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Conquering RNAi Delivery Challenges

Innovative Solutions Include Nanoparticles and Stabilized Compounds
  • Kathy Liszewski

RNA interference (RNAi) is making great strides as an indispensable strategy for target-specific knockdown of gene expression. Emerging second-generation technologies represent advances in RNAi design, efficiency, and efficacy. Yet, daunting obstacles remain, such as how to deliver RNAi compounds to the right targets in the right amounts.

Two recent meetings, Select Biosciences’ “RNAi World Congress” and CHI’s “Beyond Genome,” highlighted new and innovative strategies for overcoming delivery hurdles. Advances include utilizing tiny delivery vehicles called nanoparticles, replacing traditional ideas about RNAi compound size, and even drawing on the new field of epigenetics for changing DNA expression without altering the gene sequence.

Successful delivery of RNAi faces a number of challenges, reported Tod Woolf, Ph.D., president and CEO RXi Pharmaceuticals ( “Delivery of RNA oligos is a complicated situation but also fascinating. The first issue is one of exposure. That is, if the RNAi compound is to be injected, for instance, will the tissue targeted be exposed to the RNAi? If the RNA is injected and delivered intraperitoneally or intravenously it goes to the blood, where it is taken up by the kidneys and removed. for the most part, standard injections don’t have a prayer of turning off genes with standard RNAi compounds.”

Exposure issues also depend on the type of tissue targeted. “You can consider local delivery, such as injecting RNAi into the eye for treating age-related macular degeneration. It also can work with injections directly into the central nervous system or with routes such as inhalation or even topical.”

A second hurdle is cytoplasmic uptake. “Overcoming exposure problems doesn’t mean the RNA can get into the cytoplasm of cells,” Dr. Woolf explained. “This is a separate issue, as is the stability of the RNAi compound itself. RNA needs to be carefully designed and chemically stabilized.”

Nanotransporters used for RNA delivery can potentially solve these problems, he reported. “Our nanotransporters are chemicals of defined size that are mixed with an RNAi compound to form minute particles for delivery into target tissues. The nanotransporter has a core to which layers are added by chemical synthesis. The final layer has a positive charge allowing it to attract and bind negatively charged RNAi compounds.”

RXi has successfully utilized its nanotransporters for delivery into mouse liver as a treatment for amyotrophic lateral sclerosis (ALS). “We have been able to knockdown the gene for superoxide dismutase1 (SOD1) in a mouse model of ALS. This molecule normally neutralizes oxygen molecules that damage cells. Although ALS can be associated with more than 100 different mutations in SOD1, we found that RNAi-mediated knockdown of SOD1 had a benefit in an animal model of ALS.”

Magnetic Nanostructures

It is possible to combine nanoparticles for both efficient transfection as well as imaging studies, said Sarah Fredriksson, Ph.D., CEO and founder, Genovis ( “Our take is that you can obtain a dual transfection/labeling system using nanostructures. We employ magnetic nanoparticles with a lipid core for MRI studies. When you combine tags, such as fluorescence in combination with carrying RNAi, you can see where it works, such as in a whole cell, endosome, or liposome.”

Dr. Fredriksson pointed to stem cells as an example. “Stem cells often die before they can reach their intended use. Using RNAi combined with magnetic nanoparticles, however, you could knockdown a protein of interest in the stem cell that promotes its survival for several days to allow the cells to reach their intended target.”

The basis for Genovis’ technology is tiny superparamagnetic nanoparticles that feature iron oxide cores coated with a specific cationic lipid formulation. This facilitates particle solubility and cellular uptake.

The company’s new product, NIMT® FeOsilence, delivers short interfering RNA (siRNA). “We manufacture these particles with narrow and well-characterized size distributions that can be manufactured with high reproducibility. This is important because it greatly improves optimization of transfection efficiency for many different cell types while keeping toxicity to a minimum.”

Choosing an optimal nanoparticle size is critical. “Particle size can be important, so we provide the nanoparticles in a variety of specific sizes,” Dr. Fredriksson noted. “You might be better served with a nanoparticle of 10 nanometers instead of 100. The smaller size allows them to move more freely and get into lymph nodes. The smaller size may also help in transfecting traditionally difficult cell lines.”

Dicer-Guided Design

The RNAi pathway is populated by siRNA that consist of double-stranded RNA of ~20–25 nucleotides in length. The precursors to siRNAs are processed from longer RNA sequences by a dedicated set of enzymes and other proteins.

Exactly how big does an siRNA need to be for the most efficient and efficacious delivery? Dicerna Pharmaceuticals ( is basing its platform on Dicer Substrate Technology (or DsiRNA), a new approach that it expects will supplement or replace traditional ideas about siRNA size. “Our goal is to develop drugs that take advantage of the same cellular machinery needed for the well-known phenomenon of RNAi but with a key difference,” Roberto Guerciolini, M.D., svp and cofounder said. “We are targeting an earlier step in the RNAi process that produces a more potent knockdown of the targeted gene using a longer siRNA.”

Dicerna is harnessing the power of a key enzyme, called Dicer, involved in the processing of double-stranded RNA. “In mammalian cells, Dicer processes double-stranded RNAs that are slightly longer. So, our drug candidates are optimized to target human Dicer. By doing it this way we can attain a potency that is 5- to 100-fold higher plus a longer duration of action than that derived by traditional gene-silencing methods.”

According to Dr. Guerciolini, Dicerna’s technology approach reflects a change in thinking in the field regarding Dicer’s processing of double-stranded RNA into siRNA. “Using siRNAs longer than the traditional 21 mers was initially discouraged because the role of human Dicer was not fully appreciated. But this thinking was flawed, and we now see that longer double-strand oligonucleotides can often have superior activity and longevity.”

Dr. Guerciolini said the current challenge is to optimize the delivery modalities for the highest efficiency. In order to do that, the company is evaluating numerous commercially available reagents. Once those studies are completed, it expects to progress to animal studies.

Multiple Approaches Needed

Delivering RNA for knockdown can be quite a different creature compared to DNA delivery, warned Danny H. Lewis, Ph.D., CEO and president, Expression Genetics ( “A lot of researchers assumed you could use the same formulations for DNA as for RNA, but that’s not been the case. Our company is in an ideal situation because we have many years of experience in the drug delivery field. We’ve found that synthetic delivery systems, including polymeric systems, need to be custom designed for efficient RNA delivery and gene knockdown.”

Dr. Lewis suggested that any delivery system must efficiently function to protect the therapeutic cargo from degradation, to promote uptake by target cells, and to facilitate intracellular trafficking. “Ultimately, multiple approaches will be needed. It depends on a variety of factors, such as the disease being treated, the tissue, mode of administration, dose, and the specific siRNA or short hairpin RNA (shRNA). There’s no one-size-fits-all. Since the field is still very early, there will need to be rigorous research performed to establish clinical proof of concept.”

Expression Genetics’ lead product, EGEN-001, is now entering a second round of clinical trials. The company uses its TheraPlas® delivery technology composed of an interleukin-12 (IL-12) gene-expression plasmid and a biocompatible delivery polymer to increase the local concentration of IL-12, a potent anticancer cytokine, in patients with ovarian cancer. “We expect to use this on many types of disseminated cancers and solid tumors, such as that of the brain, as well as head and neck,” noted Dr. Lewis.

Additionally, the company will continue to seek promising formulations for RNA for both in vivo and in vitro gene knockdown. “We are continuing to develop our TheraSilence™ technology for siRNA and shRNA delivery and have several formulations that look promising to enter preclinical development this year.”

Epigenetics and Therapeutics

Epigenetics refers to stable changes in gene expression that do not involve altering the actual DNA sequence. Basically, it describes how the environment or other factors can permanently change the way a gene is expressed without the gene itself changing. Epigenetic mechanisms can control or alter gene activity in several ways, such as RNAi, DNA methylation, and modification of histones that encase DNA.

“This concept has been a real eye opener,” reported Casey Case, Ph.D., vp research, SanBio ( “Epigeneticists have known about this phenomenon for some time, but the idea to apply it to therapeutics for humans is quite new. In a broader sense, the real novelty is that you don’t need the permanent presence of a therapeutic compound for a permanent effect. So, in some cases a transient transfection of a gene can produce a permanent change.”

Dr. Case’s team has developed a cellular therapy product produced by the transient transfection of mesenchymal stem cells. Their vector encodes the Notch-1 intracellular domain.

According to Dr. Case, “Notch-1 is a powerful regulator of the fate of developmental cells. Transfection with this vector causes cells to lose their capacity to differentiate to other paths, and it causes cells to acquire neuronal precursor-like properties promoting regeneration. We are determining whether epigenetic mechanisms such as DNA methylation underlie this effect.”

Dr. Case speculated that epigenetic mechanisms may assist with the utilization of RNAi for therapeutics. “RNAi knocks down the expression of the target by destroying its mRNA. The target may be a regulatory protein that alters the expression of other genes at the transcriptional level. In some instances, some of these transcriptional effects may be locked in by epigenetic changes, such as DNA methylation, thereby allowing the RNAi effect to persist after the RNAi-inducing molecule is gone. There is much to be explored.”

Virtually every major pharmaceutical and biotechnology company is using RNAi technology to discover and validate drug targets. Although still in its infancy with many challenges to overcome, RNAi is poised to takes its place as a major therapeutic pursuit.