Glaucoma Research Foundation

Analysis of Progression in Glaucoma

Paul Habib Artes, PhD, Dalhousie University, Halifax, NS, Canada: $40,000

When patients with glaucoma are followed over time, the aim is to detect worsening of the disease and to measure its rate, so that the therapy can be optimally tailored to the individual patient’s risk of sight loss. Doctors as well as patients must be very certain that apparent change in the measurements reflect real deterioration, and that real deterioration is reflected in the measurements. Because patients with glaucoma differ greatly in how rapidly the disease progresses and how closely clinical measurements reflect these changes, the number of visits and the types of test must be adapted to each patient’s needs.

Dr Artes’ project aims to establish ways to estimate a test’s power and to identify poor test results, which will help to decide how often patients need to be seen and which tests to apply. They will establish statistical methods based on random reordering of test results to interpret change, detect outliers in the data, and estimate what kind of change could have been detected had it been present. Briefly, the idea is to take a patient’s measurements from several visits, randomly re-arrange them, and compare the real data with the randomly re-arranged data. This is done many hundreds or thousands of times until a complete distribution of the possible random change estimates emerges. The aim is to translate these findings into terms that clinicians and patients can readily understand.

Genome-wide Association in Primary Open Angle Glaucoma: The Blindness in Glaucoma Genetic Epidemiology Relative Risk Study

Jamie Craig, PhD, Flinders University of South Australia: $40,000

Finding common genetic markers which lead to glaucoma blindness.

Dr. Craig’s lab will use well-characterized cohorts of severe cases of glaucoma, and the power of high density gene screening to identify multiple genetic risk factors leading to poor outcomes in glaucoma. The strategy of concentrating analysis on the most severe disease cases in conjunction with high resolution genetic mapping has been chosen to maximize the probability of finding the most important genetic associations.

The significance to people with glaucoma is that the level of risk of progressing to blindness for any individual diagnosed with glaucoma is currently difficult to calculate. Glaucoma treatment currently balances the benefits of slowing disease progression against the risks of treatment. It would therefore be highly useful to understand genetic associations of severe glaucoma so as to lead to earlier and better treatment in those at highest risk, while freeing other lower risk individuals of the need for treatment.

Imaging the Course of Axonal Degeneration in Experimental Glaucoma

Brad Fortune, OD, PhD, Devers Eye Institute, Portland, OR: $40,000

Retinal nerve fiber layer (RNFL) defects may be one of the earliest signs of damage in glaucoma. The majority of eyes with elevated intraocular pressure already have clinically detectable RNFL defects before reproducible abnormalities first appear in the visual field. Thus RNFL evaluation has become part of the clinical standard of care for all glaucoma patients and suspects. Yet evaluation of the RNFL by conventional techniques remains a challenging clinical task, which has helped drive development of two digital imaging technologies for objective assessment of the RNFL: optical coherence tomography (OCT) and scanning laser polarimetry (SLP).

These two technologies are based on different optical principles. OCT measures the reflectance of structures within the retina and is thus able to detect the borders between various retinal layers such as the RNFL. In contrast, SLP measures what is known as “retardance” of polarized light. Retardance is caused by a tissue property called birefringence, which in the case of the RNFL, is thought to depend on the integrity of structural elements inside the retinal ganglion cell (RGC) axons, including microtubules (MT) and neurofilaments (NF). Previous studies suggest that alterations of MT and/or NF precede irreversible axon loss and manifest as altered birefringence measurements (i.e. are measurable by SLP).

Dr. Fortune’s lab plans to test the hypothesis that degradation of axonal NF and/or MT is clinically detectable and precedes complete degeneration and loss of RGC axons in an experimental model of glaucoma.

RNAi Gene Silencing of Enzymes in the Glycosaminoglycan Biosynthetic Pathway

Kate E. Keller, PhD, Casey Eye Institute, Portland, OR: $40,000

In primary open-angle glaucoma (POAG), there is increased resistance to aqueous flow through the trabecular meshwork TM, which results in increased intraocular pressure (IOP). Glycosaminoglycans (GAGs) are a likely source of outflow resistance. GAGs are long, linear sugar chains that are usually covalently attached to a protein backbone. Previous studies have shown that treatment of anterior segments with GAG degrading enzymes or with two modifiers of GAG biosynthesis, sodium chlorate and β-xyloside, resulted in an increase in outflow facility. Moreover, mRNA levels for several enzymes involved in GAG chain biosynthesis are modified in POAG eyes and in TM cells subject to TNFα or IL-1α treatment.

Dr. Keller’s lab will further investigate the role of GAG chains in outflow resistance, genes for enzymes in the GAG chain biosynthetic pathway will be silenced using RNA interference (RNAi) technology. This technique introduces short, double-stranded RNA molecules into cells to selectively inhibit expression of a target gene.

First, genes specific for different types of GAG chains will be inhibited in order to identify GAG important for outflow resistance. Second, genes for enzymes involved in GAG chain sulfation will be silenced. Double-stranded oligos for target genes will be designed and cloned into the shRNA vector pENTR/U6.These will be transfected into TM cells using the Amaxa nucleofection system, which provides high transfection efficiency. Silencing will be confirmed by quantitative RT-PCR using gene-specific primers. Extracellular matrix (ECM) synthesized by transfected TM cells will be analyzed by immunofluorescence (IF) and Western blotting to investigate the effects of silencing on ECM protein interactions.

In addition, shRNA silencing inserts will be transferred into the Block-it Lentiviral RNAi expression system so that the effects of silencing vectors can be directly assessed on outflow facility in anterior segment perfusion culture. Again, changes in ECM organization in the TM will be assessed by IF.

RNAi is an emerging technology that is well suited as a disease-modifying therapy. Results from the experiments proposed here will further investigate the role of GAG chains in outflow resistance and may lead to new therapies for POAG patients.

Development of Pharmacological Chaperone Therapy for Inherited Primary and Juvenile Open Angle Glaucoma

Raquel L. Lieberman, PhD, Georgia Institute of Technology, Atlanta, GA: $40,000

Dr. Lieberman’s lab aims to develop a new therapy for inherited glaucoma, many cases of which are caused by mutations in a protein called myocilin. Myocilin forms part of the trabecular extracellular matrix, or TEM, which is important in regulating eye pressure.

When the matrix doesn’t function correctly, eye pressure increases, leading to retinal degeneration and vision loss. Human trabecular meshwork (HTM) cells, which produce the matrix, recognize mutations in myocilin and prevent myocilin from being secreted to the TEM. Instead, mutant myocilin remains in the interior of HTM cells, causing the HTM cells to die. This results in a disrupted TEM, increased eye pressure, and eventually glaucoma.

Dr. Lieberman’s lab hopes to identify a drug molecule that interacts with mutant myocilin inside the HTM cells and restores secretion of myocilin to the TEM. The ability to secrete mutant myocilin will have two desired effects: (1) the mutant protein will not accumulate in the HTM cells and thereby help keep these cells alive and (2) the TEM will be restored and better control intraocular pressure. Taken together, this approach will retard retinal degeneration associated with glaucoma. They will accomplish their long-term goal by first unveiling the molecular structure of myocilin, and then identifying and testing drug candidates that bind to specific grooves on the surface of myocilin.

Investigation of Gene Copy Number Variants in Primary Open Angle Glaucoma

Yutao Liu, MD, PhD, Duke University Medical Center, Durham, NC: $40,000

Dr. Liu’s lab plans to investigate DNA copy number variants (CNVs), a form of genetic variation associated with inherited diseases, in patients with primary open-angle glaucoma (POAG). CNVs include small portions of DNA that are duplicated or deleted in individuals. These duplications and deletions vary in size from a few thousand DNA basepairs to millions of basepairs. CNVs that are more common among individuals with POAG compared with those without POAG will be studied as potential risk factors for glaucoma.

This study may lead to the identification of novel genetic components that increase individual risk to POAG and will improve our understanding of the inherited mechanisms of POAG.