Gene Therapy For Retinal Degeneration Research Articles

Gene Therapy for Retinal Degeneration

Biallelic mutations in the RPE65 gene are associated with inherited retinal degenerations/dystrophies (IRD) and disrupt the visual cycle, leading to loss of vision. A new adenoviral vector-based gene therapy surgically delivered to retinal cells provides normal human RPE65 protein that can restore the visual cycle and some vision. To view this Bench to Bedside, open or download the PDF.

Gene therapy using stem cells.

Viral-mediated gene augmentation therapy has recently shown success in restoring vision to patients with retinal degenerative disorders. Key to this success was the availability of animal models that accurately presented the human phenotype to test preclinical efficacy and safety. These exciting studies support the use of gene therapy in the treatment of devastating retinal degenerative diseases. In some cases, however, in vivo gene therapy for retinal degeneration would not be effective because the cell types targeted are no longer present. The development of somatic cell reprogramming methods provides an attractive source of autologous cells for transplantation and treatment of retinal degenerative disease. This article explores the development of gene therapy and patient-derived stem cells for the purpose of restoring vision to individuals suffering from inherited retinal degenerations.

Identification of Key Residues Determining Isomerohydrolase Activity of Human RPE65

RPE65 is the retinoid isomerohydrolase that converts all-trans-retinyl ester to 11-cis-retinol, a key reaction in the retinoid visual cycle. We have previously reported that cone-dominant chicken RPE65 (cRPE65) shares 90% sequence identity with human RPE65 (hRPE65) but exhibits substantially higher isomerohydrolase activity than that of bovine RPE65 or hRPE65. In this study, we sought to identify key residues responsible for the higher enzymatic activity of cRPE65. Based on the amino acid sequence comparison of mammalian and other lower vertebrates' RPE65, including cone-dominant chicken, 8 residues of hRPE65 were separately replaced by their counterparts of cRPE65 using site-directed mutagenesis. The enzymatic activities of cRPE65, hRPE65, and its mutants were measured by in vitro isomerohydrolase activity assay, and the retinoid products were analyzed by HPLC. Among the mutants analyzed, two single point mutants, N170K and K297G, and a double mutant, N170K/K297G, of hRPE65 exhibited significantly higher catalytic activity than WT hRPE65. Further, when an amino-terminal fragment (Met(1)-Arg(33)) of the N170K/K297G double mutant of hRPE65 was replaced with the corresponding cRPE65 fragment, the isomerohydrolase activity was further increased to a level similar to that of cRPE65. This finding contributes to the understanding of the structural basis for isomerohydrolase activity. This highly efficient human isomerohydrolase mutant can be used to improve the efficacy of RPE65 gene therapy for retinal degeneration caused by RPE65 mutations.

Restoration of Vision in the pde6β-deficient Dog, a Large Animal Model of Rod-cone Dystrophy

Defects in the β subunit of rod cGMP phosphodiesterase 6 (PDE6β) are associated with autosomal recessive retinitis pigmentosa (RP), a childhood blinding disease with early retinal degeneration and vision loss. To date, there is no treatment for this pathology. The aim of this preclinical study was to test recombinant adeno-associated virus (AAV)-mediated gene addition therapy in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6β deficiency that strongly resembles the human pathology. A total of eight rcd1 dogs were injected subretinally with AAV2/5RK.cpde6β (n = 4) or AAV2/8RK.cpde6β (n = 4). In vivo and post-mortem morphological analysis showed a significant preservation of the retinal structure in transduced areas of both AAV2/5RK.cpde6β- and AAV2/8RK.cpde6β-treated retinas. Moreover, substantial rod-derived electroretinography (ERG) signals were recorded as soon as 1 month postinjection (35% of normal eyes) and remained stable for at least 18 months (the duration of the study) in treated eyes. Rod-responses were undetectable in untreated contralateral eyes. Most importantly, dim-light vision was restored in all treated rcd1 dogs. These results demonstrate for the first time that gene therapy effectively restores long-term retinal function and vision in a large animal model of autosomal recessive rod-cone dystrophy, and provide great promise for human treatment.

Prospectives for gene therapy of retinal degenerations.

Retinal degenerations encompass a large number of diseases in which the retina and associated retinal pigment epithelial (RPE) cells progressively degenerate leading to severe visual disorders or blindness. Retinal degenerations can be divided into two groups, a group in which the defect has been linked to a specific gene and a second group that has a complex etiology that includes environmental and genetic influences. The first group encompasses a number of relatively rare diseases with the most prevalent being Retinitis pigmentosa that affects approximately 1 million individuals worldwide. Attempts have been made to correct the defective gene by transfecting the appropriate cells with the wild-type gene and while these attempts have been successful in animal models, human gene therapy for these inherited retinal degenerations has only begun recently and the results are promising. To the second group belong glaucoma, age-related macular degeneration (AMD) and diabetic retinopathy (DR). These retinal degenerations have a genetic component since they occur more often in families with affected probands but they are also linked to environmental factors, specifically elevated intraocular pressure, age and high blood sugar levels respectively. The economic and medical impact of these three diseases can be assessed by the number of individuals affected; AMD affects over 30 million, DR over 40 million and glaucoma over 65 million individuals worldwide. The basic defect in these diseases appears to be the relative lack of a neurogenic environment; the neovascularization that often accompanies these diseases has suggested that a decrease in pigment epithelium-derived factor (PEDF), at least in part, may be responsible for the neurodegeneration since PEDF is not only an effective neurogenic and neuroprotective agent but also a potent inhibitor of neovascularization. In the last few years inhibitors of vascularization, especially antibodies against vascular endothelial cell growth factors (VEGF), have been used to prevent the neovascularization that accompanies AMD and DR resulting in the amelioration of vision in a significant number of patients. In animal models it has been shown that transfection of RPE cells with the gene for PEDF and other growth factors can prevent or slow degeneration. A limited number of studies in humans have also shown that transfection of RPE cells in vivo with the gene for PEDF is effective in preventing degeneration and restore vision. Most of these studies have used virally mediated gene delivery with all its accompanying side effects and have not been widely used. New techniques using non-viral protocols that allow efficient delivery and permanent integration of the transgene into the host cell genome offer novel opportunities for effective treatment of retinal degenerations.

Long-term Retinal Function and Structure Rescue Using Capsid Mutant AAV8 Vector in the rd10 Mouse, a Model of Recessive Retinitis Pigmentosa

The retinal degeneration 10 (rd10) mouse is a well-characterized model of autosomal recessive retinitis pigmentosa (RP), which carries a spontaneous mutation in the β subunit of rod cGMP-phosphodiesterase (PDEβ). Rd10 mouse exhibits photoreceptor dysfunction and rapid rod photoreceptor degeneration followed by cone degeneration and remodeling of the inner retina. Here, we evaluate whether gene replacement using the fast-acting tyrosine-capsid mutant AAV8 (Y733F) can provide long-term therapy in this model. AAV8 (Y733F)-smCBA-PDEβ was subretinally delivered to postnatal day 14 (P14) rd10 mice in one eye only. Six months after injection, spectral domain optical coherence tomography (SD-OCT), electroretinogram (ERG), optomotor behavior tests, and immunohistochemistry showed that AAV8 (Y733F)-mediated PDEβ expression restored retinal function and visual behavior and preserved retinal structure in treated rd10 eyes for at least 6 months. This is the first demonstration of long-term phenotypic rescue by gene therapy in an animal model of PDEβ-RP. It is also the first example of tyrosine-capsid mutant AAV8 (Y733F)-mediated correction of a retinal phenotype. These results lay the groundwork for the development of PDEβ-RP gene therapy trial and suggest that tyrosine-capsid mutant AAV vectors may be effective for treating other rapidly degenerating models of retinal degeneration.

AAV-Mediated Gene Therapy for Retinal Degeneration in therd10Mouse Containing a Recessive PDEβ Mutation

To test AAV-mediated gene therapy in the rd10 mouse, a natural model of recessive RP caused by mutation of the beta-subunit of rod photoreceptor cGMP phosphodiesterase. One eye of a cohort of rd10 mice kept in a dark environment was subretinally injected at postnatal day (P) 14 with 1 microL AAV5-smCBA-PDEbeta. The contralateral eye was not injected. The animals were then maintained for 2 weeks in the dark before they were moved to a normal 12-hour light/12-hour dark cycling light environment for visually guided behavioral training. Three weeks after injection, treated rd10 mice were examined by scotopic and photopic electroretinography and then killed for biochemical and morphologic examination. Substantial scotopic ERG signals were maintained in treated rd10 eyes, whereas untreated eyes in the same animals showed minimal signals. Treated eyes showed photopic ERG b-wave amplitudes similar to those of the normal eyes; in untreated partner eyes, only half the normal amplitudes remained. Strong PDEbeta expression was observed in photoreceptor outer segments only in treated eyes. Light microscopy showed a substantial preservation of the outer nuclear layer in most parts of the treated retina only. Electron microscopy showed good outer segment preservation only in treated eyes. A visually guided water maze behavioral test under dim light showed significantly improved performance in one eye-treated rd10 mice compared with untreated mice. These data demonstrate that P14 administration of AAV5-smCBA-PDEbeta can prevent retinal degeneration in rd10 mice, as reflected by significant structural, biochemical, electrophysiological, and behavioral preservation/restoration. These results serve as a baseline for studying long-term retinal rescue in rd10 mice.

Preliminary Results of Gene Therapy for Retinal Degeneration

In this issue of the Journal, two groups of investigators — Bainbridge et al.1 and Maguire et al.2 — describe the first results of separate clinical trials investigating the short-term safety and preliminary efficacy of gene therapy for Leber's congenital amaurosis. Both groups present short-term data (12 months and 5 months, respectively) on three patients with Leber's congenital amaurosis in each study; the patients were enrolled in trials of recombinant adeno-associated viral delivery of the human retinal pigment epithelium-specific 65-kDa protein gene (RPE65), which was administered as a subretinal injection during vitrectomy.Originally described by Leber in 1869, . . .

AAV-Mediated Expression Targeting of Rod and Cone Photoreceptors with a Human Rhodopsin Kinase Promoter

Gene therapy for retinal degeneration requires well-defined promoters that drive expression in rod and cone photoreceptors. This study was undertaken to develop short, active derivatives of the human rhodopsin kinase (RK) gene promoter for targeting transgene expression in rods and cones. RK, also known as G protein-coupled receptor kinase 1 (GRK1), is a component of the light adaptation pathway expressed in rods and cones. Human RK (hRK) promoter and its concatemers or derivatives extending into the conserved 5' untranslated region (5'-UTR) were assayed for promoter activity in WERI retinoblastoma or Crx/Sp1-supplemented HEK-293 cells. The derivative displaying the highest activity was linked to a GFP reporter and packaged in a pseudotyped adenoassociated viral vector (AAV2/5). The AAV vector was tested in vivo by subretinal injections in wild-type mice, in the all-cone Nrl(-/-) mice, and in the cone-rich diurnal Nile grass rat (Arvicanthis niloticus). Control eyes received a similar AAV2/5 vector carrying a mouse rod opsin (mOps) promoter-controlled GFP reporter. The hRK promoter with the full 5' untranslated sequence (-112 to +180) was the most active in cell culture. Delivered by the AAV2/5 vector, RK promoter drove GFP expression specifically in photoreceptors. In rods, hRK promoter-mediated expression was as efficient as, but appeared more uniform than, mOps promoter-mediated expression. In cones, the hRK promoter drove expression, whereas the mOps promoter did not. The hRK promoter is active and specific for rod and cone photoreceptors. Because of its small size and proven activity in cones, it is a promoter of choice for somatic gene transfer and gene therapy targeting rods and cones.

Recombinant AAV-mediated gene transfer to the retina: gene therapy perspectives.

Retinal degenerative diseases such as retinal macular degeneration and retinitis pigmentosa constitute a broad group of diseases that all share one critical feature, the progressive apoptotic loss of cells in the retina. There is currently no effective treatment available by which the course of these disorders can be modified, and visual dysfunction often progresses to total blindness. Gene therapy represents an attractive approach to treating retinal degeneration because the eye is easily accessible and allows local application of therapeutic vectors with reduced risk of systemic effects. Furthermore, transgene expression within the retina and effects of treatments may be monitored by a variety of noninvasive examinations. An increasing number of strategies for molecular treatment of retinal disease rely on recombinant adeno-associated virus (rAAV) as a therapeutic gene delivery vector. Before rAAV-mediated gene therapy for retinal degeneration becomes a reality, there are a number of important requirements that include: (1) evaluation of different rAAV serotypes, (2) screening of vectors in large animals in order to ensure that they mediate safe and long-term gene expression, (3) appropriate regulation of therapeutic gene expression, (4) evaluation of vectors carrying a therapeutic gene in relevant animal models, (5) identification of suitable patients, and finally (6) manufacture of clinical grade vector. All these steps towards gene therapy are still being explored. Outcomes of these studies will be discussed in the order in which they occur, from vector studies to preclinical assessment of the therapeutic potential of rAAV in animal models of retinal degeneration.

1056. PhiC31 Integrase for Long-Term Non-Viral Gene Therapy in Rat Retina

We have developed a lentiviral vector system that can transduce terminally differentiated retinal cells in vivo. Using this system, we designed experiments to evaluate the therapeutic potentials of gene therapy for retinal degenerations in the cyclic nucleotide-gated channel CNGA3-deficiet mice, an animal model for cone photoreceptor cell degeneration, and retinal degeneration slow (rds/rds) mice, an animal model for retinitis pigmentosa. Wild type human CNGA3 and mouse Peripherin cDNAs were transduced in cone and rod photoreceptor cell-specific manner by human red pigment gene and bovine rhodopsin gene promoters in CNGA3-deficient mice and rds/rds mice, respectively. Moreover the additional neuroprotective effects of human pigment epithelium-derived factor (hPEDF) driven by cytomegalovirus promoter were also assessed. In CNGA3-deficient mice, transduction of hCNGA3 and hPEDF reduced the speed of cone degeneration, although subacute thinning of outer nuclear layer (ONL) was observed in some samples. Electrophysiological analysis revealed that hCNGA3 reconstitution improved cone response and maximal response which is derived from rods and cones. These effects were prolonged by combinational transduction of hPEDF. In rds/rds mice, transduction of mPeripherin and hPEDF partially prevented thinning of ONL until eight months after the injection. Electrophysiologically we could detect improvement of rod response and maximal response, although the additional effects of hPEDF were not observed. We conclude that subretinal lentiviral vector delivery had significant rescue effects on these two retinal degeneration animal models.

1057. Successful Gene Therapy for Retinal Degenerations by Lentiviral Gene Transfer

Our goal is to perform gene therapy by efficient integration into safe positions in the host genome. The integrase enzyme from the phage phiC31 directs site-specific integration into a small subset of native genomic sequences 1. Recent gene therapy studies using phiC31 integrase in mice have demonstrated site-specific integration of transgenes and long-term gene expression in liver, skin, and muscle (e.g., 2). Here, we demonstrate use of the phiC31 integrase as a simple and effective method for non-viral long-term gene transfer in the eye. We developed a method to achieve efficient non-viral delivery of plasmid DNA to rat retinal epithelium (RPE) in vivo. The method involves subretinal injection of plasmid DNA, followed by in situ electroporation (adapted from 3). Delivery of GFP by this method was evaluated in frozen sections using fluorescence microscopy. In subsequent experiments, a luciferase plasmid, with or without a plasmid encoding the phiC31 integrase, was delivered to rat RPE. Luciferase expression was followed over time by using in vivo luciferase imaging. Subretinal DNA injection followed by electroporation yielded abundant transgene delivery in rat RPE, as assayed by both GFP and luciferase expression. Expression was strongest 48 hours after delivery. In the absence of integrase, luciferase expression declined to near-background levels within 3-4 weeks after treatment. Co-injection of the integrase plasmid led to long-term stable transgene expression at all time points tested, up to three months post-procedure. Animals receiving integrase showed ~64-fold higher long-term expression than control animals. We conclude that subretinal injection followed by electroporation affords abundant transfer of plasmid DNA in mammalian RPE. Addition of phiC31 integrase confers robust long-term transgene expression, presumably by providing site-specific integration of the gene of interest into the genome of RPE cells. Having established non-viral permanent retinal gene transfer by site-specific genomic integration, we now plan to apply the technology to rodent models of retinal degeneration. Because site-specific integration provides strong, stable gene expression while reducing the probability of insertional mutagenesis, the phiC31 integrase system has great potential as a novel approach to non-viral gene therapy in the eye.

Is successful gene therapy for retinal disease in sight?

The correction of a genetic defect by the transfer and expression of a non-mutated version of the effected gene is the basis of most gene replacement strategies. However, predicting whether this will be sufficient to restore cell function and alter the progress of a given disease can be hard particularly when dealing with something as complex as the eye and vision. Thus, it is exciting to see the recent results of Ali and co-workers 1 Ali R.R. et al. Restoration of photoreceptor ultrastructure and function in retinal degeneration slow mice by gene therapy. Nat. Genet. 2000; 25: 306-310 Crossref PubMed Scopus (292) Google Scholar describing gene therapy for retinal degeneration by expression of the Prph-2 gene that encodes the peripherin-2 membrane protein required for the stabilization of the outer segment discs of photoreceptor cells. In this study an adeno-associated viral vector expressing peri-pherin-2 under the control of the rhodopsin promoter was administered by sub-retinal injection to Prph-2 mutant mice – a well-characterized model of retinal degeneration. Analysis of the retinas of treated mice showed structural (the formation of outer segments), biochemical (increased concentrations of rhodopsin) and physiological (electroretinal responses to light) changes consistent with correction of a significant number (approximately 30%) of photoreceptor cells. This data indicates that function can be restored to a well-differentiated cell (the photoreceptor) albeit at this stage in relatively young (10-day-old) animals. It will be interesting to see if gene transfer of the Prph-2 gene can now be used to rescue older mice with more established retinal degeneration and also to see whether long-term exogenous expression can be used to maintain or even restore vision.

Gene therapy for retinal degeneration.

Inherited retinal degenerations are a group of diseases leading to blindness through progressive loss of vision in many patients. Although with the cloning of more and more disease genes the knowledge on the molecular genetics of these conditions and on the apoptotic pathway as the common disease mechanism is steadily increasing, there is still no cure for those affected. In recent years, new experimental treatments have evolved through the efforts of many investigators and have been explored in animal models. The rationale of the different strategies for developing a treatment based on gene replacement or rescue of the diseased neuronal tissue with growth factors will be outlined and discussed in this paper.