BY GEAROID TUOHY PHD
BIO-OPHTHALMOLOGY
                                   Ancient defence turns into future medicine

Rapid advances in our understanding of how several different organisms defend themselves against infection are now opening a host of technologies that may transform medicine and ophthalmology over the coming decade.

In particular, patients with age-related macular degeneration (AMD) and diabetic retinopathy may ultimately benefit from ongoing research.Researchers believe that ribonucleic acid interference, or "RNAi" for short, is a biological process that evolved to counter viral infections and a number of other enemies.Potential applications for RNAi are so huge that they have already attracted significant attention from biotechnology investors. For instance, a recent Fortune magazine article described the impact of RNAi as "Biotech's Billion Dollar Breakthrough!" Within the last two years, biotechnology investors have collectively invested millions in "RNA" companies such as Alnylam, Sirna, Ribopharma, and Archemix.

So what is RNAi, and where did it all come from? And most importantly for ophthalmology, what impact will it have on eye diseases? To begin with, there is a rather ironic connection between RNAi and the human eye in the story of how researchers first identified the process.A decade ago, in a bid to make flowers more aesthetically pleasing to the eye with deeper petal colours, plant genetic researchers began experimenting with genes responsible for flower pigmentation. By introducing such genes into plants, researchers expected to breed varieties with more vivid colours; instead, the researchers got the opposite result, creating flowers with variegated pigmentation or no pigmentation at all! Introducing such genes, it appeared, somehow affected the normal expression of flowers' endogenous pigment genes.

Of course, the researchers became curious about the cause of the reaction. In examining the cause, researchers stumbled onto RNAi. In particular, the researchers found that when plants were attacked by certain viruses one way that the plant reacted was to rid itself of the pathogen by destroying the virus's RNA. The rationale here was that by destroying the virus' ability to translate viral RNA into viral protein, the plant could halt the infection in its tracks. At the time, researchers dubbed the phenomenon "PTGS" for "post transcriptional gene silencing." Today researchers now refer to the phenomenon as RNA interference or just "RNAi." In essence, RNAi is a biological mechanism that interferes with the information flow between RNA and protein. When researchers observed what a plant did to counteract a viral pathogen, they soon began to wonder if they could engineer plant viruses to carry other gene sequences and trick the plant into targeting artificially inserted genes for destruction. The experiment worked and lit a fuse that has today opened an entirely new frontier in the biological sciences.

Many plant viruses instead of carrying a double stranded DNA molecule carry a double stranded RNA molecule which the virus uses to mediate its infectious pathology within the plant. A key finding in these early experiments demonstrated that double stranded RNA (dsRNA) was a potent initiator of RNAi. The viral dsRNA package triggers the plants' defence system to target such complementary RNA for destruction. Soon, researchers found that artificially introducing dsRNA into either plant or animal cells initiated a similar gene silencing response by the host. With such information, research groups in the area quickly worked out the biological mechanics of what was going on: once it detected dsRNA, the plant or animal produced enzymes that chopped up the dsRNA into shorter fragments called small interfering RNAs, or "siRNAs," for short. The siRNAs then assembled into complexes with certain proteins where they guided the entire RNA-protein assembly to complementary RNAs which are then cleaved and destroyed. In essence, researchers havediscovered a highly flexible but exquisitely specific gene silencing apparatus.

The key links here for medicine are the siRNAs. If researchers could design these short molecules to specifically fish out disease-causing genes or cancer-causing genes, then medicine could leverage the natural biology of the body to destroy such targets and potentially devise realistic treatments for a range of disorders, including AMD and diabetic retinopathy.Among those leading the research into AMD is a team of scientists and clinicians in the United States at Acuity Pharmaceuticals in Philadelphia (www.acuitypharma.com) and at the F.M. Kirby Centre for Molecular Ophthalmology at the Scheie Eye Institute of the University of Pennsylvania .

As ophthalmologists know, AMD is a collection of conditions characterised by degenerating visual acuity which afflict approximately 10% of Western populations over the age of 65 years. So-called "wet" AMD is characterised by the development of abnormal blood vessel growth – called "choroidal neovascularisation" – under the centre of the retina. These new blood vessels are prone to bleeding and leakage causing the macula to bulge or lift and distort central vision. A sudden rupture can cause immediate, albeit transitory, blindness for a distressed patient. The new blood vessel growth, or "angiogenesis", is caused by the expression of a vascular growth factor known by the abbreviation of "VEGF" for vascular endothelial growth factor. Many therapeutic rationales now attempt to interfere in the biological role of VEGF in an effort to interfere with new blood vessel growth. Although VEGF as a therapeutic target is well established, siRNA as a tool to abrogate its function is a new weapon in the battle to control the disease.

Against such a background, the Acuity and Kirby Centre research team carried out experiments to answer the following questions:
1. Can siRNAs silence target genes in cells?

2. Can these siRNAs be delivered into cells at the back of the eye in a mouse model of AMD?

3. Can target genes be silenced in live animal models?

4. Can such technology inhibit neovascularisation in a standard model of AMD?

The research team generated siRNA molecules against mouse VEGF and human VEGF. They also generated siRNA molecules against an experimental research tool known as "GFP" for green fluorescent protein to monitor the effect of the siRNAs. Under specific conditions, GFP lights up like a beacon, allowing researchers to track where and when in the cell it appears.Researchers first tested siRNA against VEGF in cells. Those tests demonstrated that siRNA could target and effectively silence VEGF. The researchers then used artificial vectors – engineered viruses that can act as a Trojan horse to transport foreign DNA into a cell – to carry siRNAs and GFP molecules into retinal pigment epithelium cells at the back of a mouse retina. Those tests demonstrated that the green beacon could be turned on and off like a light switch in the absence and presence of siRNAs.

The researchers' final experiments assessed the effectiveness of siRNA in reducing both levels of VEGF in the retina and in reducing the neovascularisation characteristic of AMD. The researchers carried out those experiments in artificial animal models of AMD; those tests demonstrated that siRNA reduced VEGF concentration and choroidal neovascularisation by about 75%. "To our knowledge, this is the first time that siRNA has been shown to silence the target gene in a clinically relevant animal model of ocular disease," commented the study's supervisor, Michael J. Tolentino MD, assistant professor at the University of Pennsylvania and Scheie Eye Institute. "We are encouraged that these proof-of-principal results may translate into a new class of highly effective siRNA therapeutics for diseases like wet macular degeneration and diabetic retinopathy, the two leading causes of adult blindness."

Glossary:

Complementary RNA: DNA normally forms a double helix in which one strand is complementary to the other via "base pairing" in which the letters of the genetic code "A," "G," "C," and "T" pair up. A pairs with T, and C pairs with G. Under certain conditions, similar pairing can occur with RNA and a complementary RNA sequence.

dsRNA: RNA – unlike DNA – usually occurs as a single stranded molecule; in certain circumstances, however, RNA can base pair with complementary sequence to form a double stranded RNA. Many pathogenic viruses have dsRNA genes.

Engineering (of a gene): Modern molecular biology allows researchers to mix and match genes from different organisms; much of the biotechnology industry is based on this simple technology. Genetic engineering is conducted at the lab bench by simply cutting and pasting genes form one sample of DNA to another.

GFP-green fluorescent protein: A research tool used in molecular biology in a variety of experiments. For instance, GFP can test if a cell type is capable of being engineered with a foreign gene. GFP, under specific conditions, emits a green light (like that of a firefly) allowing researchers to track its expression.

RNA: The messenger molecule that assists in converting the instructions of DNA into a working protein which can then carry out useful functions within the cell.

RNAi: RNA interference, a newly discovered molecular phenomenon that turns off gene expression in a highly specific manner. siRNA: Stands for "short interfering RNA," which seeks out complementary RNA and tags it for destruction by an RNA-protein assembly.

Translation (of a gene): Once RNA has been transcribed from DNA in a cell, it must then be converted into protein through a process known as translation.

Vector: A lab-engineered virus in which pathogenic genes are removed and replaced with foreign genes. The vector may then be used to infect cells and thereby transport the foreign genes.