September 15, 2006 (Vol. 26, No. 16)

Combining Tools and Technologies to Improve Delivery and Efficacy of Key Compounds

The fledgling science of RNAi is making strides to take its place not only as a powerful therapeutic but also as a dynamic tool to increase the speed and efficiency of target validation and drug discovery. New advances will be highlighted at the upcoming Cambridge Healthtech Institute’s “RNAi: From Target Discovery and Validation to Therapeutic Development,” being held in October in Boston.

Current progress in the field includes delivery methods, reducing off-target effects, and coupling siRNA libraries to differential screening for identifying unique pathways in cancerous versus normal cells.

Delivery and Efficacy

Many diseases are caused by the inappropriate activity of specific genes. The ability of siRNAs to down-modulate designated genes provides an approach to treat a wide range of human diseases.

“Identification of potent siRNAs can have a very dramatic effect,” states Nagesh Mahanthappa, Ph.D., senior director business development and strategy, Alnylam Pharmaceuticals ( “We demonstrated this in vivo for treating respiratory syncytial virus (RSV) in animal models. We are investigating the first RNAi therapeutic for an infectious disease. In animals, we delivered the siRNA targeting RSV directly into the lungs using only saline. We have now completed two Phase I human trials.”

But for the full effect, the siRNAs must be appropriately and efficiently delivered. Dr. Mahanthappa says the company is pursuing an approach using liposomal formulations. “The liposomal formulations are injected parenterally,” he explains. “A study at our company demonstrated a successful liposomal strategy to deliver siRNA into liver hepatocytes. There are a number of delivery methods into liver, but being able to target hepatocytes is more unique.

“The goal was to decrease apolipoprotein B, or apoB. We found decreases in apoB mRNA, as well as the secreted protein. We also found the expected decrease in LDL cholesterol levels. So this demonstrated specific effects.

“Another consideration after delivery is duration,” says Dr. Mahanthappa. “In this same study we also found tremendous duration of the therapeutic that was dose-dependent.

“The big picture from these studies is that we now may be able to target a number of liver genes, as well as a variety of other tissues using the liposomal delivery method.

“The ultimate goal is to deliver the siRNA using the lowest dose that provides the most efficient effect,” asserts Dr. Mahanthappa. “Liposomal formulations are one way of delivering siRNA systemically, but we also can look at a number of other methods, such as polymers, and peptide complexes, as well as antibody-mediated methods.”

Lentiviral Delivery

Another approach is the use of lentiviruses to deliver short hairpin RNAs (shRNAs) for screening. “Current-generation lentiviral vectors have been engineered to allow delivery into most cell types,” explains Michele A. Cleary, Ph.D., research fellow, Rosetta Inpharmatics ( “This allows for RNAi in cells that cannot be transfected with siRNAs.”

Lentiviral delivery of RNAi can provide sustained silencing, according to Dr. Cleary. When cells are transduced with shRNA-encoding lentiviral vectors, the shRNA expression cassette becomes stably integrated into the host genome. “This is a major advantage, particularly because siRNAs silence genes for only five to seven days. But some assays need a longer time, for example, 14 days, to achieve an effect.”

Lentivirus-mediated delivery also creates cell lines capable of expressing shRNAs that target genes whose silencing is lethal to the cell. This can be done by manipulating expression by using inducible promoters that can be turned on or off as desired.

“It can also help when looking for off-target effects. We simply turn on knockdown, assess the effects by gene-expression profiling, and compare those profiles to when the shRNA is silent. Using this approach, we have learned that, like siRNAs, shRNAs have off-target effects, but the extent and magnitude is less. This may be because shRNAs enter the RNAi pathway at a different point.”

But just as with siRNAs, shRNAs must be carefully designed. “It’s critical to choose an effective shRNA core sequence using validated algorithms.”

Rosetta is utilizing this system for cancer biology, but other applications can be much broader. “Because lentiviruses can be used on nondividing cells, such as neurons that are traditionally difficult to transfect, the system has a wide repertoire of target cells. However, delivery of RNAi to living tissues via lentiviruses still needs some optimization. The use of other viral vectors is being explored to help achieve that goal.”

Reducing Off-target Effects

A vexing problem in RNAi is the off-target effect of siRNAs. This phenomenon, mediated by both the sense and antisense strands of siRNA, can unintentionally knockdown dozens of genes. Off-target effects may account for as many as 30% of positives identified in a screen.

“Despite our ability to rationally design effective siRNAs that perfectly match the targeted mRNA, we may still see significant off-target effects,” warns William S. Marshall, Ph.D., group vp of technology and business development, Fisher Biosciences ( and its business unit Dharmacon (

“Recently, we found that the 5´ end of the siRNA guide strand can act in an analogous manner to the ‘seed’ region at the 5´ end of a microRNA (miRNA),” Dr. Marshall explains. “Furthermore, we observed that we could modify this region to virtually eliminate most off-target effects.

“We found position-specific, sequence-independent chemical modifications that reduce silencing of partially complementary transcripts by almost all of the siRNAs we tested,” adds Dr. Marshall. “The key modification is the addition of a 2´-O-methyl ribosyl nucleoside at position 2 in the guide strand. When coupled with a modification that inactivates the passenger strand, the number of off-target effects decreases by up to 90 percent.

“This is a major discovery and provides us with a stronger mechanistic understanding that becomes tantamount for therapeutic development.”

Dharmacon will utilize its technology in collaboration with Abbott ( to identify therapeutic siRNAs for multiple therapeutic areas, initially focusing on oncology.

Validating Knockdown

To determine efficiency, validating siRNA-mediated knockdown is critical. “Some siRNAs work and some don’t for as yet unknown reasons,” says Katherine Keating, Ph.D., manager of R&D, EiRx Therapeutics ( “So, it is important to validate RNAi-induced knockdown at both the protein, as well as the mRNA levels.

“For example, mRNA reduction seen without a corresponding reduction in protein levels can indicate the protein turnover is slow and that a different time point may be more appropriate for the required biological assay. In contrast, protein reduction in the absence of mRNA reduction may indicate that an siRNA is mediating its effects at the translational level, thus behaving as an miRNA.

“Usually 70% knockdown is considered optimal for target validation,” Dr. Keating notes. “You don’t always want complete knockdown in validation, since therapeutic compounds seldom inhibit 100% of a target enzymes’ efficacy.

“To measure knockdown, most people use Western blots. Another option is ELISAs that can be utilized for high throughput or for less-abundant proteins.”

Western blots and ELISAs, however, require target-specific antibodies. “In the absence of available antibodies, you would measure mRNA knockdown using a reverse transcriptase quantitative PCR reaction. One of the most important aspects of this reaction is the reverse transcription stage.

“Variations in the amount of transcript generated can be affected by such things as different enzymes and different template preparations, including primers used for cDNA synthesis. So, it is critical to optimize your conditions very carefully for accurate assessment of reverse transcription.”

EiRx is focusing on developing anticancer drugs using novel regulators of apoptosis discovered using its ALIBI™ genomics platform.

MicroRNA Mimics

According to Open Biosystems’ ( CTO, Troy Moore, traditional approaches to knockdown the company’s genes of interest using shRNA methodology worked only about 20% of the time. Hence, the company uses second-generation miRNA-adapted shRNA(s) (shRNAmir), designed to mimic primary miRNA transcripts.

“We wanted to mimic what the cell does, so we took our gene-specific RNAi sequence and fused it into human primary microRNA-30 transcripts. This design allows endogenous processing of the resulting shRNAmir by Drosha and Dicer enzymes and has produced shRNAmir that are more than 80% efficient for knockdown and also show decreased off-target effects.”

According to Moore, the efficiency of the shRNAmir system makes it a viable tool for screening whole-genome libraries. “It is also useful for biotechnology applications since you can save time and money with its enhanced efficiency during target validation, high-throughput RNAi screening, etc.”

Open Biosystems provides lentiviral and retroviral shRNAmir libraries that allow transfection and infection-based applications for RNAi studies in most cell types, including primary and nondividing cells. “Most other RNAi libraries include only transcripts of known function, but we have targeted the entire transcriptome, including transcripts of unknown function, creating a resource for the ongoing discovery of gene function.”

RNAi Goes Genomic

siRNA libraries, on the other hand, can provide valuable information about gene function and enhance functional genomics approaches.

“RNAi screening is a quick and easy way to identify genes involved in particular cellular processes,” says Christina Buchanan, Ph.D., technical applications specialist, Ambion ( “In fact, RNAi is the easiest and most cost-effective reverse genetics tool for studying gene function.

“Success in RNAi experiments depends on several things,” says Dr. Buchanan. “First, you need effective siRNAs that have been designed to maximize silencing while minimizing off-target effects. Most researchers use at least three individual siRNAs per target. Second, you must determine optimal siRNA delivery to cells by testing an assortment of reagents and methods. Third, it is critical to use positive and negative controls to confirm results, determine transfection efficiency, and control for the effects of siRNA delivery. Also, it is important to have a dependable readout/assay to monitor siRNA-induced knockdown.”

Ambion provides Silencer® siRNA libraries that “span the entire human genome, target major gene classes, or are created on a custom basis,” according to Dr. Buchanan. “By screening normal and cancerous cell lines, siRNA libraries can help identify key differences in transformed versus normal cells.”

Cell-signaling Applications

Scientists at Invitrogen ( are combining RNAi methods with kinase inhibitors to dissect cell-signaling components involved in cancer. According to George Hanson, Ph.D., senior scientist, the company’s studies were designed to demonstrate how combining multiple technologies can address unanswered biological questions.

“The transcription factor activator protein-1 (AP-1) has been implicated in a wide variety of biological processes, including oncogenic transformation,” explains Dr. Hanson. “The tyrosine kinases of the epidermal growth factor receptor (EGFR) begin one signal transduction cascade that leads to AP-1 activation. These kinases are also known to control cell proliferation and differentiation.

“We combined RNAi directed toward EGFR along with small molecule inhibitors acting at several points of the pathway. We found a 10–12-fold shift toward greater potency in the IC50 observed for EGFR.

“The immediate application for such findings is that one could lower doses required for therapy,” says Dr. Hanson. “When a patient can take less of a drug, there is also a decrease in possible side-effects.”

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