October 1, 2011 (Vol. 31, No. 17)

Recounting the Journey from Biological Oddity to a Commercialized Technology

In the fall of 2006, the RNA world arrived on the big stage when it was announced that Andrew Z. Fire, Ph.D., Craig Mello, Ph.D., and Roger Kornberg, Ph.D., were selected to receive the Nobel Prizes in Physiology or Medicine (Drs. Fire and Mellow) and Chemistry (Dr. Kornberg) for their work on RNA interference (RNAi) and RNA polymerase II, respectively.

RNA therapeutics have since emerged as the next big frontier in drug development. Not since the development of gene cloning, antibodies, and PCR has a specific biotechnology had so much impact on molecular biology, biomedicine, and beyond.

RNAi, the sequence-specific gene silencing at the messenger RNA level induced by double-stranded RNA (dsRNA), is a prime example of the value of serendipity as well as research involving model organisms.

Two decades ago, plant scientists first noted the “co-suppression” phenomenon when they created transgenic petunias and found that the transgene not only failed to express properly but was silencing sequence-homologous genes in the genomes of the same plants. Similar phenomena were soon recognized in other lower eukaryotes such as the bread mold Neurospora crassa and the worm Caenorhabditis elegans.

It was in C. elegans that Drs. Fire and Mello made the seminal discovery that it is double-stranded RNA, and not single-stranded sense or antisense RNA, that is the actual trigger for the gene silencing, which the scientists subsequently labeled RNAi. The importance of that discovery was that it made RNAi a reverse genetics tool of sufficient robustness for adoption by the wider scientific community.

Dirk Haussecker, D.Phil.

Human Cells

Molecular genetics and biochemistry in plants, worms, flies, and molds quickly converged to unravel important principles of RNAi molecular biology, but one nagging question remained: would it also work in human cells?

The reason for some skepticism about the existence of the RNAi pathway in mammals was because, with a few exceptions in early development, long dsRNAs that were used to trigger RNAi gene silencing in lower eukaryotes were known to elicit the interferon response, a potent innate immune reaction that causes the nonspecific shutdown of protein translation. Obviously, the medical and economic value of RNAi would increase dramatically if it also functioned in human cells.

There were, however, hints from plant RNAi studies by the Baulcombe group and the parallel discovery of genetically related endogenous small silencing RNAs, called microRNAs, that small RNAs may mediate RNAi activity downstream of long dsRNAs. This was confirmed by elegant biochemical studies (using the fly) on the processing of long dsRNAs into 19- to 23-nucleotide small RNAs in the test tube.

Based on these results and the speculation that a type III ribonuclease might be involved in this processing, Tuschl and colleagues fully opened up RNAi to human medicine by demonstrating that synthetic 19–20 base-pair dsRNA with two nucleotide 3′ overhangs, the classical siRNA structure, mediates efficient RNAi gene silencing in mammalian cells.

From then on, the use of RNAi in basic and applied biomedical research exploded. A number of additional RNAi trigger structures and strategies soon emerged, including DNA-directed RNAi whereby the RNAi trigger is transcribed from a DNA template.

Consequently, RNAi became widely used, from single-gene reverse genetic studies to genome-wide RNAi screens for the discovery of genes involved in biological pathways of interest. RNAi was also employed in the rapid generation of transgenic animal models of human disease and the development of crops, and as a therapeutic modality in its own right, that is, RNAi therapeutics.

Clinical trials are currently under way with RNAi to treat age-related macular degeneration, which is triggered by the VEGF protein that promotes an excessive growth of blood vessels that leak behind the retina. Novel RNAi drugs have been designed to shut down the genes that produce VEGF and stop the leaking blood vessels. [BSIP/Photo Researchers]

Pitfalls, Challenges, and Solutions

As with any new technology, especially those eagerly adopted, the development of RNAi, particularly in mammals, was not without its problems. Among the important issues have been off-targeting artefacts mainly stemming from the fact that the mammalian microRNA machinery, which is the actual pathway being harnessed by experimental RNAi in mammals, has evolved for the modulation of genes with less than complete sequence complementarities.

The innate immunostimulation by certain RNAi triggers has been another source for the misinterpretation of experimental results. This cast considerable doubt on some high-profile studies and drug development programs in the areas of oncology-, antiviral-, and angiogenesis-related research.

Finally, getting the RNAi triggers into the target cells of interest (delivery) has been widely identified as a challenge for the commercial viability of RNAi therapeutics.

RNAi trigger selection still involves considerable empiricism, but the resulting variability and increased resources this requires are mainly an issue for high-throughput gene discovery applications only, and has become largely irrelevant for smaller-scale experiments. This is because pre-established RNAi trigger libraries that contain multiple RNAi triggers against each target gene are readily available at reasonable cost. Custom-made RNAi triggers have also benefitted from improved affordability.

Better affordability also facilitates the use of at least two or three effective RNAi triggers against a target gene of interest to rule out off-targeting and immunostimulatory artefacts by rogue RNAi triggers. Still, it is good practice to test for immunostimulation directly by transcript and protein analyses, especially in settings where there is concern that a phenotype could be explained by immunostimulation.

Both microRNA-like off-targeting and immunostimulation can also be effectively addressed by applying widely available oligonucleotide modifications such as 2′-O-methylation. The modification strategy is the preferred solution in RNAi therapeutic development and can further contribute to increased stability and delivery. The choice of delivery technology may also determine which innate immune receptors an RNAi trigger gets exposed to.

RNAi delivery, especially for therapeutic purposes, is often misunderstood as a singular issue and it is wrong to talk about whether it can be considered “solved” or not. As one-size-fits-all solutions that enable gene knockdown in every cell of the body are unlikely to emerge any time soon—in fact, they may not always be desirable—RNAi delivery ought to be considered in terms of which cell/tissue types a delivery technology can address, and then consider possible gene targets.

Because the identity of a specific RNAi trigger should not alter the pharmacology of such a formulation, one can then quickly expand the development pipeline. This is already happening for liver-directed gene knockdown using Tekmira Pharmaceuticals’ liposomal SNALP delivery technology where there are already almost ten development candidates in the pipeline, five of which have already made it into the clinic.

Tumor cell-directed gene knockdown, again using Tekmira’s SNALP liposomal nanoparticles, and endothelial cell-directed knockdown that relies on Silence Therapeutics’ cationic AtuPLEX lipoplex technology are not too far behind.

The reason why this concept has apparently found little appreciation is that large pharmaceutical companies are more accustomed to thinking in terms of market size and therapeutic franchises and then trying to make drug technologies fit the pharmacological demands thus posed. This may work for small molecules to a certain degree, not so well for RNAi therapeutics.

However, at a time when the healthcare reimbursement policies in many developed countries favor innovative, high-impact therapeutics, effective RNAi drugs should be profitable, especially for orphan indications.

Overcoming Negative Perceptions

Despite some of the current negativity around RNAi therapeutics, the fact remains that more than 1,000 patients and volunteers have been recipients of RNAi drug candidates with acceptable and improving safety profiles. A number of trials have involved doses where robust target gene knockdown can be expected based on rodent and nonhuman primate preclinical studies.

Important knockdown efficacy results are expected in the next two to five months from Alnylam’s ALN-TTR01 and ALN-PCS Phase I studies in TTR amyloidosis and hypercholesterolemia. Together with the safety data from studies involving RNAi candidates for cancer therapy—Alnylam’s ALN-VSP02, Silence Therapeutics’ Atu027, and Tekmira’s TKM-PLK1—these results have the potential to boost confidence in RNAi therapeutics.

Progress in the commercialization of transgenic RNAi plants and animals is more advanced due to the decreased importance or absence of innate immunostimulatory issues, lowered safety bars, and, in the case of plant RNAi, often highly potent gene-silencing outcomes.

As much as establishing stable transgenic RNAi lines and achieving potent DNA-directed RNAi in all mammalian tissue types of interest continues to present technical challenges, it is the cultural resistance to gene-modified organisms in many parts of the world that may be rate-limiting in adopting RNAi for improving human nutrition and creating cleaner environments.

With RNAi plants (including blue rose, which may soon reach the marketplace), one can expect further commercialization of RNAi crops. This will be the case especially for pathogen and environmental resistance and, following that, for the improvement of nutritional values.

RNAi techniques leading to an increase in the yield and quality of therapeutic recombinant proteins produced via plant and animal cell culture systems are also poised to become commercialized technologies over the next decade or two.

On the biomedical front, RNAi has become a critical research tool. Ubiquitously used in target discovery and validation, it is already having a great impact by increasing the efficiency of drug development. Experience with the technology and the improved affordability of RNAi reagents and transcriptomic analysis tools suggest that the quality of the results obtained from RNAi experiments should increase steadily.

In the therapeutics space, arguably the area of highest commercial potential for RNAi, one can expect continued improvements in the safety and efficacy of existing RNAi delivery platforms. Nonhuman primate data indicates that RNAi is already 100- to 1,000-fold more efficient in inducing gene silencing in the liver compared to the older RNaseH antisense technology (single-digit microgram/kg versus single-digit milligram/kg ED50s).

Moreover, one can expect the development of new delivery technologies for gene knockdown beyond applications for the liver, solid cancers, and endothelial cells. Such candidates include dermal, respiratory, and hematopoietic cells.

Efforts in targeting the related microRNA pathway for therapeutic purposes also have made steady progress and look particularly promising for oncology indications and the treatment of hepatitis C infection. In addition, interesting applications in areas such as cardiovascular disease are emerging.

Taken together, given the maturing state of RNAi technologies and their stage of clinical development, the first regulatory approvals of small silencing RNAi therapeutics should occur in five to seven years.

Dirk Haussecker ([email protected]), D.Phil., is a former post-doc at Stanford University and has been involved in RNAi-related research for over 10 years. He currently is an assistant professor at Dongguk University in Seoul, South Korea and works as an RNAi analyst/consultant. Dr. Haussecker is also the author of a blog entitled “RNAi Therapeutics.”

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