

Different compounds have been incorporated to their structure, such as, protamine sulfate [82], poly-ethylenglicol (PEG) [83] or, Arg(R)-Gly(G)-Asp(D) motif peptides [84]. SLNs have been documented to be one of the most effective lipid-based colloidal vehicles. SLNs consist of an aqueous dispersion of a layer of surfactants surrounding a solid lipid core, resulting in a submicron size of 50-1000 nm. Like liposomes, SLNs are composed by well tolerated physiological lipids, often approved in pharmaceutical preparations for human use. In addition, SLNs have shown to be stable for long-term periods of time and may be subjected to sterilization and lyophilization procedures [85]. Our research group has demonstrated the ability of SLNs to transfect retinal cells both in vitro [86-88] and in vivo [89]. Moreover, after topical application SLNs were able to transfect corneal epithelium [89]. The efficacy of SLNs can be improved by the incorporation of different components, such as, protamine sulfate [88], cell penetration peptides [87], dextran [90], chitosan [91] or hyaluronic acid [92], among others. These ligands act increasing nucleic acid protection, cell internalization or improving the trafficking inside the cells. Polymer-based vectors Many novel biodegradable polymers such as polyethyleneimine (PEI), polyesters, chitosan, hyaluronic acid, albumin, polyamidoamine dendrimers or poly-L-lysine (PLL), are being investigated as carriers for gene therapy. PEI is a cationic polymer able to bind spontaneously to nucleic acids, with high transfection capacity, whose major obstacle is the toxicity in vivo [93]. Nevertheless, small PEI of 25kD show reduced cytotoxicity in vitro [94]. In the eye, PEI-based vectors did not provide very successful results after intravitreal delivery [95]; however, topical application of a plasmid expressing basic fibroblast growth factor–PEI vector 14

induced dose-dependent corneal neovascularization, which reached a maximum on days 24–30 [96]. PEI has been modified with different components to reduce its cytotoxicity and improve the efficacy [97-99]. PEI has also demonstrated capacity to delivery antisense oligonucleotides in vitro in rat retinal Müller glial cells, and in vivo after intravitreal administration [94]. Polyesters, including poly(lactic) acid (PLA), poly(glycolic) acid (PGA) and their copolymer poly(lactic-co-glycolic acid) (PLGA), have been also used for retinal nucleic acid delivery due to their ability to bound plasmids, their nontoxic features, and rapid internalization capacity [100,101]. Chitosans (CS) are cationic polysaccharides comprising copolymers of glucosamine and N-acetylglucosamine, able to bind DNA efficiently and to protect it from nuclease degradation [91]. The mucoadhesiveness of CS makes it an attractive carrier for increasing corneal residence time and enhancing bioavailability by interacting with the negative charges of the mucus [102,103]. In addition, due to the mucoadhesive properties of hyaluronic acid (HA), both CS and HA have been combined to obtain gene delivery nanoparticles (HA-CS-NP) for ocular applications [104]. The combination of HA-CS-NPs with cationic lipids has also been proposed as an effective non-viral vector for application in eye diseases [105]. Albumin [106], dendrimers [107] and PLL [108] are polymeric compounds also proposed as nucleic acid delivery systems for ocular applications. These substances are able to protect nucleic acids and internalize them into the cell cytoplasm, improving their presence in the nucleus. 2.3. Promoter elements Vector engineering to attain tissue-selective targeting and/or regulate the extent of gene expression is a challenging issue of ocular gene therapy and demands active research. 15

On the one hand, tissue-specific elements direct the gene expression only to desired tissues, reducing secondary effects. On the other hand, regulatory elements will make the vector able to turn on and off gene expression when needed. Tissue-specific promoters for corneal and for retinal cells are described in the literature [49,53,109-113]. For instance, keratin 12 (epithelial-specific) and keratocan (keratocytespecific) promoters have been used to target polymeric micelles [109], and domains of human glial fibrillary acidic protein to target to retinal Müller cells [110]. Photoreceptor-specific gene delivery has been investigated by design of plasmids with cone and rod homeobox (CRX) [111], rhodopsin [112,113,53] or rod opsin (mOPS) promoters [49]. All these promoters show high targeting expression in rods, whereas only the rhodopsin RK promoter targets expression in cones [112]. Expression of the delivered gene only when needed can be achieved by using inducible regulatory sequences in the promoter. Some of these promoters are responsive to specific environmental signals. Inclusion of a hypoxia-responsive element that is silenced in normoxia, but induces gene expression in hypoxic regions, has been proposed as an strategy to design vectors for neovascular disorders such as diabetic retinopathy and age-related macular degeneration [114,110]. In other cases, gene expression is regulated by drug inducible systems: expression of the protein only occurs upon administration of the corresponding inducer drug. In ocular gene therapy, this strategy has been assayed by using rapamycin [115] or doxycyclin [39] inducible systems for retinal neovascularization, or glucocorticoid response elements for glaucoma [35] or corneal transplant [116]. Promoter elements may also help to obtain long-term expression. This is the case of scaffold/matrix attachment region (S/MAR) sequences, which anchor chromatin to the nuclear matrix proteins during the interphase [103]. This sequence was included in the 16

RPE65 plasmid administered encapsulated in PEGylated-PLL nanoparticles to the subretinal space of rpe65-/- mice [117]. The long expression has been related to the stability of the expression cassette, which was isolated intact 1 year post injection [118]. 3. Application of gene therapy in ocular diseases As mentioned above, gene therapy for the treatment of ocular diseases has several advantages, since the eye is very accessible, and many retinal degenerative syndromes are well characterized from a genetic point of view. Actually, many studies in animals have demonstrated the potential utility of the gene therapy for the treatment of ocular diseases. As a result, the clinical translation has started. 3.1.Glaucoma Glaucoma is the first cause of unamendable visual disability and blindness worldwide. One of 40 adults over 40 years of age suffers from glaucoma with visual loss [119]. Estimating a prevalence of 2.65% in the population over 40, the overall number of glaucomatous subjects is expected to increase in the course of the present decade, owing both to demographic expansion and population ageing, from about 60 million in 2010 to nearly 80 million in 2020 [119]. IOP management, which is currently the main treatment for glaucoma, does not address the neuronal damage that causes blindness in glaucoma patients [120]. Therefore, there is a strong demand to explore new treatment strategies that could potentially restore vision loss in glaucoma patients. Transmission of the disease occurs mostly in monogenic form in juvenile-onset open-angle glaucoma and in complex form in adults. It has been reported that 72% of primary open-angle glaucoma cases have an inherited component [119]. Gene therapy could offer improvement in the treatment of glaucoma compared to the current standard of lowering IOP. SYL040012 is a double-stranded oligonucleotide that specifically inhibits the synthesis of the ß2-adrenergic-receptor via RNAi, and it does 17

not affect the expression of other adrenergic receptors. This oligonucleotide decreases IOP by reducing local expression of the ß2-adrenergic-receptor after topical instillation in animal models [121]. In a recent study [122], SYL040012 was topically administered to healthy volunteers. It was well tolerated without side effects neither systemic nor locally, and the oligonucleotide was not detected in plasma at any time. After administering SYL040012 to 24 subjects over a period of 7 days, the IOP decreased in 15 individuals, regardless of the dose. With one of the dose assayed, the reduction in the IOP was statistically significant, and in those subjects with greater baseline IOP, the response to SYL040012 seemed to be higher. This clinical trial was the first one in which siRNA was applied in humans by topical administration to the eye. 3.2.X-linked retinoschisis (XLRS) XLRS, which results from mutations in the gene encoding the secreted protein retinoschisin, is a retinal degenerative disease affecting between 1/5,000 and 1/25,000 people worldwide [123]. The defining characteristics of XLRS include the formation of cystic cavities in the inner and outer retina and deterioration in vision caused by retinal disorganization [53]. This disease leads to mild to severe loss in central vision, radial arising from foveal schisis, splitting of inner retinal layers in the peripheral retina, and a negative electroretrinogram with a significant decrease in b-wave amplitude [124]. The progression and severity of XLRS is very variable even within the same family and secondary complications such as detachment of the retina and hemorrhages in the vitreous can happen. Women, that are carriers, are asymptomatic, but after clinical examination in detail, minor retinal alterations can be detected [125]. Currently, there is no cure for the schisis formation, and the treatment is focused to preserve the low vision.

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As the underlying cause of this recessive monogenic disease is well understood, it is an excellent candidate for gene augmentation therapy. Retinoschisin is a secreted protein, and not only the transfected cells benefit from the replacement of the gene, since once secreted the protein spreads from the site of expression. Previous studies have shown that delivery of a normal copy of the RS1 gene using a variety of AAV vectors and routes of vector administration targeting a variety of cell types, can ameliorate degeneration [50-52]. However, studies in humans are lacking. All these studies in animal models emphasize the potential for gene therapy in XLRS, highlighting the importance of careful design and optimization for specific, minimally invasive and long-lasting gene therapy. The development of non-viral vectors is a promising alternative to viral vectors for attempting the treatment of XLRS with gene therapy. Our research group is working on the design of a vector based on SLNs containing the RS1 plasmid. An in vivo study consisting in the administration of the vector containing the RS1 plasmid to knockout mice by both intravitreal and subretinal routes is currently undergoing. Preliminary results show the expression of retinoschisin in several cell types in the retina, including photoreceptors. No other paper in literature shows the development of a non-viral vector as a potential tool for the treatment of XLRS. 3.3.Stargardt disease Stargardt disease is the most frequent inherited juvenile macular degeneration. The diagnostic of this disease usually happens before the patient is twenty years old. Characteristic features include the loss of the central vision, being progressive and irreversible, and problems to adapt to the darkness. This disease has a pattern of autosomal recessive inheritance [68]. The gene involved in Stargardt disease is named ABCA4, which encodes for a protein of the ABC transporter family [126]. ABCA4 19

protein is expressed exclusively in the retinal and cone photoreceptors, where it is located to the rim and incisures of photoreceptors outer disks, present at a molar ratio of around 1:120 with rodhopsin [68]. The impairment and loss of vision in Stargardt patients can be due to hundreds of mutations in the ABCA4 gene. The mutations in this gene are also responsible for other visual diseases such as cone-rod dystrophy and autosomal recessive retinitis pigmentosa [127, 128]. Heterozygous mutations in ABDA4 may lead to the development of age-related macular degeneration [129]. At present, there is no cure for ABCA4-associated disease Gene therapy is a logical strategy for Stargardt disease, although the large size of the ABCA4 cDNA prevents its formulation as a traditional AAV (maximum capacity approximately 4,7 kb) [130]. In this sense, lentiviral vectors are an interesting alternative. In a previous study, equine infectious anemia virus (EIAV)-based lentiviral gene therapy lead in a mouse model of ABCA4-associated diseases to a substantial modulation of the disease phenotype [67]. No obvious side effects were detected in the treated mice. The EIAV vector also demonstrated to be able to induce the expression of the gene in both cone and rod photoreceptors in addition to the retinal pigment epithelium with high efficacy in adult macaques after subretinal injection [68]. The biodistribution, shedding and toxicity of the vector was analyzed over a period of six months after the subretinal administration to macaques and rabbits, and the study showed that the vector was well-tolerated and safe. Moreover, the vector was shown to be located in the application site. These preclinical data supported for the first clinical trial in humans in patients with Stargardt disease

that

is

currently

underway

(StarGen,

Clinical-Trials.gov

number,

NCT01367444). Large genes can be incorporated in non-viral DNA nanoparticles, contrary to viral victors, which have a limited capacity to accommodate large genes. In a recent study 20

[131], optimized DNA nanoparticle technology was used to inject ABCA4 to ABCA4deficient mice by subretinal route. After administration, the expression of the transgene was detected for up to 8 months, and a significant correction of functional and structural Stargardt phenotypes was observed, including improvement of recovery of adaptation to darkness and decrease of lipofucsin granules (in Stargardt disease patients, accumulation of lipofucsin granules in the RPE occurs). 3.4.Choroideremia Choroideremia is an X-linked chorioretinal dystrophy, primarily affecting male subjects, with an estimated prevalence of 1 in 50,000. It is characterized by progressive atrophy of the choroid, RPE, and photoreceptors [132]. CHM is the gene associated with this disease; it encodes a ubiquitously expressed protein named Rab escort protein (REP-1) that enables post-translational isoprenyl modification of Rab proteins, which control vesicle formation, movement, docking, and fusion. Mutation in the CHM gene cause defects in intracellular membrane traffic pathways, including melanosome movement and phagosome processing [133]. In patients with this ocular disease, visual acuity is generally good until the affectation of the fovea; although in childhood underlying alterations in the retina can be detected, and are associated with clinically marked reduction in parafoveal retinal sensitivity as measured with psychophysical testing [134]. Accordingly, before the onset of loss in visual acuity, retinal sensitivity may be an effective marker of the functional effects of gene therapy in patients with choroideremia. Previous studies carried out in humans [135] indicate that in the later stage of the disease, the loss of visual acuity may have a reversible component. The successful of gene therapy should not be measured only in terms of improvement of visual function, but also on finishing or slowing the degeneration rate. In a multicenter clinical trial [136], six male patients with choroideremia were treated with a vector 21

(AAV.REP1) by subfoveal injection. In all individuals, the increment in retinal sensitivity was correlated with the vector dose administered. The results obtained in this clinical trial are consistent with improve cone and rod function that conquer the negative effects of retinal detachment. These findings support further investigation about the application of gene therapy for the treatment of choroideremia and other ocular diseases, for which therapy should ideally be administered before the onset of retinal thinning. 3.5.Retinitis pigmentosa Retinitis pigmentosa is the most common subtype of retinal degeneration, responsible for loss of vision in one in 4,000 people worldwide, one in 1,000 in the People’s Republic of China, and one of 930 in India [137]. Retinitis pigmentosa can result from defects in any of more than 60 genes inherited as autosomal dominant (30%-40% of cases), autosomal recessive (50%-60%), or X-linked (5%-15%), and it can occur either alone or together with other systemic disorders. Notwithstanding, mutation in 30%-35% of patients cannot be identified [137]. The disease onset and progression may vary significantly among patients, even within the same family. The patients frequently experience night blindness in the early phase of the disease, followed by loss of vision starting in the mid periphery and progressing towards the center which results in tunnel vision [138]. On the cellular level, these phenotypes correlate with a mainly affected rod photoreceptor system. In a later phase, cone photoreceptors may be further affected causing ultimately complete blindness. The affected photoreceptors undergo apoptosis, which leads to a reduction in the outer nuclear layer thickness within the retina, and lesions and/or retinal pigment deposits in the fundus. Before the loss of visual acuity, the patients might lose a marked portion of the photoreceptors [139].

22

Gene therapy for retinitis pigmentosa is being evaluated in animal models, and the results provide expectations for future clinical application. The aim of gene therapy is to slow down or stop the progress of retinal degeneration in retinitis pigmentosa. However, translation to clinic needs a deeper knowledge of the mutations and mechanisms that provoke visual defects, and a better evaluation of side effects as well. AAV is the most widely used vector for ocular gene delivery in general, and for retinitis pigmentosa in particular. The reason is its ability to transducer various retinal cell types in vivo efficiently, due to its small size relative to other viral vectors [137]. One example of gene therapy in animal models that mimics the human autosomal recessive retinitis pigmentosa is directed at mutations in the genes encoding the two rod cyclic nucleotide-gated (CNG) channel subunits. Knockout of CNGB1 in mice results in a phenotype that recapitulates the principal pathology of retinitis pigmentosa patients. In a recent study [140], CNBG1-deficient (CNGB1-/-) mice were used to evaluate AAVmediated gene therapy as a potential treatment of retinitis pigmentosa caused by rod photoreceptor-specific gene mutations. The therapy was able to restore the normal expression of rod CNG channels and rod-driven light responses in the mice retina. Consequently, a marked delay of retinal degeneration and long-term preservation of the morphology of the retina was induced. Additionally, treated CNGB1-/- mice behaved markedly better than untreated animals in a rod-dependent vision-guided behavior assay. Other animal model of retinitis pigmentosa is a dog deficient in a GTPase regulator interacting protein 1 (RPGRIP1) [141]. This animal model exhibits a severe cone-rod dystrophy similar to that observed in humans. The subretinal administration of AAV vectors encoding the canine Rpgrip ameliorated photoreceptor survival in transduced zones of the treated retinas. The function of cones was markedly and stably improved in all the treated eyes (18-72% of those recorded in normal eyes) up to 24 23

months post-injection. Rod function was improved as well (22-29% of baseline function) in four of the treated dogs up to 24 months post-injection. In untreated contralateral eyes, cone function was not detected. More importantly, the treatment maintained bright- and dim-light vision. The good results obtained in this large animal model of cone-rod dystrophy raise expectations for human applications. Rhodopsin gene (RHO), which encodes the photosensitive pigment in rod photoreceptors, is another gene whose mutations (>100) have been identified in individuals with retinitis pigmentosa [137]. Suppression and replacement for RHOlinked retinitis pigmentosa has been attempted with the therapeutic strategy named "mutation-independent suppression and replacement", which comprises both gene suppression and gene replacement. The suppression in a site independent of the mutation leads to the suppression of both mutant and wild-type alleles [142]. Moreover, a codon-modified replacement gene refractory to suppression is provisioned. In a previous study [143], both in vitro and in vivo validation of suppression and replacement for RHO-linked retinitis pigmentosa was undertaken. In vivo, approximately 90% suppression of RHO in photoreceptors was obtained by using RNAi with AAV for delivery. Expression of RHO-replacement genes was also demonstrated. Evidence of therapeutic benefit from AAV-delivered siRNA suppression and replacement therapy was obtained in transgenic P23H mice. 3.6.Age-related macular degeneration Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in people aged 50 years or older in the developed world. More than 9 million Americans have AMD, and cases are expected to nearly double by 2050 to 17.8 million [144]. Macular degeneration affects central vision and distance visual acuity, near visual acuity, color discrimination, contrast sensitivity, and other sense functions. It affects the 24

ability to read, to recognize people’s faces, to choose clothes, to view pictures, and to play or view sports [145]. Depending on the histopathological characteristics, AMD can be classified into several categories: early, intermediate and, advanced AMD [146]. Many patients with intermediate AMD eventually progress to advanced forms. Advanced AMD includes geographical atrophy and neovascular AMD. Early and intermediate AMD, as well as geographic atrophy, are generally referred to dry AMD. Only minimal visual acuity impairment occurs in early and intermediate AMD; however, advanced AMD is the leading cause of blindness worldwide. In some patients, the two forms of advanced AMD may co-exist in the same eye, probably because both forms have common risk factors. The risk for AMD involves many factors, but strong genetic contribution is well accepted. The AMD Gene Consortium recently published the most complete analysis of AMD risk, in which more than 17,000 cases and more than 60,000 controls were included [147]. This study has provided very useful information, including identifying genes and processes underlying AMD risk; however, the knowledge of the genetic drivers of AMD progression is still insufficient. Currently, there is no cure for AMD; however, recent advances in clinical research have led to a better understanding of its pathophysiology, which, in turn, has led to new treatment strategies and drug therapies. Improved treatments of AMD under study include

human

retinal

transplantation,

artificial

vision,

retinal

prosthesis,

neuroprotection, and gene therapy [145]. In a phase I clinical trial involving 28 patients with advanced neovascular AMD, Ad vector-mediated intravitreal gene transfer of pigment-epithelium-derived factor (PEDF), an antiangiogenic cytokine, appears to help arrest the growth of neovascular AMD [37]. Gene therapy for AMD can also be attempted with RNAi. PF-04523655 is a siRNA to inhibit the expression of the hypoxia-inducible gene, RTP801; it is being developed for 25

the treatment of choroidal neovascularization secondary to AMD [148]. Expression of the RTP801 gene is upregulated in response to hypoxia and/or oxidative stress, which leads to the induction of neuronal cell apoptosis. In a multicenter phase I clinical trial, PF-04523655 was administered as a single intravitreal injection at doses ranging from 50 to 3000 µg. It was well tolerated without any significant safety issue. However, the limited sample size was not sufficient to statistically evaluate efficacy [149]. 3.7.Leber congenital amaurosis (LCA) LCA is an autosomal recessive disease resulting from mutation in at least 15 genes [150]. Patients with this severe retinal disease suffer from a marked impairment of the visual acuity at birth or during the first six months of life, sensory poorly reactive pupils and severely diminished or non-detectable electroretinogram activity [151]. Prevalence of LCA is 1 in 35,000 newborn of all blind children [151]. Up to know, there is no any successful treatment for LCA, and gene therapy is being widely investigated. The most common mutation that provokes LCA, which affects to at least 10% of patients with LCA from Northern European and North-American descent, is an intronic mutation in CEP290 that results in the inclusion of an aberrant exon in the CEP290 mRNA [152]. Autosomal recessive CEP290-associate LCA is a good candidate for gene replacement therapy. In a recent study [153], a lentiviral vector containing CMV-driven human full-length CEP209 has been developed. In cultures of photoreceptor precursor cells derived from LCA patients, lentiviral delivery of CEP290 rescued the ciliogenesis defect (CEP290 is involved in ciliogenesis). In other study using antisense oligonucleotides (AONs) immortalized lymphoblastoid cells of patients with LCA homozygously carrying the intronic CEP290 mutation were transfected with several AONs that target the aberrant exon that is incorporated in the mutant CEP290 mRNA.

26

RNA isolation and reverse transcription-PCR analysis showed that that a number of AONs were able to almost fully redirecting normal CEP290 splicing [152]. Mutations in the RPE65 gene may also provoke LCA. This gene encodes for all-transretinyl-ester hydrolase, a 65 KDa enzyme that in retinal pigment epithelium is critical for the production of 11-cis-retinal. This compound is transported to the photoreceptors where it binds to apo-rhodopsin; the apo-rhodopsin-11-cis retinal complex reacts with a photon to produce a change in membrane potential, which generates a nerve signal that travels to the visual cortex for image formation and recognition. Deficiency of all-transretinyl-hydrolase due to mutation in RPE65 gene happens in about 6% of LCA cases in humans [151]. Canine, porcine and rodent models have been used to show that AAV vectors can deliver functional all-trans-retinyl-ester hydrolase with the goal of preventing retinal degeneration and resolving visual function. In these three animal models, the administration of the vector led to improved visual function and remarkable functional rescue as well [150]. Gene therapy for human RPE65-LCA has been assayed in four independent clinical trials [154-157], and it has resulted in marked improvement of the visual function within days to weeks after treatment, which is maintained for as long as 3 years [15, 158]. The good results obtained in these clinical trials demonstrate the potential utility of gene therapy to improve the severe and lifelong visual impairment in the patients with RPE65-LCA. Although gene therapy provokes marked and durable vision improvement, it does not slow the photoreceptor degeneration. 3.8.Gene therapy for diseases in the cornea The cornea is the transparent avascular tissue at the most anterior surface of the primate eye. It is mainly composed of an external stratified epithelium, an endothelium made up of a cuboidal monolayer of epithelial-like cells, and a thick collagenous stroma 27

separating the two. The cornea is an ideal target for gene therapy. It is easily accessible, usually transparent, and the general circulation and the systemic immune system are limited. By using an appropriate vector system, it is possible to deliver a gene in the cornea either locally or systematically, with the subsequent expression of a transgenic protein [64]. Different proteins may be expressed, structural such a collagen, or functionally active such as cytokines, enzymes or growth factors, which may modulate congenital or acquired diseases. Contrary to gene therapy for the treatment of disorders of the retina due to monogenic inherited diseases, gene therapy in the cornea has been extensively studied in animal models but clinical trials in humans are scarce. Corneal diseases that might potentially be amenable to gene therapy include the monogenic lysosomal storage disorders (LSDs) that affect the cornea such as mucopolysaccharidosis (MPS) type IV (Maroteaux-Lamy syndrome) and MPS type VII (Sly syndrome), corneal scarring, corneal neovascularization, some anterior and stromal dystrophies that are linked to genetic mutations, corneal graft rejection, and the maintenance of corneal endothelial cell density in the eye-banked corneas [64]. Serratrice et al. [159] have developed a novel gene transfer platform based on helperdependent canine adenovirus type 2 (HD-CAV-2) vectors for corneal transfection. The authors compared transduction efficacy of this vector in vivo in healthy mouse and nonhuman primate corneas, and ex vivo in healthy human and canine corneal explants by intrastromal injection. Additionally, they tested the potential of the system for corneal therapy in MPS VII dog cornea. Vectors efficiently transduced keratocytes throughout the stroma via a single injection. Moreover, HD-CAV-2 vector harboring a human βglucuronidase expression cassette corrected the pathology of the canine MPS VII cornea explants. Yet, this correction will be dependent on β-glucuronidase diffusion in the thick collagenous stroma. These encouraging data support the continued evaluation of 28

HD-CAV-2 vectors to treat corneal disease associated with MPS VII and possibly other LSDs. Gene therapy has a potential for the treatment of infections of herpes simplex type 1 (HSV-1). In developed countries, infections due to HSV-1 are responsible for most cases of corneal blindness and rejection of corneal grafts [160]. The rates of HVS-1 range from 50% to 90% worldwide [161]. Gene transfer to the cornea applied locally is rather preferred to avoid systemic effects, or ex vivo before transplantation to improve the graft survival in those patients with herpetic keratitis history. Different approaches have been applied to target certain components of the viral envelope, protein, replication process, or the resultant inflammation [160], being this later the most studied. Several studies have explored the capacity of gene therapy to deliver specific anti-inflammatory mediators, including IL-10, IL-2, IL-4, interferon (IFN)-β, IFN-α1, and matrix metalloproteinase, among others [160]. The pretreatment of human corneas in eye banks with meganuclease-encoding vectors would provide herpetic keratitis patients who have received medicated corneas with higher resistance to the infection and common graft rejection. Using recombinant AAV2/1 vectors, Elbadawy et al. [162] have shown meganuclease expression in endothelial cells, and decrease of the total viral load in the media and in the endothelial cells of corneas infected with HSV-1. Corneal transplantation is one of the most commonly performed organ transplantation. Although corneal allografts enjoy the privilege of being among the most successful solid organ transplants, their two-year graft survival rate of over 90% in ‘‘low-risk’’ avascular host beds significantly decreases over time [163]. Nevertheless, immunologic rejection remains a leading cause of graft failure. The prevalence of graft rejection varies from 5% to 40%, depending upon vascularization of the recipient cornea and prior episodes of graft failure, with rejection rates approaching 70% in vascularized 29

“high-risk” beds, even with maximal local and systemic immune suppression [163]. Gene therapy is a promising approach to promote graft survival and prevent allogeneic graft rejection. Orthotopic corneal transplantation presents an unique platform providing ease of vector delivery to both the donor graft ex vivo, and the recipient bed in vivo, prior to transplantation. In ex vivo gene therapy, several advantages include the accessibility of the corneal endothelium, a monolayer of somatic cells, the greater inherent safety of ex vivo gene transfer compared with systemic or direct loco-regional transfer, the blood-eye barrier that limits any systemic spillover of the vector, the ease with which a corneal graft can be visualized and replaced at need and the continuous production of the transgenic protein following a “one-shot” therapy [64]. Gene therapy in corneal graft survival can be focused to decrease neovascularization in the recipient corneal bed, for instance by using RNAi to silence genes involved in vascularization. Tang et al. [164] demonstrated in a BALB/c mouse model of suture-induced corneal neovascularization that knockdown of neuropilin-2 with RNAi improves corneal graft survival by selectively inhibiting lymphangiogenesis in vascularized beds before transplantation. Other strategies are the modulation of the immune response through the expression of pro-inflammatory cytokines that mediate graft rejection, or the inhibition of apoptotic pathways [163]. Ex vivo gene therapy of the donor cornea has been demonstrated to prolong corneal allograft survival in various animal models. Ad vectormediated expression of ovine IL-10 or the IL-12p40 subunit has shown to prolong ovine corneal allograft survival significantly, but does not produce indefinite graft survival in every recipient [165]. Although lentiviral vectors show some promise for corneal gene therapy, a recent study has revealed that they are less efficient than Ad vectors [166]. 4. Gene therapy clinical trials for ocular disease

30

The eye has been at the forefront of translational gene therapy largely due to appropriate disease targets and its suitable anatomic features. These advantages have fostered research that has culminated in various clinical trials of gene therapy for ocular diseases. The registered clinical trials from the database of Gene Therapy Clinical Trials Worldwide, provided online for the Journal of Gene Medical Clinical Trial [22], restricted to “ocular diseases” and shorted by indication are summarized in Table 1. Currently 33 clinical trials are reported to have been approved, to be in progress, or have been completed. As it can be seen in the table, viral vectors are the most used gene delivery systems for ocular diseases. In fact, 22 from de 33 ongoing clinical trials use viruses as vectors, being the AAVs the most used, specifically in 16 trials. Gene silencing through siRNA delivery has been employed only in 5 of the 33 studies, confirming the gene replacement therapy as the most widely used in clinical approach.

Table 1. Clinical trials of gene therapy for ocular diseases. Indication AMD

Phase

Gene

Vector

Administration

Trial

Date

Status

I/II

NA

AAV

NA

Australia

2006

Open

I/II

sFLT01

AAV

Subretinal

Australia

2011

Open

I

PDGF

Adenovirus

Intraocular

USA

2001

Open

I

Endostatin Angiostatin

Lentivirus

Subretinal

USA

2010

Open

I

siRNA (Bevasiranib/Cand5)

-

Intraocular

USA

2004

Closed

I

siRNA (Sirna-27)

-

Intraocular

USA

2005

Closed

II

siRNA (Sirna-27)

-

Intraocular

USA

2006

Open

31

WET AMD

II

siRNA (Bevasiranib/Cand5)

-

Intraocular

USA

2005

Closed

Diabetic Macular Edema

II

siRNA (Bevasiranib/Cand5)

-

Intraocular

USA

2006

Open

MD

II

CNTF

Naked/Plasmid DNA

Intraocular

USA

2005

Open

I

sFLT01

AAV

Intraocular

USA

2008

Open

SMD

I/II

ABCR

Lentivirus

Subretinal

USA

2010

Open

Macular Telangiectasia Type 2

II

CNTF

Naked/Plasmid DNA

Intraocular

USA

2013

Open

Choroideremia

I

REP1

AAV

NA

Canada

2014

CA

I

REP1

AAV

Subretinal

Canada

2014

Open

I

REP1

AAV

NA

UK

2009

Open

I

REP1

AAV

NA

UK

2011

Open

I/II

ND4

AAV

Intravitreal

China

2011

Open

I/II

ND4

AAV

Intraocular

France

2013

Open

I/II

myo7a

Lentivirus

Intraocular

France

2013

Open

I/II

myo7a

Lentivirus

Subretinal

USA

2011

Open

LCA

I

hRPE65

AAV

Subretinal

Israel

2009

Open

MERTK Mutations

I

VMD-2 MERTK

AAV

Subretinal

Saudi Arabia

2011

Open

Superficial Corneal Opacity/Corneal Scarring

I/II

dnG1 Cyclin

Retrovirus

Instillation

USA

2002

Open

Retinitis pigmentosa

I

CNTF

Naked/Plasmid DNA

Intraocular

USA

2003

Open

II

CNTF

Naked/Plasmid DNA

Intraocular

USA

2006

Open

II

CNTF

Naked/Plasmid DNA

Intraocular

USA

2006

Open

p21

Adenovirus

Intraocular

USA

2003

Open

LHON

RP Usher1B

Glaucoma

I

WAF-1/Cip1 RPE65 Mutations

RPE65 Mutations. LCA Type 2

I

RPE65

AAV

Subretinal

USA

2004

Open

I

RPE65

AAV

Subretinal

USA

2005

Open

I

RPE65

AAV

Subretinal

USA

2008

Open

III

RPE65

AAV

Subretinal

USA

2009

Open

III

RPE65

AAV

Subretinal

USA

2010

Open

Abbreviations: NA: Not Available; CA: Conditional Approval; AAV: Adeno-associated virus; AMD: Age-related Macular Degeneration; MD: Macular Degeneration; REP1: Rab Escort Protein 1 LHON: Leber Hereditary Optic Neuropathy; RP Usher1B: Retinitis Pigmentosa Associated with Usher Syndrome Type 1B; LCA: Lebers Congenital

32

Amaurosis; PDGF: Platelet-derived growth factor; CNTF: Ciliary neurotrophic factor; SMD: Stargardt´s Macular Degeneration; ABCR: Retina-Specific ABC Transporter; ; siRNA: short interfering RNA

5. Conclusions Ocular gene therapy is a hopeful approach to treat, cure, or prevent diseases by changing the gene expression in the eyes. Gene therapy is still starting, and current therapies are primarily experimental, with most human clinical trials still in the research states, although beginning to show encouraging results. A greater understanding of the mutations and mechanisms that cause visual defects along with the development of more efficient clinical grade vectors will be crucial to the development of effective treatments for the future.

Acknowledgements This work was supported by the Basque Government’s Department of Education, Universities and Investigation (IT-341-10) and by the Spanish Ministry of Economy and Competitiveness (SAF2010-19862). We thank the University of Basque country (UPVEHU) for the grant awarded to Paola S. Apaolaza. References [1] B. Qin, R.S. Shukla, K. Cheng, Delivery of nucleic acids for ocular gene modulation, in: A.K. Mitra (Ed.), Advances in Ocular Drug Delivery, Research Signpost Kerala, India, 2012, pp. 87-114. [2] P. Colella, G. Cotugno, A. Auricchio, Ocular gene therapy: current progress and future prospects, Trends Mol. Med. 15 (2009) 23-31. [3] C. Bloquel, J.L. Bourges, E. Touchard, M. Berdugo, D. BenEzra, F. Behar-Cohen, Non-viral ocular gene therapy: Potential ocular therapeutic avenues, Adv. Drug Deliver. Rev. 58 (2006) 1224–1242. [4] A. Urtti, Challenges and obstacles of ocular pharmacokinetics and drug delivery, Adv Drug Deliver. Rev. 58 (2006) 1131–1135. 33

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Graphical abstract

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Treatment of ocular disorders by gene therapy.

Gene therapy to treat ocular disorders is still starting, and current therapies are primarily experimental, with most human clinical trials still in r...
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