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Despite new and improving diagnostic and therapeutic options for glaucoma, blindness from glaucoma is increasing and glaucoma remains a major public health problem. The role of heredity in ocular disease including glaucoma is attracting greater attention as the knowledge and recent advances of Human Genome Project and the HapMap Project have made genetic analysis of many human disorders possible.
Glaucoma offers a variety of potential targets for gene therapy. All risk factors for glaucoma and their underlying causes are potentially susceptible to modulation by gene transfer. As genetic defects responsible for glaucoma are identified and the biochemical mechanisms underlying the disease are recognized, new methods of therapy can be developed. Genetic tests are indicated for treatment, diagnosis, prognosis, counseling, and research purposes; however, there is significant overlap among them. One of the important genetic tests for glaucoma is OcuGene. Therefore, it is of utmost importance for the glaucoma specialists to be familiar with and understand the basic molecular mechanisms, genes responsible for glaucoma, and the ways of genetic treatment.
Recently, several promising genetic therapeutic approaches had been investigated. Some are either used to stop apoptosis and halt further glaucomatous damage, wound healing modulating effect or long lasting intraocular pressure lowering effects than the conventional commercially available antiglaucoma medications.
The literature was searched on the Medline database using the PubMed interface. The key words for search were glaucoma, gene therapy, and genetic diagnosis of glaucoma.
The identification of the molecular events responsible for glaucoma has been difficult because of a general lack of knowledge about the cellular and biochemical events necessary for the normal regulation of intraocular pressure (IOP) and retinal ganglion cell (RGC) function. Access to human diseased tissue is also difficult, and animal models have only recently been developed and characterized. The advantage of a genetic approach is that the responsible protein can be identified without access to diseased tissue. The identification of genes (and their protein products) that can cause or contribute to glaucoma will help define the underlying pathophysiology, as well as lead to the development of new DNA-based diagnostic tests and novel therapeutic approaches.
The availability of predictive tests would provide a mechanism for early detection and treatment. Those individuals at risk who are identified early in the course of the disease and who begin therapy prior to significant damage to the optic nerve will have the best chance of maintaining useful vision.
In the broad sense, a genetic test is any clinical or laboratory maneuver that has the potential to increase or decrease the likelihood that a patient has an inherited disease. Thus, an abnormal electroretinogram in an asymptomatic 10-year-old child of a parent with autosomal-dominant retinitis pigmentosa is as much a genetic test as a molecular investigation of the rhodopsin genes.
A knowledgeable clinician is arguably the single most important component of the genetic testing process. Indeed many physicians, laypeople, and regulatory agencies often have a much narrower view of a genetic test. They tend to see it as the performance of one or more laboratory techniques that result in a black-and-white answer about the presence or absence of a disease-causing mutation. At least four features of a genetic test are of interest to a clinician and patients as well; the cost, the turn-around time, the report, and the likelihood that the test will assist in the management of the patient. Many tests are available for inherited eye diseases such as glaucoma [Table 1].[2,3] These tests are available at different laboratories in many schools of medicines in USA, Canada, and at the National Institute of Health, USA.
The indications for genetic testing can be divided into five broad categories; treatment, diagnosis, prognosis, counseling, and research. However, there is significant overlap among them. One of the important genetic tests for glaucoma is OcuGene.
InSite Vision (Alameda, CA) recently released a diagnostic kit for primary open-angle glaucoma (OcuGene) based on the TIGR/MYOC mt1 variant in the promoter region of the gene. OcuGene is the first commercialized genetic test that screens for the presence of this promoter region mutation and several coding region mutations of the TIGR/MYOC gene. The presence of mutations in the coding region has been associated with an increased probability of developing the disease. This is a noninvasive in-office test in which individual DNA sample is collected using cheek brushes. Some patients appear predisposed to development of aggressive form of the disease. OcuGene offers the clinician the mean to identify people at risk, particularly genetically predisposed nonglaucomatous family members who cannot be diagnosed with current glaucoma testing. The test is positive in 15–20% of POAG patients and 99% sensitive. It is useful for both diagnostic and prognostic purposes. Negative test is reassuring for both the patient and the doctor, whereas positive test indicates aggressive disease and necessitates treatment and closer monitoring of the patient (internal data, InSite Vision).
At present, glaucomatous optic neuropathy can be diagnosed only when damage in the form of either optic nerve axonal loss or visual function deficits is already present. The use of genetic tools makes possible both early diagnosis, before any irreversible damage has occurred, and early treatment. In addition, ruling out the existence of glaucoma can eliminate unnecessary treatment, lifelong follow-up, and perhaps, just as important, lifelong anxiety with its effect on the individual’s quality of life. Genetic testing may be particularly relevant to the following groups, who pose difficult diagnostic and therapeutic problems:
Current therapeutic options for ameliorating glaucoma involve lowering of IOP. Although neuroprotective drugs as therapy for glaucoma are promising, the use of this potential treatment option could be enhanced by early diagnosis. Patients already diagnosed with glaucoma could benefit from genetic screening if the test results could predict the severity and rapidity of disease progression. Treatment options range from mild (a β-blocker) to aggressive (initial trabeculectomy), predicting future disease behavior could help tailor therapy to the needs of an individual.
Gene therapy of ocular disorders benefits from the accessibility of the eye, the ability to visualize the diseased tissue, and the large number of specific gene defects known to be responsible for many inherited eye disorders. Mutations in the DNA sequence of a particular gene can result in a protein product that is not produced, works poorly, or has adopted a novel function that is detrimental to the cell.
Gene therapy is a technique for correcting defective genes responsible for disease development. Theoretically, a normal copy of the gene can physically take the place of the flawed gene and restore the gene function of the cell. In practice, however, actually replacing the flawed gene with a normal gene is a difficult task. Currently, the aim of gene therapy is to add a useful gene to the cell or tissue that suffers the consequences of the flawed gene. In some cases, the new gene may code for an entirely different protein whose function compensates for the protein encoded by the flawed gene.
In general, most of the current approaches to gene therapy are aimed at repairing the somatic cells of the particular tissue affected by the disease gene. Gene delivery may be tailored to the somatic cells affected by the disorder. Specific treatment of the diseased cells does not affect the other cells of the body, which include the germline cells. Because the germline cells continue to carry flawed copies of the gene, the disease may still be passed to offspring of the affected patient. Gene therapy targeted to germline cells as well as the diseased somatic cells results in successful treatment of the disease in the affected individual and prevents transfer of the disease to any offspring.
A general approach to gene therapy is to use an altered (recombinant) virus to carry the gene of interest to the desired tissue. Using genetic engineering techniques, the viral DNA is modified so that the viral genes required for virus proliferation are removed and the therapeutic gene is put in their place. Such a virus may invade the diseased tissue, become incorporated into the host DNA, and express the desired gene. Because the modified virus does not have the viral genes required for viral replication, the virus cannot proliferate, and the host cell does not die.
Diseases caused by mutations that create a gene product destructive to the cell need to be treated using a different approach. In these cases, genes or oligonucleotides that may inactivate the mutated gene are introduced into the cell. This is called “antisense therapy,” and it is proving to be a useful approach for diseases caused by the “gain of function mutations.” A number of different viral vectors likely to be useful for gene therapy are currently under investigation. In addition, evaluation of nonviral mechanisms for the introduction of therapeutic genes into diseased tissue is ongoing.
There are several approaches for correcting faulty genes:
Gene therapy works by delivering the therapeutic gene to the patients’ target cells through the carrier molecule called a vector. Currently, the most common vector is a virus that has been genetically altered to carry normal human DNA. Once the vector enters target cells, it unloads its genetic material containing the therapeutic human gene. The generation of a functional protein product from the therapeutic gene restores normal cell function [Figure 1].
Four basic prerequisites should be met for any genetic therapy targeted to an ocular disease: (1) An efficient and nontoxic gene delivery technique. (2) Sufficient characterization of the genetic basis of the disease to select an appropriately matched therapeutic approach. (3) Proper control of the expression of the therapeutic gene. (4) The availability of an animal model of the disease for preclinical testing. Glaucoma is a disease in which some of these conditions can be met. Strategies now exist that use gene therapy to modulate aqueous production and outflow and prevent RGC death.[16,17]
The clinical application of gene transfer can be accomplished in either of two ways: in vivo or ex vivo. During in vivo gene transfer, the foreign gene is injected into the patient by viral and nonviral methods. The in vivo gene delivery systems are summarized in Table 2. In contrast, an ex vivo gene transfer involves a foreign gene transduced into the cells of a tissue biopsy, outside the body, and then resulting genetically modified cells are transplanted back into patient.
Because of their role in aqueous production, drainage, and pathogenesis of glaucomatous damage, these are appropriate target structures or cell types for glaucoma gene therapy: trabecular meshwork (TM), ciliary epithelium (CE), ciliary muscle (CM), RGCs, and Muller cells (MCs). To date, six delivery systems have been tested for ability to deliver genes to the relevant tissues or cells. These are summarized in Table 3 and include adenoviruses (Ads), adeno associated viruses (AAVs), herpes simplex viruses (HSVs), lentiviruses (LVs; Feline Immunodeficiency Virus [FIV] and Human Immunodeficiency Virus [HIV]), Liposomes (LPs), and Naked DNA.
Several studies have established that adenovirus vectors (Ad vectors) can deliver transgenes very efficiently to the TM after intracameral injection. Because of the natural flow of aqueous humor, intracameral delivery of vectors carries the viruses directly to the TM. Recombinant Ads transduce all TM cell types with high efficiency in all species so far investigated (rabbits, mice, rats, dogs, monkeys, and anterior segments from postmortem human donors). Adeno associated virus (AAV) vectors appear to be unsuitable for anterior segment delivery.[19–21]
HSV vectors are capable of efficient gene delivery to structures and cells relevant to glaucoma. In monkeys and rodents, intracameral injection results in efficient delivery to cells in the TM and CE.[22,23] Only the CMV promoter has been tested with HSV vectors, and gene expression has lasted only 10 days, at most. Vector DNA persists at least 1 month after transduction, suggesting that a promoter shutoff is involved.
Delivery with LV vectors based on HIV and FIV has also been tested. Efficient delivery occurs in the TM with both types of vectors. One potential advantage of LV vectors is the ability to integrate into the host cell genome of nondividing or slowly dividing TM cells, which may result in increased duration of expression. The major drawback to integrating vectors is the potential for insertional mutagenesis, which although rare, is a serious concern.
Liposome-mediated plasmid delivery has also been achieved. TM cells are transduced, but the efficiency is low. Toxicity due to certain types of liposomes or specific constituents has also been seen. Transfer of naked DNA to anterior chamber structures has not been reported to date. Successful delivery of naked DNA to cells at the time of glaucoma filtration bleb surgery has also been achieved; efficient gene delivery to the CM in vivo has not been reported to date.
It is now well established that intravitreal delivery is the preferred route to deliver genes to the RGCs. Intravitreal injection of Ad vectors results in efficient delivery to MCs. For reasons not yet clear, Ad gene transfer to the RGCs is very limited. MCs, however, appear to be important sources of neurotrophic factors, and delivery of such secreted factors or modulation of their expression in these cells could be important in neuroprotective strategies to protect RGCs. AAV appears to have selective, stereotype-specific tropism for the RGCs.
Intravitreal injections in the rat result in up to a 72% transduction efficiency, with optimal expression occurring between 2 and 4 weeks after injection in rodents. This high efficiency may be due to the expression of membrane associated heparin sulfate proteoglycans in RGCs, which mediate attachment of AAV to cells. Heparin sulfate proteoglycans are also receptors for HSV. Delivery of HSV results in efficient transduction of RGCs. Efficiencies of up to 50% can be achieved with a single intravitreal injection in rats. Table 4 shows the glaucoma relevant tissues and vector system available.
Direct toxicity to transduced cells does not appear to be a problem with the vectors tested to date. The most common negative response has been the induction of an inflammatory response composed predominantly of monocytic cellular infiltrates in the anterior chamber. The most likely explanation for the effect is the induction of proinflammatory cytokines by the vector viruses. Vehicle components and injection per se do not appear to be involved, because inflammation is not seen in control eyes. The induction of inflammation is dose dependent, because reducing the amount of vector with Ad and HSV vectors eliminates inflammation. However, this reduces transduction efficiency, particularly with HSV vectors. Both AAV and LV vectors do not appear to induce inflammation, but most of these studies have been performed in rodents, and it is clear that species differences also play a role.
Although the genetic basis of most glaucomas remains unknown, the transfer, modulation, and expression of genes encoding IOP lowering and/or neuroprotective gene products may serve to modify the physiology of relevant cells and block the pathogenesis of the disease. As with other chronic diseases, the use of genes to treat glaucoma will provide improved efficiency and a longer duration of effect. Table 3 shows the potential target genes and tissues that could be used therapeutically to treat glaucoma.
Lowering IOP by manipulating the tissues of the anterior segment with gene therapy could represent the first immediate treatment of glaucoma. The TM, CE, and CM are all potential targets. The TM’s juxtacanalicular region and inner wall of Schlemm’s canal constitute the primary barrier to aqueous humor before it leaves the eye. Manipulation of the biochemistry of the cells and extracellular matrix in these regions has the potential to modulate outflow resistance and lower IOP. Investigators have successfully transferred genes to this tissue with different vectors and through different routes of administration [Table 4].
Several investigators deliver potential physiologically relevant genes, in addition to reporter genes. Kee et al. have demonstrated that an Ad vector carrying the metalloproteinase stromelysin can be transduced to TM cells of rats after intracameral injection. In human postmortem perfused organ cultures, Ad vectors carrying wild-type myocilin and genes that affect the cytoskeleton increase outflow facility.[29,30] Lentivirus vector was also used with high efficiency to transduce the human trabecular meshwork (HTM) with overexpression of metalloproteinases, green fluorescent proteins (GFP) for at least 10 months after injection. In addition, AAV had been used by Borras et al. and demonstrated genes and mechanisms which lead for the first time to efficient AAV transduction of the HTM.
The final common outcome of glaucoma is RGC death. The stimulatory mechanism or mechanisms causing RGC death are unclear, but several strategies to block RGC apoptosis are available. The goal of gene therapy in glaucoma, therefore, is to slow the rate of RGC death. There are a number of strands of evidence that suggest brain-derivied neurotrophic factor (BDNF) as a potential neuroprotective agent in glaucoma. RGC are trophically dependent on BDNF, retrogradely transported from target areas in the brain to ganglion cell bodies in the retina.
Several investigators are now exploring the use of gene transfer to deliver a neurotrophic factors. Ad-mediated intravitreal delivery of BDNF has been shown to protect RGCs in a rat optic nerve transection model. MCs are transduced by Ad and enhancement of RGC survival is due to secretion of BDNF from these cells. Even though the expression of the transgene was prolonged to 1 month by the use of immunosuppressants, the protective effect was transient. Furthermore, AAV-mediated TrkB gene transfer into RGCs combined with exogenous BDNF administration markedly increases neuronal survival.
In addition, AAV incorporating cDNA was used for BDNF to transfect RGC in a rat model of glaucoma. A high proportion of RGC were transfected after a single intravitreal injection. After 4 weeks of experimental glaucoma, eyes that received intravitreal AAV-BDNF 38% of RGC were rescued by the AAV-BDNF.[33,34] Other studies have shown that AAV can mediate transgene expression in RGC for at least 1 year. Isenmann et al. also found protection of the RGCs after Ad-mediated delivery of BDNF, and protection was increased by the combined systemic administration of the free radical scavenger N-tert-butyl-(2-sulfophenyl)-nitrone (S-PBN). Similar RGC survival results were obtained recently with Ads containing the ciliary neurotrophic factor (CNTF).
Another approach to gene therapy in glaucoma has recently been reported by McKinnon and coworkers. They have taken a different approach, involving the modulation of pathways responsible for apoptosis in RGC. The final common pathway of RGC apoptosis involves the activation of caspase enzymes that participate in the mechanisms of cell suicide. McKinnon et al. injected an AAV vector coding for human baculoviral IAP repeat-containing protein-4 (BIRC4), a potent caspase inhibitor, into one eye of rats. BIRC4 promoted RGC survival, presumably by inhibiting the enzymatic completion of apoptosis.[38,39]
Gene therapy could also be used to prevent the proliferative wound-healing response that follows glaucoma filtration surgery. Using an Ad vector encoding the cell cycle inhibitor p21 (rAd-p21). Perkins et al. inhibited proliferation of Tenon’s fibroblasts in rabbits with a single administration of rAd-p21 (by applying a vector-soaked sponge for 5 min). This resulted in the maintenance of functional filtration blebs at 30 days after surgery without the severe tissue effects observed in the mitomycin-treated eyes. Other investigators have used naked DNA to transfer the reporter gene chloramphenicol acetyltransferase to the same cells.
Antiproliferative gene therapy has been used adjunctively with glaucoma surgery in animal models. A recombinant adenovirus (rAd) was used to introduce the human gene for p21WAF-1/cip-1 (p21) into rabbit Tenon’s fibroblasts in a normotensive model of glaucoma surgery. This transgene product normally blocks entry of dividing cells into S phase of the cell cycle by antagonizing the activity of several cyclin-dependent kinases. The expression of p21 in the rabbit model persisted for more than 2 weeks after transduction and was as effective as mitomycin C (MMC) at reducing IOP and preventing fibroproliferation over a 4-week period. Unlike MMC, however, the p21 gene therapy produced no toxic effects in rabbits.
Gene therapy using naked plasmid DNA and a simple collagen shield delivery vehicle or injected directly subconjunctivally may be useful for regulating wound healing after glaucoma surgery. Transfection of filtration tissues is enhanced by absorption of naked DNA into a collagen shield soaked in a solution containing the plasmid. Furthermore, transfection is localized to the fibroblasts and inflammatory cells of the filtration bleb site.
An interesting poster presented in the ARVO meeting by Jimenez et al. 44about the therapeutic use of siRNA (small interfering RNA) in OHT and glaucoma treatment. siRNA holds great therapeutic promise for gene silencing in a nontoxic and highly effective way, as it is naturally used by cells to regulate gene expression. In this study, the effect of siRNAs targeting different isoforms of ATPases and cyclooxygenases on IOP was investigated in rabbits. Latanoprost and dorzolamide were used as control drugs. The results showed that IOP decreases by siRNA were comparable with those by commercially available drugs. The IOP-lowering effect of siRNA lasted longer (about 100 h) than that of commercially available drugs.
The following are limiting factors on the use of gene therapy:
Despite the above limitations, the future hold promises for gene therapy in glaucoma. Gene therapy could be used in two ways in glaucoma: as a drug delivery system, and as a basis for developing new therapies and treatment end points based on the genetic mutations that cause glaucoma. If indeed the TIGR/MYOC gene or separate genes could be shown to be a risk factor for earlier onset or more progressive disease, a patient’s therapeutic end points could be modified based on his or her genetic profile. In the future, such a patient may start therapy earlier in life and be maintained at a lower IOP to help prevent glaucomatous progression and visual loss. Consequently, the genetic profile may help individualize patient therapy to better ensure a stable glaucomatous disease course.
Source of Support: Nil
Conflict of Interest: None declared.