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

response (1,2). Thus, systemic administration of large molecules, such as CNTF and other NT factors, is simply not an effective approach for CNS and ocular disorders.

There are two other options for delivering proteins to the CNS or the retina: bolus injection of purified recombinant proteins and gene therapy. Bolus injection is clinically impractical because it requires repetitive injections for long-term therapy. Gene therapy, on the other hand, can achieve sustained expression of a given protein. However, the doses of therapeutic protein are difficult to control due to the fact that no reliable means is available to regulate the expression levels of the transgene. Furthermore, it is impossible to reverse the treatment once the gene is delivered.

The ECT is a delivery system that uses live cells to secrete a therapeutic agent. This is usually achieved by genetically engineering a specific type of cells to overexpress a particular agent. The engineered cells are then encapsulated in semipermeable polymer capsules. The capsule is then implanted into the target sites. There are several advantages of ECT. First, it allows potentially any gene encoding for a therapeutic protein to be engineered into the cells and therefore has a broad range of applications. Also, the therapeutic protein is freshly synthesized and released in situ so that a relatively small amount of the protein is needed to achieve a therapeutic effect. The long-lasting output assures that the availability of the protein at the target site is not only continuous, but also long-term. Furthermore, the output of an ECT implant can be controlled to achieve the optimal dose for treatment. Finally, the treatment by ECT can be terminated if it becomes desirable by simply retrieving the implant. Thus, ECT is a very effective means of long-term delivery of biologically active proteins and polypeptides to the CNS and the retina. In fact, ECT is now proven to be an excellent choice for retinal degenerative diseases, especially considering the limited distribution volume, easy access to the eye, and the chronic nature of the diseases.

The therapeutic efficacy of growth factors delivered by ECT has been demonstrated in a number of animal models of neurodegenerative diseases, including CNTF in the rodent and primate models of the Huntington’s disease (3,4), Glial cell-line derived neurotrophic factor (GDNF) in rat model of Parkinson’s disease (5,6), and nerve growth factor (NGF) in rodent and primate models of Alzheimer’s disease (7–9). Furthermore, studies have shown that growth factors produced by mammalian cells, synthesized de novo, are more potent than purified recombinant ones expressed in Escherichia coli (10,11). And transplantation of encapsulated mammalian cells delivers therapeutic agents to the target site in the CNS to produce therapeutic effects at lower dosage than are required with other means of delivery (10).

An intraocular implantable device prototype of ECT has been developed by Neurotech for long-term delivery of therapeutic agents to treat ophthalmic disorders (Fig. 1) (12). The device consists of genetically modified cells packaged in a hollow tube of semipermeable membrane that prevents immune molecules, e.g., antibodies and host immune cells, from entering the device, while it allows nutrients and therapeutic molecules to diffuse freely across the membrane. The encapsulated cells secrete therapeutic agents continuously, and derive nourishment from the host milieu. The device is designed for implantation through a small incision in pars plana and a small titanium wire loop on the device allows it to be anchored to the sclera. The current device is about 6.0 mm in length (including the titanium loop) and approx 1 mm in diameter. These dimensions assure that the device is outside the visual axis in the human eye.

Fig. 1. Schematic illustration of the ECT device. The device is constructed with a section of semipermeable polymer membrane and supportive matrices to accommodate live cells. The two ends of the polymer section are sealed and a titanium loop is placed on the anchoring end. The loop allows the device to be anchored to the sclera. The total length of the device is 6 mm. (Reproduced from ref. 12, with permission.)

THERAPEUTIC EFFICACY OF THE NT-501 DEVICE

FOR PHOTORECEPTOR PROTECTION

Ophthalmic disorders represent a rapidly growing disease area that is associated with an increase in aging population (13–18). Patients suffering from potentially blinding diseases have become one of the largest segments of the health care field with more than 50 million patients in the United States alone. Their sight is threatened by agerelated macular degeneration (AMD) (19–21), diabetic retinopathy (22–26), glaucoma (27–30), or retinitis pigmentosa (RP) (31–34). Apart from AMD and RP, glaucoma is now considered to be a retinal degenerative disease because control of intraocular pressure (IOP) alone does not prevent ganglion cell degeneration.

Few effective treatments for retinal degenerative disorders are available to date. Newly discovered NT factors provide a great promise for treating these diseases. However, without a practical delivery system, the realization of this promise would be very difficult. The ECT device for intraocular implantation is specifically designed to overcome this obstacle. The first such device is NT-501, an ECT-CNTF product that consists of encapsulated cells that secrete recombinant human CNTF. NT-501 is manufactured to be sterile, nonpyrogenic, and retrievable. It is intended to deliver CNTF intraocularly for treating photoreceptor degenerations. NT-501 has been tested for preclinical efficacy, pharmacokinetics, and toxicology in dogs, pigs, and rabbits.

CNTF and RP

The promise of growth factors as potential therapeutics for photoreceptor degeneration was first demonstrated in 1990 (35). Since then, many growth factors, NT factors, and cytokines have been tested in a variety of photoreceptor degeneration models, mainly by intravitreal injection of purified recombinant proteins in short-term experiments (35–38). Among them, CNTF has been shown to be the most effective one in almost every model (38). However, the chronic nature of RP (years) makes repetitive intraocular injection of purified recombinant CNTF (which is only effective for short duration) impractical. In fact, the obstacles of intraocular delivery have prevented the initiation of

clinical trials and its further development. Other factors that have shown protective effect in animal models of retinal degeneration include brain-derived NT factor (BDNF), NT-4, Axokine, basic fibroblast growth factor, insulin-like growth factor II, transforming growth factor-β2, IL-1β, tumor necrosis factor, NGF (36), pigment epithe- lium-derived factor (PEDF) (39), GDNF (40), lens epithelium-derived growth factor (41,42), and cardiotrophin-1 (CT-1) (43).

RP affects approx 100,000 Americans. It is a group of retinal degenerative diseases that have a complex molecular etiology. More than 100 mutations in several genes, including rhodopsin, peripherin, and phosphodiesterase (PDE)β, are believed to be responsible for RP, although the genotypes of the majority of RP patients are unknown. Despite the genetic heterogeneity, the phenotypes are very similar. Typically, a patient experiences a decrease in night vision early in life as a result of the loss of rod photoreceptor. Although the genetic defects affect only rods, cone photoreceptors eventually degenerate, leading to a progressive decrease in patient’s visual field and eventually to total blindness. The similarity in phenotypes and perhaps also in pathogenesis pathways enables medical intervention with a common approach without the need to identify the genotype of a patient.

The NT-501 device has been developed for intraocular delivery of CNTF for RP. Proof-of-principle experiments were conducted in two animal models of photoreceptor degeneration, the S334ter-3 transgenic rat and the rcd1 mutant dog. Pharmacokinetics studies were performed in the rabbits. The data from these studies indicate that CNTF delivered by NT-501 is not only effective in protecting photoreceptors, but also longlasting. These preclinical studies of NT-501 paved the way for human clinical trial, which is currently ongoing.

Protective Effect of NTC-201 in a Rat Model of Photoreceptor Degeneration

NTC-201 cells are genetically engineered human retinal pigment epithelial (RPE) cells to overexpress human CNTF. In culture, these cells secret CNTF at a rate of 100 ng/million cells/d. We first assessed the efficacy of these cells in the heterozygous S334ter-3 rats carrying the rhodopsin mutation S334ter. Photoreceptor degeneration in these animals begins soon after birth (postnatal day 8 [P8]) and progresses rapidly. By P20, more than 90% photoreceptors are degenerated (44). The size of rat eyes makes it impossible to accommodate the NT-501 device so that only unencapsulated CNTF secreting cells were used. NTC-201 cells (approx 105 in 2 µL phosphate buffered saline [PBS]) were injected into the left eyes intravitrealy at P9. Control animals were injected with untransfected parental cells (NTC-200). The contralateral eyes (right eyes) were untreated. In addition, a group of animals were treated with 1 µg of purified recombinant CNTF protein (in 1 µL of PBS, intravitreal injection to the left eye at P9) for comparison. Eyes were collected at P20, and processed for histological evaluation.

In untreated eyes of S334ter-3 transgenic rats, severe photoreceptor degeneration was observed by P20. The outer nuclear layer (ONL) contained only 1 row of nuclei (Fig. 2A), reduced from 10 to 12 rows of a normal animal. The NTC-201 injected eyes had five to six rows of nuclei in the ONL (Fig. 2C), whereas in the control eyes that were injected with untransfected cells (NTC-200), only one to two rows of nuclei remained (Fig. 2B). No evidence of retinal inflammation was observed in any of the treated or control eyes. In animals treated with a single intravitreal injection of purified

Fig. 2. Photoreceptor protection by CNTF secreting NTC-201 cells. Sections of retina from transgenic rats carrying the rhodopsin mutation S334ter were examined at PD 20 by light microscopy. (A) Untreated eye, (B) NTC-200 parental (control) cell treated eye, and (C) NTC-201 cell (CNTF secreting) treated eye. Eyes were injected with cells on P9. The ONL of untreated eye contained only one row of photoreceptor nuclei (A). In the retina treated with control cells, the ONL had one to two rows of nuclei, whereas in the retina treated with CNTF secreting cells, the ONL contained five to six rows, indicating significant protection by CNTF released from those cells. Brackets denote ONL. Plastic embedded sections stained with toland blue. (Reproduced from ref. 12, with permission.)

human recombinant CNTF, the ONL had two to three rows of nuclei (data not shown). These results clearly demonstrate that continuous delivery of CNTF delivered via mammalian cells protected against retinal degeneration in this model.

NT-501 Device Protects Photoreceptors in the rcd1 Dog RP Model

The efficacy of NT-501 devices was investigated in the rcd1 dog model. These dogs carry a mutation on the PDE6B gene encoding the β-subunit of the rod cGMP PDEβ (kindly provided by the Retinal Disease Studies Facility, Kennett Square, PA). The retinal

degeneration of this model is well characterized (45,46). Photoreceptor degeneration begins 3.5 wk after birth in these animals and continues for 1 yr, with 50% photoreceptor loss at 7 wk of age. NT-501 devices secreting 1 to 2 ng/d of CNTF were surgically implanted into the left eye of each rcd1 dog at 7 wk of age, the earliest time point that the surgical procedure can be performed without disruption of the retina (the eyes of younger dogs would be too small to accommodate an earlier version of NT-501 of 10 mm in length). In a normal dog the ONL contains 10 to 12 layers of photoreceptor nuclei, but in these animals at 7 wk of age, only 5 to 6 layers of nuclei in the remain. The contralateral eye was not treated.

At the endpoint of the experiment (14 wk of age), the devices were explanted and assayed for CNTF output and cell viability, and the eyes were collected and processed for histological evaluation. The ONL in untreated eyes contained only two to three rows of photoreceptor nuclei. In contrast, the ONL in the NT-501 treated eyes still had five to six rows remaining, similar to the number of nuclei rows present at the time when the treatment began (Fig. 3). The protection of photoreceptors was evenly distributed throughout the retina and not localized near the implant site. No apparent adverse effects were found in the retina. All explanted devices contained viable cells.

Photoreceptor Protection by NT-501 Devices is Dose Dependent

To determine the minimum effective dose and the optimal therapeutic dose of CNTF, a dose-ranging study was conducted. Thirty-one rcd1 dogs were included in this study. Devices that released different levels of CNTF were implanted into one eye of an animal at 7 wk of age. The contralateral eye was not treated. The level of device CNTF output (ng/d) was defined as follows: <0.1 (n = 4), 0.2–1 (n = 8), 1–2 (n = 7), 2–4 (n = 9), 5–15 (n = 3). The devices were explanted at 14 wk of age and assayed for CNTF output and viability, and the eyes were processed for histological evaluation. As shown in Fig. 4, photoreceptor protection by the NT-501 devices in the rcd1 dog model was dose dependent. Complete protection was achieved at the highest dose tested (5–15 ng/d of CNTF), and minimal, but statistically significant, protection was observed at levels as low as 0.2–1 ng/d of CNTF. CNTF delivered less than 0.1 ng/d had no protective effect. No cellular evidence of an immune reaction, inflammation or damage to the retina was observed. Evaluation indicated that all devices contained healthy, viable cells.

PHARMACOKINETICS OF NT-501 DELIVERED CNTF

To evaluate the pharmacokinetics of CNTF in the vitreous humor and the long-term function in vivo, NT-501 devices were implanted into rabbit eyes and explanted at different time points and vitreous samples harvested. The CNTF output from the explanted devices and CNTF levels in vitreous samples were determined by enzymelinked immunosorbent assay (ELISA).

As shown in Fig. 5, the explanted NT-501 devices produced a consistent amount of CNTF up to 12 mo in vivo. CNTF was readily detectable in the vitreous. Data from these pharmacokinetic and long-term device function studies indicate that the CNTF secreting function of the NT-501 device last at least for one year when implanted into

Fig. 3. Photoreceptor protection by CNTF secreting ECT device NT-501. Sections of retina from rcd1 dog model of retinitis pigmentosa were examined by light microscopy. A device was implanted into one eye at 7 wk of age and explanted at 14 wk of age. The contralateral eye was not treated. (A) Treated eye, (B) untreated eye. The ONL of treated retina contained five to six rows of nuclei. In contrast, the untreated retina had only two to three rows. Thus the NT-501 device provided significant protection to photoreceptors in the rcd1 dogs. Brackets denote ONL. (Reproduced from ref. 12, with permission.)

the eye. The released CNTF was throughout the vitreous, readily available for retinal cells. The functional results are confirmed by the histological evaluation showing that all devices contained healthy, viable cells.

POTENTIAL APPLICATION OF ECT FOR OTHER RETINAL DISEASES

Neuroprotection in Glaucoma

Glaucoma is a leading cause of blindness worldwide and the second cause of irreversible blindness in the United States. Approximately 2 million people in the United States have glaucoma, although roughly half are not even aware of it. Glaucoma is a group of diseases characterized by abnormal IOP and progressive death of retinal ganglion

Fig. 4. Dose–response protection of photoreceptors in rcd1 dogs. Data are presented as rows of nuclei in the ONL in ECT-CNTF treated eyes vs nontreated eyes (Mean ± SEM). The levels of CNTF output (ng/dev/d) were: <0.1 (n = 4, p = 0.744), 0.2–1 (n = 8, p = 0.0009), 1–2 (n = 7, p = 0.0001), 2–4 (n = 9, p = 0.0004), and 5–15 (n = 3, p = 0.043). The preservation of photoreceptor nuclei in the ONL depends on the amount of CNTF out put of the device. (Reproduced from ref. 12, with permission.)

Fig. 5. Time courses of CNTF output and vitreous CNTF levels in rabbit eyes after NT-501 implantation. The NT-501 devices were implanted into rabbit eyes. At indicated time points, the devices were explanted and vitreous samples were collected. The CNTF output of devices (ng/device/d) and CNTF vitreous levels (ng/mL) were assayed by ELISA. Data are presented as Mean ± SEM.

cells. The pathologic hallmark of glaucomatous optic neuropathy is the selective death of retinal ganglion cells associated with structural changes in the optic nerve head. Glaucoma is still considered to be a disease associated with abnormal IOP, and most current approaches for treating glaucoma are directed toward pressure control. However, in some cases the progress of retinal ganglion cell death continues even when

IOP is under control, indicating IOP independent mechanisms associated with the development of glaucomatous optic neuropathy. In fact, it is now recognized that glaucoma is also a retinal degenerative disease. Investigators in the glaucoma research field are actively seeking new approaches aimed at protecting retinal ganglion cells in patients with glaucoma.

Many neurotrophic factors have been found to protect retinal ganglion cells, including GDNF (47,48), BDNF (49,50), and CNTF (49,51–53). All these factors are deliverable by the intraocular ECT device. The fact that CNTF also protects retinal ganglion cells indicates that NT-501 could be used for glaucoma as well.

Neuroprotection in AMD

AMD is the leading cause of irreversible blindness in people over the age of 50. About 15 million people in the United States alone and 25–30 million people worldwide are affected by AMD.

The pathogenesis of AMD is not clearly understood. The disease affects the central region of the retina, the macular, hence the name. Cone photoreceptor and RPE cell degeneration is evident in patients with AMD. In some cases, newly formed blood vessels from the choroid (choroidal neovascularization [CNV]) invade the affected retinal area. This usually results in severe complications and loss of central vision.

Approximately 85–90% of the cases of AMD are of the “dry” form, characterized by soft drusen, retinal pigmentary disturbance, and/or focal retinal atrophy. Vision loss is generally not severe in patients with the dry form of AMD. Delivery of CNTF by NT-501 or other NT factors may help in photoreceptor protection in these cases.

Another form of AMD, the exudative or “wet” form, occurs in only 10–15% of all AMD cases. The wet form is characterized by CNV as new blood vessels originating from the choroid penetrate Bruch’s membrane to enter the retina. As a result, the normal architecture of the retina is destroyed with devastating complications, including hemorrhage, retinal detachment, disciform scar formation, and loss of central vision.

Currently, there are two Food and Drug Administration-approved therapies (Visudyne® and Macugen) for CNV in wet AMD aimed at inhibiting neovascularization. Visudyne (Novatis) is a photosensitizer used in photodynamic therapy to block new blood vessels with a laser beam. The other approved therapy, Macugen (Eyetech) is an aptamer that binds and inhibits vascular endothelial growth factor. Although the most important goal for treating wet AMD is to inhibit neovascularization, photoreceptor degeneration is still a problem that needs to be addressed. Again, ECT delivery of NT factors could be helpful in protecting the visual function for patients with wet AMD.

ECT Delivery of Antiangiogenic Factors for Ocular Neovascularization

There are two major neovascular diseases that affect the retina, exudates AMD and diabetic retinopathy. As mentioned previously, the existence of CNV is a characteristic of exudates AMD. CNV originates from choriocapillaris and invades the retina, causing blinding complications. In diabetic retinopathy, neovascularization in the retinal vasculature leads to hemorrhage and other complications, leading to blindness.

Neovascularization research, pioneered by Dr. Jodah Fokman of Harvard University, has lead to the discovery of many polypeptide anti-angiogenic factors, including