Jeffrey S. Buguliskis Ph.D. Technical Editor Genetic Engineering & Biotechnology News

A GEN 35th Anniversary Retrospective

Only five short years after the launch of GEN magazine—1986 to be precise—in the early heydays of genetic engineering, molecular geneticist Richard Jorgensen, Ph.D., was working for a then small biotech company called Advanced Genetic Sciences, which later became DNA Plant Technology Corp. In what Dr. Jorgensen and his colleagues hoped would have been a tantalizing display of their control and understanding of plant genetics, the researchers attempted to create an extremely dark purple petunia, to garner the attention and financial backing of some venture capitalist groups.

Dr. Jorgenson knew which gene controlled purple pigment in the flower, and he was aware that petunias are very amenable to DNA transfection. The researchers logically surmised that additional copies of the purple gene should make petunias that were shades darker. However, to the investigators’ surprise and dismay, after they added an extra version of the purple gene to a petunia, the plant did not produce dark purple flowers—instead it bore stark white flowers devoid of pigment.

After Dr. Jorgenson and his colleagues verified that the gene they had placed into petunias was correct, these scientists—along with a horde of molecular biologists—spent the next several years trying to figure out what went wrong. During this time, they had little idea they were doing work that would prove to be so significant. It not only won Nobel Prizes for two scientists, but it also led to a discovery that would spark a molecular revolution.


Simple, But Elegant

In the early 1990s, researchers Andrew Fire, Ph.D., and Craig Mello, Ph.D., were investigating gene-expression mechanisms in the classical genetic animal model Caenorhabditis elegans. They found that separate injections of either sense or antisense messenger RNA (mRNA) that encoded for various muscle proteins produced no phenotypic or behavioral changes. However, when the researchers injected sense and antisense mRNA together, the worms responded with a unique and characteristic twitching effect. Interestingly, these erratic movements were similarly found in worms that lacked a functional gene for a muscle protein that corresponded to one of the experimental mRNAs.

After some years investigating this phenomenon, Drs. Fire and Mello surmised that in their original experiments, injecting single-stranded sense or antisense mRNA led these molecules to pair up with their “mirror image” counterparts in the cell, resulting in no observable changes. However, through a series of elegant experiments, the researchers, and their colleagues found that the addition of double-stranded RNA (dsRNA) had the capacity to silence the endogenous genes that matched the sequence of the injected RNA. In every experiment, injection of dsRNA carrying a homologous genetic sequence led to the silencing of that gene and subsequent reduction in the accompanying protein levels.

Curiously, the research team found that the induced RNA interference—or RNAi as it is now called—was capable not only of spreading from cell to cell within an organism but of passing to the organism’s offspring. Moreover, RNAi genetic silencing required only minuscule amounts of dsRNA to achieve the desired effect—which led Drs. Fire and Mello to propose that RNAi was a catalytic process. The researchers published their findings in Nature on February 19, 1998, clarifying contradictory results seen previously—in Dr. Jorgenson’s petunias, for example—and starting the race to uncover the molecular mechanisms that regulated RNAi within the cell.



New Opportunities Emerge

The years following the initial RNAi publication saw a massive influx of research describing RNAi mechanics with new terms being introduced into the biological lexicon such as Dicer, RISC, and Argonaute to explain the RNA-dependent gene silencing process. At the same time, many investigators had fixed their gaze toward clinical applications of this new gene-silencing technique. The potential for this method to treat and possibly even cure diseases as diverse as blindness and cancer was viewed, at that time, to be limitless.

The continued study of RNAi allowed researchers to discover that dsRNA sequences approximately 70 nucleotides in length, called short-hairpin RNAs (shRNAs), may be cleaved by the Dicer enzyme into smaller molecules. These molecules, which are 21 nucleotides long, are functional. They are dubbed small interfering RNAs or siRNAs. The antisense strand of siRNA then merges with RISC (RNA-induced silencing complex) to guide it to the siRNA-complementary mRNA molecule, signaling mRNA’s demise by cellular machinery. Researchers found that this molecular pathway and many of its components were highly conserved across mammalian species, a finding that encouraged RNAi research to move rapidly across species and gear up for the eventual jump into human trials.

In the early years of the new millennium (ca. 2002), a few enterprising biotechnology companies decided to go “all in” with RNAi, believing firmly in the science’s potential for significant impact on disease. One such company was Ribozyme Pharmaceuticals. The company, which changed its name in 2003 to Sirna Therapeutics to reflect its renewed commitment toward gene-silencing technology, was later bought by Merck & Co. in 2006. In the company’s Sirna days, it developed an RNAi therapy against age-related macular degeneration (AMD). The therapy was put into clinical trials, but Phase II results were less than spectacular, causing the trials to be shut down toward the end of 2008.

The Sirna outcome is one of the dozens of examples of RNAi-based companies that were (and still are) looking to make their mark on history. While the AMD clinical trial failed, early clinical successes using refined versions of RNAi technology have increased over the past several years. Moreover, an even greater understanding of the basic molecular biology behind RNA-induced gene silencing has led to technological improvements as a laboratory tool and clinical therapeutic. However, despite gains made, the perception of RNAi as a clinically useful tool have yet to be realized and may suffer an even more critical blow with the genome-editing tool CRISPR setting the molecular biology field ablaze with excitement over the last several years.

Time will tell what the future holds for RNAi in the clinical arena, but its impact on the biological sciences has been nothing short of remarkable. Opening new avenues of research in the areas of gene silencing and regulation has led to entirely new business endeavors being created to address the needs of RNAi researchers and those seeking to utilize the technique therapeutically. All told, it's not difficult to see why Drs. Fire and Mello were awarded the 2006 Nobel Prize in Medicine for their seminal research. Who would have thought the petunia could have so much impact on modern science?







































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