Ординатура / Офтальмология / Английские материалы / Retinal Degenerations biology, diagnostics, and therapeutics_Tombran-Tink, Barnstable_2007

endostatin, angiostatin, and PEDF. These factors could be delivered by ECT. In addition, these anti-angiogenic factors could be combined with neurotrophic factors in a single device.

ECT-Based Delivery of Anti-Inflammatory Factors in Uveitis

Uveitis is inflammation of the uvea. Approximately 80% of the cases in humans are autoimmune related and chronic, whereas the remaining 20% are caused by infectious processes. Topical and systemic corticosteroids and immunomodulators are the current standard of care. Studies have demonstrated the possible use of anti-inflammatory cytokines for treating uveitis. For example, IL-10, a 35-kD homodimeric cytokine synthesized by monocytes, B cells, and Th2 lymphocytes, induces mainly immunosuppressive effects through the down regulation of macrophage functions and the inhibition of the synthesis of pro-inflammatory cytokines, such as IL-1β, IL-6, IL-8, interferon-γ, and granulocyte/macrophage colony-stimulating factors, produced by Th1 lymphocytes or monocytes. Because of its short half-life, daily injections of recombinant IL-10 are required to produce therapeutic effects. Repetitive injections of IL-10 inhibit experimental autoimmune uveoretinitis (EAU) (54) and endotoxin-induced uveitis (55). Inhibition of EAU by systemic and subconjunctival adenovirus-mediated transfer of the IL-10 gene has been reported (56), indicating sustained availability of IL-10 would be beneficial. Obviously, ECT would be a good choice for intraocular delivery of IL-10 for uveitis.

SUMMARY

Preclinical development of ECT has demonstrated the therapeutic efficacy, longterm delivery, and relative safety in the animal eyes. Based on this data, a clinical phase I safety study of NT-501 has been initiated at the National Eye Institute to treat RP. If safety and consistent delivery are demonstrated in clinical trials, ECT could potentially serve as a delivery system not only for RP, but also for a number of ophthalmic diseases for which no effective therapies are currently available.

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University Hospital (Zurich, Switzerland) (11). Currently, there is no effective treatment for this congenital and further progressive blinding disease.

RPE65 is a 61-kDa microsomal protein expressed almost exclusively in the RPE (12,13). The gene spans 20 kb and is divided into 14 exons (14) and is localized to chromosome 1p31 (14,15). The transcript consists of about 0.1% of the total messenger RNA isolated from the RPE and is approx 2.9 kb. The 533-amino acid protein is highly conserved between various species. RPE65 is associated with the smooth endoplasmic reticulum and is necessary for the synthesis of the 11-cis-retinal chromophore of the photoreceptor cell visual pigments (16). In an RPE65 knockout mouse, 11-cis-retinoids cannot be generated and there is an accumulation of all-trans-retinyl esters in the RPE, forming lipoid inclusion bodies (16).

LCA-Like Disease of Dogs

The Briard dog is affected by a hereditary retinal dystrophy (17) and has been shown to be a very suitable animal model for LCA (18,19). The molecular defect that underlies this canine disorder is a 4-bp deletion in the RPE65 gene (20,21). Dogs homozygous for the RPE65 null mutation are congenitally night blind, and show severe visual deficits in daylight. Most, but not all, cases show a fast-quivering nystagmus. Their resting pupillary size is slightly larger than in normal dogs in accordance with their reduced sensitivity to light (22). ERG studies are already diagnostic at the age of 5 wk (19). There are no, or barely recordable, scotopic responses, and photopic ERGs are usually of low amplitudes, most clearly observed in 30 Hz flicker recordings. Ophthalmoscopic examination reveals normal fundus appearance until about 3 yr of age, then there is a generalized vascular attenuation and a slight paling of the fundus. In most affected older animals, grayish to white spots, mainly in the central tapetal and nontapetal fundus, appear. These spots increase slowly with age and spread peripherally with time (Fig. 1). Thus, the disease is slowly progressive clinically. Large lipoid-like inclusions accumulate primarily in the RPE of the central fundus, but they become more generalized at later ages. There is also disorganization of photoreceptor outer segments (OS) at an early age, followed by degenerative changes and later by rod, and then cone loss, with a gradient going from the peripheral retina to the central parts with increasing age (18,23). There are obviously close similarities between the clinical characteristics of the diseases resulting from RPE65 gene mutations in dogs and in humans (24).

Retinal Gene Therapy

Gene therapy is a promising technology for the treatment of inherited genetic disorders in which the genetic defect is known. The retina is an especially good target for gene therapy because it is easily accessible and because it is an immunoprivileged site. The gene therapy vector can be injected either into the vitreous or the subretinal space that lies between the neuroretina and RPE with only minimal local side effects. Additionally, in the advent of an unexpected adverse reaction to the therapy, the eye can be removed surgically.

Immune rejection of the transgene is a potential problem in gene replacement therapy, because many patients express a truncated form of the protein. Therefore, the new transgene may contain epitopes that are recognized as foreign by the patient’s immune

Fig. 1. (A) Minor ophthalmoscopic signs of the RPE65 null mutation are illustrated in this 3.5-yr-old affected dog, treated by subretinal gene transfer 1 yr previously (area of injection not shown in this fundus photograph). Some grayish spots are seen in the fundus and there is a slight generalized vascular attenuation. (B) Shows the fundus in the same dog 3 yr later. The aberrant spots have increased markedly as well as the vascular attenuation.

system. However, like other internal structures in the eye, the subretinal space displays two important immune privilege features: (1) it will tolerate tissue grafts without immune rejection, and (2) it promotes the acquisition of systemic immune deviation to antigens placed within it (25). Therefore, long-term transgene expression is possible in the retina without clearance of transduced cells by the immune system.

Viruses are naturally occurring gene therapy vectors and can be highly efficient in delivering their DNA or RNA to the target cells. Vectors based on a parvovirus called adeno-associated virus (AAV) have been highly successful for transduction of the retina (26). AAV virions are comprised of 60 protein molecules surrounding a 4.7-kb singlestranded DNA. Of this, approx 4.4 kb can be used to express the transgene. The only AAV DNA in the recombinant AAV is the 145-bp terminal repeats located at each end. These terminal repeats contain the AAV origin of replication and the packaging signals. When AAV infects a cell, the single-stranded DNA becomes converted to doublestranded DNA and the transgene is then expressed. AAV vector DNA can persist for many years in cells as either long concatemers or circular intermediates. Vector DNA can also integrate randomly over time and can potentially express the transgene over the lifetime of the patient. Expression of therapeutic levels of RPE65 has been observed in treated animals for over 3 yr.

Epithelial cells such as in the RPE are excellent targets for AAV vectors. By injecting the recombinant AAV into the subretinal space it is possible to place the virus in close contact with the potential target. This allows for extremely efficient transduction of the RPE layer within the bleb. AAV vectors can also transduce the RPE layer when injected into the vitreous. This suggests that the vector can migrate through the neural retina and reach the RPE. Currently, eight different serotypes of AAV have been used for gene therapy (AAV1-8). We have used the most widely studied strain, AAV2, in all experiments described here. AAV4 and AAV6 vectors appear to transduce the RPE more specifically than AAV2 (27,28). The AAV6 result is somewhat surprising because AAV1 vectors transduce ganglion cells and photoreceptors in addition to RPE cells (29). AAV6 derives

from a recombination of AAV2 and AAV1 such that the capsid proteins of AAV1 and AAV6 are highly homologous.

Because the effects of loss of RPE65 function appear to be restricted to the retina, localized gene therapy appears to be a promising approach to treat LCA. Acland et al. (30) observed a significant improvement in vision in three 4-mo-old dogs treated with the AAV vector that expressed RPE65 from the β-actin promoter. In-depth experiments to assess the efficacy of AAV-mediated gene therapy in reversing the effects of the RPE65 mutation in RPE65–/– dogs were conducted in our laboratories (31–34). A canine RPE65 complementary DNA (cDNA) was cloned into a recombinant AAV2 vector. We have used a strong, constitutive promoter from cytomegalovirus (CMV) to drive RPE65 expression. Ancestors of the group of dogs used in these experiments were originally discovered in Sweden and transported to the University of Missouri where gene transfer surgeries were initiated in 2001. During the past 4 yr, 20 RPE65–/– dogs have undergone intraocular gene therapy. Treated animals have been studied using various clinical and experimental techniques. Some of the affected animals have been followed for more than 3 yr postoperatively with some very dramatic and exciting results.

MATERIALS AND METHODS

Preparation of the Gene Construct

Development of our gene construct has been described (31) and, in short, was performed as follows: Normal RPE65 dog cDNA was cloned into a pCl vector (Promega, Madison, WI) carrying the human CMV promoter and the late SV40 polyadenylation signal (poly A). Following transfection into Cos-7 cells (ATCC, Rockville, MD), the expression of RPE65 protein was analyzed by Western blotting. Subsequently, the RPE65 expression cassette was cloned into pSSV9 (35) in between the AAV2 terminal repeats. A control vector with green fluorescent protein (GFP) cDNA in place of the RPE65 cDNA was also constructed. Human embryonic kidney (HEK 293) cells (ATCC, Rockville, MD) were transfected with pAAV.CMV.GFP or pAAV.CMV.RPE65 and assessed for the expression of the transgenes by fluorescence microscopy and Western blot analysis, respectively. The excision and replication of the rAAV.RPE65 was assessed by Hirt analysis (36). Large-scale production of recombinant viruses was done at the Vector Core Facility of the University of North Carolina Gene Therapy Center (Chapel Hill, NC) (37). AAV vectors are typically assessed for their ability to transduce target cells (e.g., HeLa) and for the number of DNA-containing virions by quantitative polymerase chain reaction (PCR). The titer of the rAAV.GFP preparations were approx 2 × 1010 transducing units per milliliter and ranged from 5.5 × 1011 to 8.6 × 1011 singlestranded vector genomes per milliliter. The four rAAV.RPE65 preparations were approx 1012 vector genomes per milliliter. Purity of the virus preparations were evaluated by silver-staining sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis gels. Unfortunately, two out of four rAAV.RPE65 preparations were contaminated with nonvector proteins, which resulted in an inflammatory reaction in some treated animals (see Immediate Postoperative Results section). The purity

of more recent batches was approx 95% and we have not observed any inflammatory reactions using these batches.

Methods for Dog Experiments

A majority of dogs that underwent gene transfer were treated surgically at the age of 4 mo, whereas two of the RPE65–/– dogs were 2.5 and 4 yr old, respectively, at the time of gene transfer. From age 5 to 7, wk baseline clinical studies were performed. Initially, their visual behavior was tested by merely watching the dogs walk in unknown surroundings in the dark and in daylight conditions. Their ability to follow a strong beam of light in both lighting conditions and see falling cotton balls was also studied. Direct and indirect pupillary light reflexes were tested in both conditions and indirect ophthalmoscopy, slit-lamp biomicroscopy, and fundus photography were performed regularly (31). Baseline ERGs were obtained at least twice before surgery (31,32,34). Follow-up studies after surgery were performed in similar ways, but extended with maze testing for objective visual behavior (34), multifocal ERG (mfERG) studies (38,39), and imaging of the treated and untreated fundi using Fluorescein (FL) and Indocyanine Green (ICG) angiography. Fundus images were obtained by confocal scanning laser ophthalmoscopy (SLO; Heidelberg, Engineering Retina Angiograph, Heidelberg, Germany), equipped with an argon blue laser (488 nm), a green laser (514 nm), and an infrared laser (835 nm) for visualization of fluorescence and fundus during the angiography. All electrophysiological studies were performed under general anesthesia using propofol (Diprivan, 1%, Astra Zeneca Pharmaceutical LP, Wilmington, DE, 6 mg/kg iv) and isoflurane (Isoflurande USP, Abbott Laboratories, North Chicago, IL). Imaging studies were performed in deep sedation using medetomidine (Domitor vet., Vetpharma, Lund, Sweden, 0.01 mg/kg IM) and ketamine (Ketalar, Park-Davies, Morris Plains, NJ, 2.5 mg/kg IM).

Gene transfer was performed under microscopic visualization using routine aseptic methods by two trained vitreo-retinal surgeons. In most cases, both eyes were treated with subretinal injections: one eye with rAAV.RPE65 and the contralateral eye with rAAV.GFP (31). One hundred microliters of each construct was injected subretinally in most of the dogs.

A lateral canthotomy was performed and conjunctiva and Tenon’s capsule were dissected so that access to the sclera was obtained. Two sclerotomies were performed in the temporal sclera about 4 mm apart and 6–8 mm from the limbus. A fiberoptic light was inserted into the vitreous cavity through one sclerotomy and a custom-made glass micropipet through the other opening and guided toward the fundus, under direct visualization through an operating microscope and a Machemer flat lens (Ocular Instruments, Bellevue, WA) on the cornea. For subretinal injections, the inferior-nasal central part of the neuroretina was perforated and a bleb was obtained with the injection of one of the constructs. The neuro-retinal detachment encompassed approximately 30% of the total fundus area. After the subretinal deposition, the micropipette and light guide were withdrawn and the sclerotomies sutured using 7/0 Vicryl. The conjunctiva, Tenon’s capsule and subcutaneous tissues were sutured in a routine manner. A subconjunctival injection in each eye of decadrone phosphate (Dexamethasone, Butler, Columbus, OH, 1 mg in 0.25 mL) was given postoperatively.