Ординатура / Офтальмология / Английские материалы / Retinal and Vitreoretinal Diseases and Surgery_Boyd, Cortez, Sabates_2010

Abstract

Both gene replacement therapy and alteration of host gene expression are playing increasingly important roles in the treatment of ocular diseases. Ocular gene therapy may provide alternatives to current treatments for eye diseases that are either greatly invasive and thus run the risk of complications, that offer only short-term relief from disease symptoms, or that are unable to directly treat vision loss. The recent success of three separate Phase I clinical trials investigating a gene therapy intervention for the treatment of the retinal degenerative disorder Leber’s congenital amaurosis (LCA) have unveiled the therapeutic

potential of gene therapy. Preliminary results have demonstrated ocular gene transfer, using non-pathogenic recombinant adeno-associated viral (rAAV) vectors specifically, to be a safe, effective, and long-term treatment for LCA, a previously untreatable disorder. Nonpathogenic rAAV vectors offer the potential for long-term treatment. Many of the genes implicated in human ocular diseases have been identified, and animal models for such diseases have been developed, which has greatly facilitated the application of experimental rAAV-mediated gene therapy. This review highlights the key features of rAAVmediated gene therapy that make it the most suitable gene therapy treatment approach for ocular diseases. Furthermore, it summarizes

the current progress of rAAV-mediated gene therapy interventions/applications for a wide variety of ophthalmologic disorders.

Ocular Gene Therapy:

An Introduction

Visual function is directly dependent on the health of the numerous tissues that comprise the eye, and these tissues are often implicated in a variety of ocular diseases. Disease can affect external, anterior structures of the eye, including the cornea and lens, as well as the innermost regions of the retina and the optic nerve. Many of the inherited dystrophies and acquired disorders that affect ocular structures have been demonstrated to be amenable to rAAV-mediated gene therapy in pre-clinical models. Some examples of diseases treatable with rAAV-mediated gene therapy include neovascularization of the retina, choroid, and cornea, as well as the maternally inherited disorder primarily affecting the optic nerve, known as Leber’s hereditary optic neuropathy. The inherent structural features of the eye also make it well-suited as a target organ for rAAVmediated gene therapy. Both the small size and compartmentalized structure of the eye reduce the rAAV vector-encoded gene necessary to obtain a substantial therapeutic effect, and confine rAAV vector delivery to only the affected area of the eye. Additionally, the accessibility of the eye facilitates multiple administration routes for therapeutic rAAV vectors and allows diverse types of ocular cells to be transduced.1

The development of rAAV vectors has made it possible for gene therapy to overcome the challenges of efficiency and prolonged gene transduction; rAAV vectors have been shown to sustain gene transduction for up to year in a wide array of cell types. Also, rAAV vectors are particularly well-suited for gene therapy interventions of the eye as they are the only viral vectors able to efficiently transduce both photoreceptor and RPE cells and can also transfect Müller and retinal ganglion cells.2,3 In addition, rAAV vectors can target specific ocular cell types, which both increases transduction efficiency as well as decreases the chances for targeting cells unaffected by disease or damage. These characteristics of rAAV vectors further demonstrate the potential that rAAV-mediated gene therapy holds for the treatment of ocular diseases.

A major limitation to rAAV-mediated gene therapy is the small packaging capacity of the rAAV vector,3 with the transgene to be packaged often exceeding the vector’s 5kb capacity. However, the recent development of hybrid rAAV vectors has expanded the genomic size limitation to fit transgenes up to 9kb. This will be discussed in the context of various specific rAAV vector and ocular disease applications. Additionally, the slow onset of transgene expression following rAAV-mediated transduction constitutes another limitation of this gene therapy vector. The challenge presented by slow onset of transgene expression is most pronounced in treatment of early onset disorders, such as rapidly degenerating retinal dystrophies.

Despite such limitations, rAAV-mediated gene therapy offers a multitude of treatment options for ocular diseases. The recent discovery of novel rAAV vector serotypes has given way to the development of the aforementioned hybrid vectors. The novel rAAV serotypes of hybrid vectors modulate the cellular tropism of the vector, increasing both its specificity and efficacy. rAAV-mediated gene therapy in the eye also has numerous administration routes; both non-invasive and invasive delivery methods can be used depending on the ocular tissue target.

The prospects for rAAV-mediated gene therapy to treat various ocular diseases continue to be studied in numerous in vitro, in vivo, and ex vivo experimental models. Ocular disease models have been created in vivo in rats, monkeys, mice, and dogs, among others. Efficient rAAV-mediated gene transduction of corneal cells has been demonstrated in vivo in animal models, as well as in ex vivo organ cultures. The extensive pre-clinical studies of rAAV-mediated gene therapy applications for ocular diseases have culminated in the first successful ocular gene therapy clinical trial. Three different Phase I clinical trials were conducted to investigate the therapeutic potential of rAAV-mediated gene therapy in Leber’s congenital amaurosis (LCA). Thus far, results from all three clinical trials have proven quite promising, as some of the patients enrolled in the study are experiencing marked improvements in their vision. As data continues to be collected from the LCA clinical trials, advancements in pre-clinical

Ocular Gene Therapy

665

studies of rAAV-mediated gene therapy for other ocular diseases persist. Although ocular gene therapy with rAAV vectors is only in its initial stages, the future looks bright for the use of rAAV-mediated gene therapy as a long-term therapeutic for the treatment of ophthalmologic disorders.

rAAV-Mediated Ocular Gene

Transfer

Ocular function is entirely dependent upon many internal specialized structures, which capture an image in the form of visible light and transduce the light into neural signals. Specifically, the photoreceptor cells of the retina transduce photons into electrical impulses that are transmitted to the brain via the optic nerve. A variety of ocular tissues that contribute to visual function may be involved in inherited and acquired diseases, previously untreatable, for which ocular rAAVmediated gene therapy approaches are being developed.

The rAAV vector is a non-pathogenic, humanparvoviruswithalinear,single-stranded DNA genome. The vector construct consists of two reading frames, rep and cap, enclosed between two symmetric T-shaped palindromic terminal sequences called inverted terminal repeats (ITRs). To generate the non-replicating rAAV vector, the rep and cap reading frames are deleted from the genome and the therapeutic gene is inserted between the ITRs.4

666

The rAAV vector does present some limitations, given its small packaging capacity of 5kb and a slow onset of transgene expression following transduction.3 These limitations, however, are far outweighed by the efficacy of rAAV vector-mediated gene therapy as a treatment for ocular diseases. Low immunogenicity is among the many optimal features of the rAAV vector that allow it to sustain transgene expression for up to a year and induce little to no ocular inflammation(unlikeadenoviralvectors);present minimal risk of insertional oncogenesis (unlike retroviral vectors); and transduce a wide array of ocular cell types (unlike lentiviral vectors). rAAV vectors are the only viral vectors able to efficiently transduce both photoreceptor and retinal pigment epithelial cells, and they can also transfect Müller and retinal ganglion cells.2,3

In addition, structural features of the eye make it well-suited as a target organ for rAAV-mediated gene therapy. The small size of the mammalian eye reduces the amount of vector and/or gene needed to observe a therapeutic effect, and its highly compartmentalized structure facilitates accurate delivery of the therapeutic vector. Also the blood-retina barrier of the eye constrains the rAAV vector to the site of delivery, which prevents any systemic leakage of the vector. In addition the transparency of the external layers of the eye facilitates noninvasive imaging of ocular tissues in vivo.5,1

Specificity of rAAV-Mediated

Ocular Gene Therapy

The tropism rAAV vectors demonstrate towards specific ocular tissues is derived from the rAAV serotype of the vector as well as the site of vector and/or gene delivery within the highly compartmentalized anatomy of the eye.6 The rAAV serotype dictates the expression kinetics of a given rAAV vector; a variety of rAAV vectors are available because of the numerous rAAV serotypes that exist. rAAV serotypes are differentiated based on the structural capsid protein sequences encoded within the cap gene of the rAAV vector construct. Numerous hybrid vectors containing novel rAAV serotypes have been developed to modulate cellular tropism of an rAAV vector to increase its efficacy. The pseudo typing strategy used to design hybrid vectors was first developed with the rAAV2 serotype, the most abundant serotype in the human population, which has long been used in designing rAAV vectors and has been studied extensively.5 To generate hybrid vectors using the AAV2 serotype, an expression cassette containing the AAV2 genome (including the therapeutic gene bordered by the ITRs on either side) was packaged into the capsid protein of another serotype. The resulting hybrid vectors have demonstrated varied kinetics of transgene expression and improved tropism for a broad range of cell types because of their novel serotypes. The rapid-onset rAAV-2/1, -2/5, and -5/5 vectors

can demonstrate transgene expression 3 to 4 days after vector delivery. The transgene expression for rAAV-2/2 vector has a delayed onset, reaching a stable level of expression 2 to 4 months after vector delivery.6 Hybrid vectors have also been shown to transduce

Ocular Gene Therapy

667

a wide array of ocular tissues, including retinal pigment epithelial cells; corneal cells; photoreceptors cells; the bipolar, amacrine, and Müller cells of the inner retina; retinal ganglions cells; and cells of the anterior ocular structures (Figure 1; Table 1).

Routes of Administration

The route through which rAAV vector will be delivered depends on which of the numerous ocular cell types it will transduce. The various administration routes described here are illustrated below in Figure 2. When rAAV vectors are used to transduce photoreceptors and retinal pigment epithelial cells, which are predominantly implicated in inherited retinal degenerative disorders, the vectors are administered via subretinal injection. Since the vector suspension is delivered between the retina and the retinal pigment epithelium, subretinal injections cause temporary separation of the retinal layers.6 Figure 3 shows a subretinal injection of rAAV2-CB-hRPE65 in a LCA clinical trial patient. When rAAV-mediated gene therapy is used to transduce retinal ganglion cells and other cells of the inner retina that are com-

Ocular Gene Therapy

669

monly implicated in optic neuropathies such as optic neuritis and glaucoma, the vector is injected intravitreously.4 Intravitreal injection has been shown to reach the photoreceptor cells of the outer retina and could therefore provide a less invasive delivery alternative to subretinal injection. Furthermore, intravitreal injection allows for a larger vector dosage to be delivered; repeated injections within the vitreous body; and vector delivery in conjunction with a pharmacological agent.9 Topical application is a common route of administration for therapeutic vectors used to treat diseases involving the conjunctival and corneal epithelia.10,11 Periocular injection is the route of administration for vectors treating neovascular and corneal diseases.12,1 Delivery of rAAV vectors by anterior chamber injection has been used in vivo to transduce rabbit corneal endothelium as a model for disorders of the corneal endothelium.11

Efficiency of rAAV-mediated

Ocular Gene Therapy

For target organs other than the eye, transient gene transfer is a disadvantage of rAAV-mediated gene therapy in dividing cells. However, this drawback is largely overcome when the target organ is the eye, since it is composed of an array of primarily nondividing cells. As a non-integrating vector, the rAAV vector DNA does not integrate into the host genome, thus the duration of transgene expression is not affected, making rAAV vector transduction in the eye more efficient. The efficiency of ocular vector transduction is dependent upon a number of factors, including the routes of vector administration discussed above and the promoter sequence

of the vector, which provides the impetus for transgene expression.2 Furthermore, efficiency of rAAV vector transduction can be enhanced. In 2008, Zhong et al. demonstrated the highefficiency transduction of rAAV-2 vectors at lower doses. Through site-directed mutagenesis of the surface-exposed tyrosine residues on the capsid proteins of rAAV-2 vectors, the vectors were able to avoid phosphorylation. Subsequently, the tyrosine-mutant rAAV-2 vectors escaped ubiquitination and thereby circumvented proteasome-mediated degradation. These results indicated an increase in transduction efficiency of the tyrosine-mutant rAAV-2 vectors, as well as improved intracellular trafficking to the nucleus.13 Table 2 summarizes rAAV-mediated gene therapies for ocular diseases.