Friday, December 16, 2011

DELIVERY - siRNA/RNAi | RNAi No Longer Blue Sky

Neil Canavan

Despite significant delivery hurdles, researchers make progress toward genetic therapies

The use of double-stranded RNA to deliberately “interfere” with gene expression was first described in the journal Nature in 1998.1 Though the work was performed in Caenorhabditis elegans, the potential was immediately clear: RNAi made for an excellent research tool and likely represented a new class of therapeutic agents.
In either application, a recent advance may revolutionize the optimal selection of RNA inhibitors.
“The potential for RNAi demands that you have to have the right trigger,” said Christof Fellmann, a graduate student at Cold Spring Harbor Laboratory, N.Y. Algorithms to successfully predict an optimal RNAi sequence, in this case for short hairpin RNAs (shRNAs), remain elusive, and current methods involve extensive time-consuming screens. To overcome this barrier, a “sensor assay” was developed that rapidly identifies optimized shRNAs at large scale.
For this method, a library of 20,000 expression vectors was created, each consisting of (a) a unique shRNA sequence, (b) a known target sequence (the sensor), and (c) a fluorescent reporter gene. Once expressed in transfected cells, the components were free to interact (or not) with resulting levels of fluorescence inversely relating to the degree of target-gene suppression. This innovative screen was able to identify shRNAs not only of exquisite potency, but also of unprecedented specificity, thereby greatly reducing off-target effects.2
The sensor assay is already bearing fruit at the bench, as demonstrated by Cold Spring Lab’s recent identification of a potential therapeutic target for acute myeloid leukemia, and the technology will soon be commercialized by Mirimus Inc., a Cold Spring Harbor spin-off.3

Enhanced In Vivo Survival

Working to optimize their proprietary siRNA platform for in vivo use, investigators at Ambion, a Life Technologies company in Austin, Texas, have been working on chemical modifications to synthetic oligos. “About three years back, we came out with a technology for high-throughput screening for siRNAs called Silencer Select,” said Nitin Puri, PhD, senior manager of R&D, Ambion. “With chemical and/or sugar modifications to the oligo we have now managed to both enhance specificity, while decreasing the immunogenicity of the siRNA.”
Along with other proprietary chemistries, the use of RNA analogues (so-called locked nucleic acids) in the Ambion process lends rigidity to the RNA structure that confers enhanced hybridization-to-target behavior, while at the same time blocking destructive ribonuclease activity. This last point is critical, because this dynamic has a marked effect on serum stability—a major issue for in vivo applications. “We have increased serum stability more than 150-fold in rat and mice serum,” said Dr. Puri. Unprotected, siRNA half-life is roughly five minutes; with the Ambion modifications, the half-life is about 24 hours.
Life Technologies has made other recent strides toward enhancing RNAi selection, quantification, and delivery: a method described by Cheng and colleagues, based on stem-loop real-time polymerase chain reaction technology, allows for easier quantification of siRNAs in vivo.4 A new vehicle reagent for RNAi duplex delivery, Invivofectamine 2.0, which enables high transfection rates in the liver, is also now available.
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CASE STUDY: RNAi May Replace Standard Stent Coatings

Angioplasty revolutionized the treatment of coronary artery disease; however, there are still improvements to be made. First-generation bare metal stents, scaffolds set in situ to maintain the balloon-opened artery, were vulnerable to rapid restenosis via the buildup of scar tissue; secondgeneration appliances, the so-called drug-eluting stents, prevent scar tissue proliferation but at the same time delay endothelial healing, which can lead to eventual thrombosis.1
CASE STUDY: RNAi May Replace Standard Stent Coatings
Extended recovery times require the prolonged use of expensive antithrombotic agents and are clearly suboptimal. “We had the idea to use siRNA because we think that we can have a faster re-endothelialization while stopping the inflammation,” explained Andrea Nolte, PhD, research scientist at the Children’s University Hospital in Tuebingen, Germany.
In her study, Dr. Nolte and colleagues conducted an in vitro assay using endothelial cells (ECs) exposed to a complex of polyethylenimine (PEI) and siRNA targeted to E-selectin, a molecule that plays an important role in the inflammatory response. The PEI/siRNA complex was mixed with a gelatin solution and then fixed to the bottom wells on culture plates. ECs and culture media were added, and after cells reached confluence, an inflammatory response was induced with the addition of TNFα. Results showed a 70% knockdown of expression of expected inflammatory factors.2,3
Along with validation of the approach, there was a happy surprise, and a challenge to be considered. “Normally, when you put a PEI/RNAi complex on ECs, not a whole lot happens,” said Dr. Nolte, but the construct fixed in gelatin sustained therapeutic efficacy even in the presence of serum. “We were very happy to see that.”
On the downside, the release of siRNA from the gelatin coating was too rapid; a different matrix will have to be found for the approach to be viable for a coated stent. “We’re looking for a release profile of about four weeks,” said Dr. Nolte, and added that they are exploring the use of poly(lactic acid-co-glycol acid) films. A sustained release over time should prevent initial stent-induced scarring while posing little hindrance to vascular healing.
Dr. Nolte’s use of siRNA-coated surfaces has caught the attention of numerous players in the industry. “They’re interested because there are other applications that would be useful with a coating,” she said. Beads coated with siRNA could be inserted into tumors, and topical formulations could be applied to chronic inflammatory conditions of the skin. “There are numerous settings that require the immobilization of the siRNA.”
Dr. Nolte’s work has, in fact, been done in partnership with industry. The stent application is being developed with Qualimed, of Hamburg, Germany.


  1. Zhao FH, Chen YD, Jin ZN, Lu SZ. Are impaired endothelial progenitor cells involved in the processes of late in-stent thrombosis and re-endothelialization of drug-eluting stents? Med Hypotheses. 2008;70(3):512-514.
  2. Nolte A, Walker T, Schneider M, Deniz O, Avci-Adali M, Ziemer G, et al. siRNA eluting surfaces as a novel concept for intravascular local gene silencing [published online ahead of print July 22, 2011].
  3. Walker T, Saup E, Nolte A, Simon P, Kornberger A, Steger V, et al. Transfection of short-interfering RNA silences adhesion molecule expression on cardiac microvascular cells [published online ahead of print June 20, 2011]. Thorac Cardiovasc Surg.

RNAi Delivery

Delivering enough therapeutically active molecules to the intended target remains the most vexing suite of obstacles for RNAi researchers. Challenges include achieving persistence in systemic circulation and effective reuptake by target cells.
Delivering enough therapeutically active molecules to the intended target remains the most vexing suite of obstacles for RNAi researchers. In brief, the challenges are achieving persistence in systemic circulation (no rapid clearance), localization of active moiety to target-tissue cell surface, efficient uptake by target cells, and rapid release of the internalized RNAi cargo from the transporting endosome into the cytosol.
It would take an entire textbook to delineate these issues. However, one author of a recent review of the field, Xudong Yuan, PhD, assistant professor in the division of pharmaceutical sciences at Long Island University in Brooklyn, N.Y., touches on some of the vehicular approaches that have recently caught his eye.5
“The good thing about (PLGA [D, L-lactide-co-glycolide]) is that it’s biodegradable, biocompatible, and, most importantly, it’s already approved by FDA,” Dr. Yuan said. Release of cargo from PLGA alone is too rapid, however. Dr. Yuan and colleagues are experimenting with PLGA combined with polyethyleneimines (PEIs). “This gives you better RNAi loading and also facilitates endosomal release through the proton sponge effect,” whereby cationic PLGA/PEI nanoparticles induce osmotic swelling, rupturing the endosome.6 With the same goal and mechanism in mind, Dr. Yuan is also working with chitosin, a biodegradable, linear polysaccharide, an approach that should have the added advantage of minimized toxicity.7
Currently not on his bench top but certainly on his radar are a few more applications for which Dr. Yuan also sees great promise:
  • RNAi-loaded nanoparticles composed of cyclodextrin, specifically, CALAA-01, the first targeted siRNA nanoparticle administered to humans, which is currently in clinical trials;8 and
  • “Lipidoids,” a combinatorial approach to constructing RNAi-bearing particles being developed by the Massachusetts Institute of Technology in collaboration with the RNAi therapeutics company Alnylam.9


  1. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391(6669):806-811.
  2. Fellmann C, Zuber J, McJunkin K, Chang K, Malone CD, Dickins RA, et al. Functional identification of optimized RNAi triggers using a massively parallel sensor assay. Mol Cell. 2011;41(6):733-746.
  3. Zuber J, Shi J, Wang E, Rappaport AR, Herrmann H, Sison EA, et al. RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia [published online ahead of print August 3, 2011]. Nature.
  4. Cheng A, Vlassov AV, Magdaleno S. Quantification of siRNAs in vitro and in vivo. Methods Mol Biol. 2011;764:183-197.
  5. Yuan X, Naguib S, Wu Z. Recent advances of siRNA delivery by nanoparticles. Expert Opin Drug Deliv. 2011;8(4):521-536.
  6. Nel AE, Mädler L, Velegol D, Xia T, Hoek EM, Somasundaran P, et al. Understanding biophysicochemical interactions at the nano-bio interface. Nat Mater. 2009;8(7):543-557.
  7. Yuan X, Shah BA, Kotadia NK, Li J, Gu H, Wu Z. The development and mechanism studies of cationic chitosan-modified biodegradable PLGA nanoparticles for efficient siRNA drug delivery. Pharm Res. 2010;27(7):1285-1295.
  8. Eifler AC, Thaxton CS. Nanoparticle therapeutics: FDA approval, clinical trials, regulatory pathways, and case study. Methods Mol Biol. 2011;726:325-338.
  9. Siegwart DJ, Whitehead KA, Nuhn L, Sahay G, Cheng H, Jiang S, et al. Combinatorial synthesis of chemically diverse core-shell nanoparticles for intracellular delivery. Proc Natl Acad Sci U S A. 2011;108(32): 12996-13001.

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