April 1, 2010 (Vol. 30, No. 7)

Kathy Liszewski

Delivery and Specificity Offerings Are Increasing Potential of Young Field

Inappropriate gene activity is being linked to a growing number of diseases. The ability to modulate or silence such activity via RNA interference (RNAi) technologies could revolutionize the way we treat disease. RNAi, as a new class of drugs, has already been convincingly demonstrated in a number of preclinical models of diabetes, hepatitis, oncology, influenza, macular degeneration, Parkinson and Huntington diseases, and others.

RNAi therapeutics have enormous potential. Because they inhibit gene expression at the level of translation, they offer a unique opportunity to rapidly identify and potently inhibit disease targets. The total market for RNAi therapeutics could reach $580 million by 2012, according to Market Research. CHI’s recent “From Tools to Therapies” meeting highlighted challenges facing this young field, exciting new applications, delivery, and cutting-edge platforms.

The best RNAi product in the world is useless if it cannot be delivered effectively to the target. “Delivery of RNAi therapeutics is one of the biggest challenges the industry faces,” reported Ian MacLachlan, Ph.D., CSO of Tekmira Pharmaceuticals. “Nucleic acids are comparatively large molecules that also are unstable in the blood. Additionally, they cannot readily cross membranes into target cells. To reach the maximum therapeutic potential of RNAi-based drugs, effective delivery is crucial.”

While the mechanism is called RNAi, the therapeutic agents are the so-called short interfering RNAs (siRNA) that consist of ~21 nucleotides of double-stranded RNA. Dr. MacLachlan said that nanoparticles coated with lipids encasing the payload siRNA represent an emerging new approach for delivery. “In the early days, researchers were using naked siRNA or chemically modified versions. Although initially there were some good in vivo results, further studies were less encouraging. We found, however, specially coated nanoparticles can potentiate delivery by 1,000-fold.”

Tekmira’s siRNA delivery technology platform is called SNALP for stable nucleic acid-lipid particles. SNALP are specialized lipid nanoparticles that encapsulate and systemically deliver a variety of nucleic acid molecules such as siRNA.

“Our preclinical studies have shown them to be effective in delivering the drug to target organs and into cells where the nucleic acid-based drug can carry out its desired effect while minimizing systemic toxicity,” Dr. MacLachlan reported. “Tekmira and its partners will have five product candidates in clinical trials by the end of 2010.”

One application targets hypercholesterolemia. The technology, called ApoB SNALP, targets ApoB, a protein that is essential to the assembly and secretion of very low density lipoprotein, a precursor to low density lipoprotein (LDL), both of which are required for the transport and metabolism of cholesterol.

In animal studies, the ApoB SNALP are delivered with high efficiency into liver hepatocytes, the cells that produce ApoB. The payload siRNA acts to knock down the precursor mRNA coding for ApoB protein resulting in significant reductions in LDL.

Although the nanoparticle technology is currently used for systemic application, according to Dr. MacLachlan the field is moving toward a targeted modality. “A variety of research is under way to see how RNAi can be delivered to specific target cells. That’s the race for the future.”

Recombinase-mediated cassette exchange between a modified ROSA26 locus and the shRNA construct allows introduction of the shRNA vector into ES cells, which facilitates development of RNAi mouse lines at TaconicArtemis.

Cancer Cell Vulnerability

RNAi can be directed against various types of cancers. But one of the key challenges for cancer therapeutics is the ability to destroy only the tumor cell and not normal ones. A novel way to specifically attack cancer cells is to take advantage of their “oncogenic addictions and associated dependencies,” reported Roderick L. Beijersbergen, Ph.D., group leader, division of molecular carcinogenesis at The Netherlands Cancer Institute. 

“It turns out that cancer cells can be highly dependent on the mutated proteins associated with their cancerous behavior. For example, breast cancer cells are dependent on the continuous activation of  Her2. Inhibition of this receptor blocks the growth of those tumor cells when normal cells are not affected.

“Many cancerous proteins cannot be inhibited with drugs, but it is likely that these cells are dependent on other proteins for their survival. A major challenge is to identify those proteins we should attack to eliminate only those cancer cells that carry a specific genetic alteration.”

According to Dr. Beijersbergen, RNAi screening can help. “It is still impossible to predict those specific dependencies in tumor cells. We perform large-scale functional screens using genome-wide RNAi collections to determine for each gene, one by one, whether inactivation results in the specific killing of cells carrying a tumor-associated mutation. To achieve this we have built panels of cell lines that are either normal or contain a mutated gene frequently present in cancer cells.

“By comparing the reduction in cell viability between the normal and mutant cells, we are able to identify, in an unbiased, genome-wide scale, those proteins that can be explored as drug targets for cancer therapy. These screens involve thousands of individual experiments and can only be done in a highly automated system using robotics.”

Dr. Beijersbergen believes that RNAi methodologies are well suited for seeking such novel therapeutic targets, although the field still has many challenges to overcome. “We are in the early days. What is most needed is to obtain better insights into why proteins show this functional phenotypic partnership. If we can better understand these relationships, maybe we can identify signaling networks and other proteins that may be even more efficient targets. This would help determine what is most druggable and then to develop improved strategies for attack.”

Novel Mouse Models

Animal models can provide invaluable tools for assessing therapeutics. TaconicArtemis has developed in vivo technology platforms to help researchers gain insights into drug and target-related disease mechanisms. Christine L. Olsson, Ph.D., commercialization scientific director, reported on the company’s inducible/ reversible RNAi technology.

“We have developed mouse models that are transgenic for custom shRNAs that can be turned on and off because of an inducer element cloned along with the shRNA. This allows us to see changes in protein levels based on RNAi message levels as a measure of phenotypic parameters.”

Such models allow for the mimicking of how drugs function. “The RNAi can decrease the amount of a specific protein in different tissues, to a different degree,” Dr. Olsson said. This differential decrease in target is similar to the action of an antagonistic drug. These drugs decrease the amount of activity of their target depending upon bioavailability, etc.

“In the RNAi case, we are determining if the anticipated drug target is an actual drug target. One can reduce the amount of protein in the cells by giving the inducer, measuring the in vivo effect, removing the inducer, giving the drug of interest, and seeing if you have the same effect.”

According to Dr. Olsson, the transgenic technology begins by cloning a specific shRNA into an inducible cassette and then placing it into modified embryonic stem cells (ESC). “As in typical transgenic technologies, these are injected into normal mouse blastocysts. During embryogenesis, the ESC incorporate into the blastocyst by an unknown mechanism to create a chimeric mouse. Further genetic screening of subsequent offspring creates strains for specific RNAi.”

The process can be complicated since not all tissues have the same level of expression. “The levels of knockdown can vary. That’s why the inducer is valuable. We can change the level of induction using a tetracycline derivative that can be given in the feed and water, for example, depending on the study. You can also vary the duration of knockdown in that way.”

Taconic has also created in vivo models for reversible kinase switches that provide a way to identify biological roles of specific kinases and possible side effects that result from their inhibition. “The novelty of both technologies is in their ability to preferentially allow inhibition or induction. They are valuable models whose uses are only limited by the imagination of the investigator.”      

Cilia and Cellular Phenotypes

High-throughput RNAi (HT-RNAi) screening provides a means to discover new therapeutic targets. Pedro Aza-Blanc, Ph.D., director of functional genomics resources at the Sanford-Burnham Medical Research Institute, heads a core facility that performs siRNA screens.

“HT-RNAi screening allows forward-genetic approaches in tissue culture cells by providing rapid genetic screens for cellular phenotypes. This can be applied to multiple fields including cancer, virology, stem cell biology, and metabolism. You can address any cellular phenotype as long as you have a high-throughput amenable method to detect it. To perform a screen, individual siRNAs are transfected into cells in individual wells in a high-throughput manner. The effect is measured in an assay system such as a high-throughput microscope or a plate reader.”

According to Dr. Aza-Blanc, there are two major applications for HT-RNAi screening. “The most common application is target discovery toward the treatment of diseases that can be associated with cellular phenotypes. An emerging application is to profile compound activity in the same way that genetic screens in yeast have been used in the past.

“Although it is unclear how widely applicable it will be, one can envision this technology being used to classify compounds such as those directed against the same molecular target but displaying different activities in live cells. This will help in the drug discovery pipeline as you can get an early snapshot of the compound’s mechanism of action.”

Dr. Aza-Blanc described work performed in collaboration with Joon Kim and Joseph Gleeson to understand the dynamics of primary cilia. “In recent years, we’ve seen the importance of these organelles in regulating intracellular signals involved in diverse processes from embryonic development to cancer. The disruption of their structure or function can have profound phenotypic consequences.

“We have used siRNA-screening techniques to identify modulators of cilia formation. This work exemplifies the use of siRNA libraries. Such screening can cover the entire genome as long as you have an assay to measure an effect. Other types of screens may employ library subsets of the genome such as proteases or kinases.”

HT-RNAi screening will continue to emerge as a major player in therapeutics on several fronts, Dr. Aza-Blanc said. “HT-RNAi screening can identify genetic networks involved in disease, as well as help identify an agent’s target and detect unexpected off-target activities. Overall, this approach allows one to make better informed decisions early on in the drug discovery process.”

RNAi therapeutics possess enormous potential. Researchers are just beginning to scratch the surface of this young and vibrant field. Many challenges remain, but progress continues to encourage further development. 

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