GENE THERAPY IN GLAUCOMA
Dr Haimanti Choudhury, MS
Glaucoma is a progressive optic neuropathy and a leading cause of blindness, effecting about 70 million people worldwide. It is characterized by a particular pattern of optic nerve head and visual field damage resulting from a number of diseases that affect the eye. Many of these are associated with elevated intraocular pressure (IOP), which is the most common known associated risk factor .The final common pathologic event is retinal ganglion cell (RGC) death.
Studies have emphasised that a significant proportion of glaucoma cases are genetic1. As the understanding of the genetics of glaucoma advances, treatment of glaucoma by gene therapy is now a distant possibility. Gene therapy in the anterior and posterior segment tissues may have the potential to favourably influence aqueous hydrodynamics and retinal ganglion cell biology, thereby preventing, delaying, or minimizing glaucomatous damage to the optic nerve2. In recent years, impressive progress has been made in the molecular genetics including discovery of three genes, Myocillin,Optineurin and CYP1B1. Mutations in the myocilin gene cause autosomal dominant juvenile primary open-angle glaucoma and approximately 3% of cases of adult-onset open-angle glaucoma1. Events leading to RGC damage and death are targets for genetic modulation.
Gene therapy can be used in two ways: as a drug delivery system and as a basis for developing new therapies and treatment based on the genetic mutation which cause glaucoma3.
Target Tissues and Delivery Systems
Target structures or cell types for glaucoma gene therapy include trabecular meshwork (TM), ciliary epithelium (CE), ciliary muscle (CM), Retinal ganglion cells, and Müller cells (MC). To date, six delivery systems have been tested for ability to deliver genes to the relevant tissues or cells4.
These include adenoviruses (Ads), adenoassociated viruses (AAVs), herpes simplex viruses (HSVs), lentiviruses (LVs; feline immunodeficiency virus [FIV] and human immunodeficiency virus [HIV]), liposomes (LPs), and naked DNAs. Borras et al5 showed that single doses of 107, 106 and 105 p.f.u.(plaque forming unit) do not affect outflow facility and retain positive gene transfer. These findings indicate that adenovirus, at effective doses, could become useful vectors for gene therapy of glaucoma. Ad vectors can deliver transgenes very efficiently to the TM after intracameral injection. 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 CE4. One potential advantage of LV vectors is the ability to integrate into the host cell genome of non dividing or slowly dividing TM cells, which may result in increased duration of expression. 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 muller cells. AAV appears to have selective, stereotype-specific tropism for the RGCs. Delivery of HSV results in efficient transduction of RGCs. The most common negative response has been the induction of an inflammatory response composed predominantly of monocytic cellular infiltrates in the anterior chamber. This occurs more often in primates than in rodents, at least with HSV vectors.
Target Genes
The transfer 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. 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.
Kee et al.6 have demonstrated that an Ad vector carrying the metalloproteinase 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.
Aqueous humor, which contains numerous factors that can signal the cells of the TM and modulate their resistance characteristics, is produced and secreted by the CE. Efforts to identify relevant genes have revealed that the CE has neuroendocrine activity, releasing hormones and regulatory peptides. Manipulation of the concentration of such CE factors may be an attractive target for glaucoma gene therapy.
The final common outcome of glaucoma is retinal ganglion cell death. Repeated intraocular injections of neurotrophic factors temporarily rescue RGCs from axotomy-induced death. Ad-mediated intravitreal delivery of brain-derived neurotrophic factor (BDNF) has been shown to protect RGCs in a rat optic nerve transection model. AAV-mediated TrkB gene transfer into RGCs combined with exogenous BDNF administration markedly increases neuronal survival. Isenmann et al.7 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).
Conjunctival wound healing after glaucoma filtration surgery is a major determinant of the long-term clinical success of the procedure. A major goal of glaucoma filtration surgery is to identify the molecule(s) that play key roles in regulating conjunctival scarring and to develop agents that selectively and controllably inhibit excess scarring and bleb failure. A unique aspect of glaucoma filtration surgery healing is the bathing of wound tissues by aqueous humor. The presence of multiple growth factors in normal aqueous humor and tears, especially latent transforming growth factor(TGF-ß2) suggests that components in aqueous humor can influence conjunctival scarring.8 Recently, repeated injections of a recombinant monoclonal neutralizing antibody to human TGF-ß2 significantly inhibited conjunctival scarring in a rabbit model of filtration surgery. Gene therapy using naked plasmid DNA and a simple collagen shield delivery vehicle may be useful for regulating wound healing after glaucoma surgery9. The cellular localization of gene expression predominantly in fibroblasts is an important finding.
In Site Vision has recently released a diagnostic kit for Primary open angle glaucoma based on TIGR/MYOC mt1 variant in the promoter region of the gene3.
Future of gene therapy
Several systems are available for gene delivery to tissues relevant for glaucoma gene therapy. It is clear that multiple therapeutic strategies are available to affect aqueous production and outflow to modulate IOP and to protect retinal ganglion cells from apoptosis. New therapies could be derived from glaucoma related genes. Enzymes or proteins produced by abnormal gene could be identified; their action prevented or turned off. Thus, the field of glaucoma gene therapy is poised to provide significant advances in alleviating blindness due to this disease.
References:
- Brahmbhatt. Genetics as a basis of glaucoma. Ophthalmology Today2005;6(3),85-87
Kaufman PL et al. A perspective of gene therapy in the glaucomas. Surv Ophthalmol.1999 Jun;43 Suppl 1:S91-7 - M. Yanoff, J.S.Duker. Ophthalmology.2nd edition
- Borras et al. Gene Therapy for Glaucoma: Treating a Multifaceted, Chronic Disease Investigative Ophthalmology and Visual Science. 2002;43:2513-2518
- Borras et al. Adenoviral reporter gene transfer to the human trabecular meshwork does not alter aqueous humor outflow. Relevance for potential gene therapy of glaucoma. Gene therapy 1999;6(4),515-524
- Kee, C, Sohn, S, Hwang, JM. (2001) Stromelysin gene transfer into cultured human trabecular cells and rat trabecular meshwork in vivo Invest Ophthalmol Vis Sci 42,2856-2860
- Isenmann, S, Klöcker, N, Gravel, C, Bähr, M. (1998) Short communication: protection of axotomized retinal ganglion cells by adenovirally delivered BDNF in vivo Eur J Neurosci 10,2751-2756
- Stroman, GA, Stewart, WC, Golnik, KC, Cure, JK, Olinger, RE. (1995) Magnetic resonance imaging in patients with low-tension glaucoma Arch Ophthalmol 113,168-172
- Angella et al. Enhanced Short-Term Plasmid Transfection of Filtration Surgery Tissues Investigative Ophthalmology and Visual Science. 2000;41:4158-4162
