January 15, 2011 (Vol. 31, No. 2)

Emerging Field Simultaneously Holds Both Promise and Problems

RNA interference (RNAi), an exciting yet evolving gene-silencing technology, holds great promise for drug discovery and as a novel therapeutic approach. But troubling doubts have recently emerged as to how quickly that promise will deliver. Perhaps ominously, in November, Roche terminated its multimillion-dollar RNAi therapeutics programs.

One of the main bottlenecks vexing the field is functional delivery. Select Biosciences recent “RNAi Asia” conference discussed the latest developments and innovations, such as development of specialized nanoparticles for delivery, improvements in high-throughput screening, and new in vivo studies.

Delivery problems could be overcome with the use of specialized nanoparticles that entrap small interfering RNAs (siRNAs), suggested Andrew D. Miller, Ph.D., professor at King’s College London. He has an optimistic point of view about RNAi therapeutics.

“RNAi should be the closest thing to a magic bullet for the biopharmaceutical industry,” he said. “In principle, one can get high-quality knockdown with very few side effects or off-target effects. The problem is that RNAi effectors need to be delivered to the target site in a potent form using the smallest possible amount of material. At present, doses are too high. That problem can only be solved with better delivery technologies and improved methodologies.”

A possible solution envisioned by Dr. Miller is the use of cationic, lipid-based, self-assembly nanoparticles that entrap siRNAs into lipoplex nanoparticles, which themselves need to be further stabilized.

“Stabilized nanoparticles with improved circulation half-lives are realized using a polyethylene glycol (PEG) polymer layer to shield the nanoparticle surface. The best stabilized nanoparticles should then be those that are stable until triggered to release entrapped siRNAs by local changes in condition such as pH, or through an external trigger such as from a laser,” he said.

“Our approach is to use a Lego building block-like process to derive stabilized nanoparticle delivery systems in which purpose-designed components are allowed to self-assemble. We have designed RNAi delivery systems according to this paradigm and successfully formulated specialized nanoparticles for the delivery of siRNAs to in vivo targets such as liver and tumors. These are tailor-made delivery solutions.”

The nanoparticle systems are composed of nucleic acids condensed within functional concentric layers of lipid components, surrounded by a biocompatibility layer and equipped with an optional biological recognition/targeting layer, he explained.

There are still significant hurdles to be overcome. “We have achieved proof-of-concept for functional delivery in vivo, but not yet proof-of-therapy,” Dr. Miller said. “We still need time to make significant improvements to the delivery process. We believe that we may be facing a situation in general in which academics still need to solve basic delivery issues before biopharma finds RNAi as attractive as it did several years ago. Delivery will work eventually, but it is likely to take more time and effort than is commonly appreciated,” he concluded.

Progress in Cancer Treatments

RNAi for cancer treatment relies on the specific downregulation of target genes that support cancer cells. A number of issues remain, however.

“Effective RNAi therapy requires delivery to each and every cancerous cell in order to directly and specifically eliminate the cancer,” reported Nigel A. J. McMillan, Ph.D., associate professor, principal research fellow and deputy director, Diamantina Institute, University of Queensland. “However, this is unlikely with today’s technology. Currently, only a handful of studies have shown in vivo efficacy in siRNA delivery using vehicles such as cationic lipids, nanoparticles, collagen, and others.”

Dr. McMillan said that he has developed simple, easily prepared lipid particles that he has utilized in animal studies. “Now, we are looking at the immune response after delivery of these particles. We want to know what kind of inflammatory response occurs and if one can unmask an antigen and use that to attack the tumor,” he explained.

“To augment RNAi treatment, some studies seek to silence suppressive immune response regulatory factors, while other studies utilize specific siRNAs to enhance the innate response by upregulating cytokines such as interferons.

“We took a nonbiased approach asking how to silence genes as well as to activate the innate immune response. Certain immune cells can sense and recognize siRNAs in a sequence-specific manner. The systemic delivery of siRNAs will likely recruit immune cells. Although immune activation is of concern, it may also be therapeutically beneficial to enhance the immune response,” according to Dr. McMillan.

“The use of bi- and tri-functional siRNAs—that is, siRNAs that target a gene, activate innate immunity, and/or unmask antigens—allows one to create siRNAs that can combine gene silencing and immunostimulation. This represents a potentially powerful two-pronged attack to kill cancers,” he asserted.

“RNAi doesn’t work like most people think it works,” Dr. McMillan concluded. “We need to more fully understand exactly how it interacts with the immune system before we can realistically target the clinic.”

SiRNA treatment eliminates tumor burden in mouse models. Mouse tumors treated with bifunctional siRNA (bottom two mice) do not develop while those in untreated controls (top) do. [University of Queensland]

siRNA-Based Screening

Scientists at Merck & Co. have utilized siRNA screening to identify novel drug targets. “Recent advances have allowed our scientists to test the effects of knocking down not only each individual gene separately but also multiple genes simultaneously,” noted Jeremy Caldwell, Ph.D., vp, RNA therapeutics.

Merck is applying siRNA-based screening across several disease states. In cancer, for example, its researchers have used siRNA screening to search for genes that enhance the activity of chemotherapy and eradicate tumor cells—but not normal cells—when they are knocked down.

Dr. Caldwell agrees that the two biggest challenges with developing siRNA-based therapeutics are delivery and establishing safety. “Delivery of siRNA is currently possible for some solid tumors and some accessible organs, such as the liver and the eyes. However, systemic delivery to other cells, tissues, and organs is more challenging and will likely require multiple approaches optimized for each target.”

Merck is focusing on optimizing lipid nanoparticles (LNP) for systemic delivery of RNAi therapeutics to the liver. “The safety profile using the LNP delivery approach remains a key challenge. Chemically modifying siRNA can minimize off-target activity; however, as with conventional targeted therapies, inhibiting the properties of one particular target may have difficult-to-predict effects on the activity of other genes in a pathway or network.

“Another potential concern is that introducing siRNA into the bloodstream can trigger the body’s innate immune response. This effect also can be minimized by chemical modifications to siRNA, but this may not be equally effective in every case.”

Dr. Caldwell reports that Merck has made progress in optimizing lipid nanoparticles for systemic delivery of RNAi-based therapeutics to the liver. “We’ve demonstrated that optimization of cationic lipids in the lipid nanoparticles improves potency by greater than 10-fold. The extent of PEGylation affects particle size, efficacy, and inflammation. We’ve also found that the larger lipid nanoparticles are suboptimal relative to smaller-sized particles with respect to elevation of liver toxicity markers. Finally, the lipid nanoparticles are rapidly absorbed into the liver and maximally induce the silencing complex within six hours.”

Advances in miRNA

Melanoma is associated with abnormal patterns of microRNA (miRNA) and large noncoding RNA (lncRNA) gene expression. Ranjan Perera, Ph.D., associate professor at Sanford-Burnham Medical Research Institute, is deciphering how miRNAs and lncRNAs impact development of this disease.

“As master regulators of hundreds of RNAs, miRNAs have been implicated in a number of malignancies. Because they may be either upregulated or downregulated, we are examining the possibility of using miRNA and lncRNAs as biomarker and discovery tools. As biomarkers, they may be useful for early prognostics or diagnostics. As discovery tools, they could be employed as therapeutics targeting certain genes or even to assist with gene-function discovery.”

Dr. Perera first looked at the miRNA signatures between normal and malignant melanocytes and melanoma cell lines. “Our gene-expression studies identified a handful of miRNAs that were downregulated. One of these is miR-211. While normal cells expressed miRNA-211, malignant cells did not. Thus, our basic hypothesis is that miRNA-211 downregulation in melanocytes induces human melanoma.”

By putting miR-211 back into melanoma cells, Dr. Perera and his team found that it blocked tumor growth and invasion. To begin to dissect the mechanism, he performed functional studies. “We found that miR-211 targets the KCNMA1 gene that codes for the so-called potassium large conductance calcium-activated channel, subfamily M, alpha member 1,” he said.

“Next, we found that miR-211 resides in the intron of TRPM1, which encodes a calcium-channel protein called the transient receptor potential cation channel subfamily M member 1 protein. The expression of TRPM1 is inversely correlated with melanoma aggressiveness, suggesting that it suppresses the metastasis of melanoma cells.”

Finally, Dr. Perera’s team found that the expression of miR-211 is regulated via TRPM1 gene that is regulated by MITF, which encodes the microphthalmia-associated transcription factor. He summarized his findings: “When we utilized siRNAs to knockdown the expression of MITF, we found that both TRPM1 and miR-211 were also knocked down.”

“We are now able to build a new signal-transduction pathway that may ultimately provide information for early melanoma prognostics,” Dr. Perera explained. “My team and others are evaluating patient samples and have already demonstrated that indeed miR-211 is associated with human melanomas.”

The regulation of miR-211 in melanomas

Cell Fate Architecture

The earliest events in human embryonic development are orchestrated by a complex, yet poorly understood, regulatory architecture that involves miRNAs. Previous work from the group of Paul Robson, Ph.D., group leader in stem cell and developmental biology at Genome Institute of Singapore, applied advanced single-cell gene-expression analysis to the developing mouse embryo.

“This clearly indicated that removal of particular transcripts was a key event in cell fate-decisions. The primary candidate to control such a mechanism is microRNAs,” he said.

Now Dr. Robson’s group is seeking to unravel these early events in human development utilizing a human ES cell (hESC) model. “We employed next-generation transcriptome analysis methods to characterize the dynamics of gene expression upon hESC differentiation.”

Dr. Robson focused on examining the differentiation of cells of trophoblast lineage. Trophoblasts are the first cells of the fertilized egg to differentiate. They constitute the outermost layer of the embryo, assist in uterine implantation, and develop into the fetal component of the placenta.

Dr. Robson’s team profiled, in tandem, polyadenylated and small RNA transcriptomes using strand-specific RNA-sequencing.

“There were several surprises in our study,” he stated. “In addition to identifying the expression of many known microRNAs not previously described in the cells of trophoblast lineage, we also saw the expression of many novel microRNAs. We found that many well-known genes are poorly annotated in their 3´ untranslated regions and are much longer than anticipated, thus greatly expanding the potential target sites of microRNAs.”

These studies have clinical potential relevance, according to Dr. Robson. “The application of next-generation sequencing technology will likely identify many new microRNAs in the human genome, some of which may be associated with diseases of placental lineage. This coupled with new studies that fetal-derived microRNAs transfer into the maternal circulation may allow fetal-derived microRNAs to serve as biomarkers in mother’s blood that could be used to noninvasively test for the early indications of diseases such as pre-eclampsia and intra-uterine growth restriction.”

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