RNA interference as a gene silencing therapy for mutant MYOC protein in primary open angle glaucoma

Diagnostic Pathology Hypothesis RNA interference as a gene silencing therapy for mutant MYOC protein in primary open angle glaucoma Mao Li 0 Jianjiang Xu 0 Xueli Chen 0 Xinghuai Sun 0 0 Address: Department of Ophthalmology and Vision Science, Eye and Ear Nose Throat Hospital, Shanghai Medical School, Fudan University , No. 83, Fenyang Road, Shanghai , PR China Background: Primary open-angle glaucoma (POAG) is the most common form of glaucoma which is an irreversible blind leading disease and lacks effective remedies. In recent years, POAG has been linked to the gene MYOC encoding myocilin that has been identified to harbor causal mutations. A variety of studies show that the mutant myocilin acts by gain of function. The mutant MYOC protein induces endoplasmic reticulum (ER) stress and the resultant unfolded protein response (UPR) induces apoptosis in the trabecular meshwork cells, which then leads to an increase in resistance to aqueous humor outflow, elevated intraocular pressure (IOP), and, ultimately, glaucoma. Culturing human trabecular meshwork (HTM) cells at a condition facilitating protein folding promotes secretion of mutant myocilin, normalizes cell morphology and reverses cell lethality. Presentation of the Hypothesis: We speculate that a complete elimination of mutant myocilin expression in trabecular meshwork cells is safe and that gives the possibility of avoiding the POAG phenotype. Testing the Hypothesis: We propose RNA interference (RNAi) as a gene silencing therapy to eliminate the mutant myocilin proteins in the trabecular meshwork cells, either in a mutationdependent or mutation-independent way due to the different engineering of the small interfering (si) RNA. Implications of the Hypothesis: The RNAi strategy can reverse the pathological process of trabecular meshwork cells and thus treat the POAG caused by myocilin gene mutation. This strategy can also be applicable to many protein-misfolding diseases caused by gain-of-function mutant proteins. - Background Glaucoma is a group of progressive optic neuropathies that have in common a slow progressive degeneration of retinal ganglion cells and their axons, resulting in a distinct appearance of the optic disc and a concomitant pattern of visual loss[1]. Without adequate treatment, glaucoma can progress to irreversible visual disability and eventual blindness [1]. Of the many types of glaucoma, primary open angle glaucoma (POAG) is perhaps the most common, particularly in populations of African ancestry and European [2-4]. In most cases of POAG, increased resistance to the outflow of aqueous humor results in a rise in intraocular pressure (IOP), which eventually leads to loss of retinal ganglion cells[5]. Though Increased IOP has been proven to be the only treatable risk factor for glaucoma, the biological basis of the disease is not yet fully understood[1]. This may underlies that the present treatment of POAG directed at lowering IOP[6] does not seem to halt all cases of progression [7-10]. To date, six loci (GLC1A-E) have been linked to POAG alone. Among them, the gene MYOC encoding myocilin has been identified as harboring causal mutations, which are responsible for 3-4% adult-onset POAG cases [11]. This warrants the gene therapy which holds the promise for curing the disease. Current investigations of gene therapy mainly focus on the transfer and expression of genes encoding IOP-lowering, neuroprotective gene products, and/or, wound healing inhibitors[12], but do not really emphasize a specific elimination of the causal mutant proteins. Here we propose RNA interference (RNAi) as gene silencing therapy for complete elimination of mutant myocilin from human trabecular meshwork (HTM) cells. Presentation of Hypothesis The human myocilin gene encodes a 504 amino acid glycoprotein expressed extra- or intracellularly in almost every ocular tissue[13,14] but predominantly in the trabecular meshwork [15]which is a tissue provides major resistance to the aqueous humor outflow pathway, and the tissue involved in elevated IOP associated with glaucoma. However, MYOC-null individuals (no matter mice [16] or human beings [17,18] have normal eyes and intraocular pressure. These individuals are both viable and fertile as well [16-18]. Overexpression of wild type (WT) MYOC in transgenic mice does not lead to any glaucomatous phenotype[19,20]. All these facts suggest that haploinsufficiency of MYOC is not a critical mechanism in the pathogenesis of POAG [16-20]. A variety of recent studies show that the mutant myocilin acts in the HTM cells by gain of function[16,21-27]. It was observed that, in culture media, very little to no mutant myocilin associated with the development of glaucoma was secreted out of the HTM cells, while WT and polymorphism variant myocilin were secreted normally[26]. Further investigations found that the disease-causing myocilin mutants were actually misfolded, were highly aggregation-prone and accumulated in large aggregates or formed heteromeric complexes with WT myocilin in the endoplasmic reticulum (ER)[5,22,24,28]. The ER is responsible for the synthesis, modification and delivery of correctly folded proteins to their proper target sites. Increased expression of mutant, folding-incompetent proteins causes ER stress and an ER stress response, called the unfolded protein response(UPR). The UPR is an adaptive mechanism to return the ER to its normal physiological state by upregulating the ER folding capacity and downregulating the biosynthetic load of the ER. When the UPR does not remedy the stress situation, apoptosis is initiated in higher eukaryotic organisms, presumably to eliminate unhealthy cells [29]. In the case of MYOC-associated POAG, the aggregates induced the unfolded protein response proteins BiP and phosphorylated endoplasmic reticulum-localized eukaryotic initiation factor-2_ kinase (PERK) with the subsequent activation of caspases 12 and 3 and expression of C/EBP homologous protein (CHOP)/ GADD153, leading to abnormal HTM cell morphology and cell apoptosis[24,28]. The progressive loss of HTM cells due to apoptosis should accordingly diminish the phagocytotic capacity of the remaining TM cell population to clean the outflow drainage and eventually results in humor outflow resistance and elevated IOP[24]. This is consist with the electron microscopic findings from biopsies of POAG patients showing a reduction of TM cells as well as thickened trabeculae and accumulation of sheathderived plaques [30-33]. Since the mutant myocilin induces ER stress and eventually leads to the apoptosis of HTM cells and hence the pathogenesis of Myoc- associated glaucoma, a complete elimination of mutant myocilin in HTM cells may disrupt or even partly reverse the disease. In support of this proposal are two major previous investigation findings: (1) Culturing HTM cells at a condition facilitating protein folding promotes secretion of mutant myocilin, normalizes cell morphology and rever (...truncated)