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Delivering the siRNA Promise: Solutions to Intracellular Delivery and Why They Are Only Part of the Answer

Delivering the siRNA Promise: Solutions to Intracellular Delivery and Why They Are Only Part of the Answer

Apr 11, 2010

In 1989, researchers at the DNA Plant Technology Corporation in Oakland, CA attempted to over-express the enzyme responsible for the violet color of petunias.  They soon came to find that, instead of observing an even deeper violet color, their flowers had turned white.  The following years would be filled with further elucidation of a very mysterious effect.  Their discovery was firmly based in the central dogma of biology, defined by the transcription of DNA to mRNA and translation of mRNA to protein.  When they delivered the gene for calchone synthase (the color-generating enzyme), the mRNA produced from the gene bound to (hybridized) the natural mRNA, leading to degradation and enzyme loss and thus producing the first evidence for ribonucleic acid interference (RNAi).

Over the past decade, there has been an exponential growth in the pursuit of small interfering ribonucleic acid (siRNA) based therapies.  Accompanied by an increase in basic research on biological mechanisms of action and design of therapeutics, siRNA has shown the potential to address a wide array of diseases.  RNAi with siRNA as one of its primary effectors has failed to deliver on that promise because of the challenge of intracellular delivery1.

siRNA are small (20-25 nucleotide) pieces of ribonucleic acid that, when delivered to the cytoplasm of cells, can effectively eliminate the expression of a pathogenic protein.  Once inside the cytoplasm, siRNA is recognized by the RNA-induced silencing complex (RISC), a set of proteins responsible for messenger RNA (mRNA) cleavage.  RISC uses the anti-sense strand of the siRNA to recognize the target mRNA.  Since the cellular machinery for siRNA activity is already in place, the only trick is delivering siRNA to the cytoplasm of the target cells.  Unfortunately, siRNA are too large to permeate across the lipid membranes of cells and thus depend on specific cellular processes to access the cytoplasm.  Primarily, cellular uptake for large particles takes place through pinocytosis, a process by which the cell membrane envelops extracellular material.  However, these internalized compartments, called endosomes, become acidified and exposed to degradative enzymes that can destroy the siRNA, necessitating escape from the compartment into the cytoplasm.

There are several siRNA-based therapeutics currently in clinical trials2; however most of them are for specialized applications that sidestep targeting and delivery issues.  Targeting refers to the ability of a therapeutic to be preferentially distributed to or retained in a particular tissue.  For siRNA therapeutics administered by intravenous injection, the primary issue is the transport across cell membranes.  A recent publication from the labs of Pat Stayton and Allan Hoffman at the University of Washington describes one of the most recent and innovative approaches to the intracellular delivery of siRNA3.

As a test case, they knocked-down the expression of polo-like kinase 1, a signaling protein that leads to resistance to anthracyclines, and were able to sensitize drug-resistant ovarian cancer cells to a loaded chemotherapeutic.  To achieve this, they built a polymeric nanoparticle containing a diblock copolymer based on poly(dimethylaminoethyl methacrylate), which is positively charged, and poly(butylmethacrylate).  The copolymer forms micelles in water (spherical structures with a hydrophobic core and hydrophilic shell).  The micelle cores were loaded with doxorubicin, a chemotherapeutic, and complexed with negatively charged siRNA through electrostatic interactions.  Finally, the net-positively-charged particle was further coated with the pH-responsive, endosome-disrupting, poly(styrene-alt-maleic anhydride).  The resulting nanoparticles were tested on cells in vitro.

Polymer-based siRNA delivery systems have become popular and successful research endeavors among multiple labs at MIT.  A team working with Dan Anderson and Institute Professor Robert Langer at the David H. Koch Center for Cancer Research was able to deliver siRNA in vivo using a library of diverse lipid-like polymers based on random combinations of primary or secondary amines and alkyl chains4.  The cationic amine groups are important in the formation of electrostatic attractions between polymers and siRNA.  After lead variants were selected using an in vitro assay, they were tested for silencing of Factor VII in mice and monkey livers.  Factor VII is an important component in initiation of the coagulation cascade, is produced only in the liver, and has a short plasma half-life, making it a useful marker of liver-specific siRNA delivery.  Substantial and comparatively persistent in vivo silencing was achieved at low doses.  However, since the clearance mechanism for large molecular species and particles is the liver, the results may be misleading for non-scientists, because a far more substantial dose may be required to silence protein expression in other organs (such as epidermal growth factor receptor in a breast cancer model).  In short, while the results are promising in that the lipidoid system appears capable of enhancing intracellular delivery both in vitro and in vivo, the current formulation has not been shown to specifically target a tissue independent of the route of clearance.

Sangeeta Bhatia’s group, of the MIT-Harvard Division of Health Sciences and Technology program, used a different polymer particle design to achieve siRNA delivery in vivo5.  Here, they used amine-functionalized magnetic nanoparticles to build “nanoworms” consisting of an average of seven iron particles per unit.  The worms were then functionalized with generation five polyamidoamine (PAMAM) dendrimers and siRNA.  Dendrimers are branched polymers, where the generation refers to the number of levels of branching. The PAMAM variety consists of primary and tertiary amines that serve to buffer endosomal compartments and compromise their stability due to osmotic pressure build-up as the cell attempts to acidify the compartment.  The final complex structure was referred to as a “dendriworm”.  Studies showed that dendriworms were capable of mediating calcein release into cells as well as knock-down of EGFR in a glioblastoma cell line.  In vivo experiments were designed to complement those done in vitro by treating a mouse model of glioblastoma with the same siRNA.  However, clinical needs for tissue targeting were circumvented by intracranial infusion of the therapeutic to the central nervous system.  After examining these examples of siRNA delivery, it’s clear that all show merit with respect to the ability to potentiate intracellular delivery.  The unique ability of the Stayton and Hoffman approach to deliver two species is especially appealing.  In disease settings in which resistance to a therapeutic may arise, siRNA knock-down of the gene product responsible for the resistance might prove to be an elegant solution, enabling sustained use of an effective drug.  Technologies developed by the Stayton/Hoffman and Anderson/Langer teams are currently under development by the biotechnology companies PhaseRX (Seattle, WA) and Alnylam (Cambridge, MA), respectively.

The challenging hurdle of achieving tissue and cell-specific targeting of siRNA therapeutics still remains largely unsolved.  Research into the biodistribution and general pharmacokinetics of nanoparticles suggests that non-vascular applications, as their characteristic biodistribution cannot be altered by molecular recognition agents such as antibodies6.  Because of their size, they largely resist extravasation and escape from the blood stream, leading to accumulation in sites of enhanced vascular permeation (like tumors) and the liver (due to clearance).  This reduces their potential targets to diseases of the liver, or vasculature.  To expand the range of applications for siRNA therapeutics, an effective vehicle of a much smaller scale could be envisioned.  A therapeutic retaining the intracellular delivery properties achieved thus far, with a molecular weight, plasma retention, and targeting similar to that of an IgG may optimize tissue specificity, paving the way for an effective, systemically delivered siRNA therapeutic.


  1. Molecular Pharmaceutics (2009) 6
  2. http://clinicaltrials.gov/ct2/results?term=siRNA
  3. Benoit, DSW. Henry, SM. Shubin, AD. Hoffman, AS. Stayton PS.  pH-responsive polymeric siRNA carriers sensitize multidrug resistant ovarian cancer cells to doxorubicin via knockdown of polo-like kinase 1.  Molecular Pharmaceutics (2010) Online ahead of Print.
  4. Love, KT. Mahon, KP. Levins, CG. et al.  Lipid-like materials for low-dose in vivo gene silencing.  Proceedings of the National Academy of Science (2010) Online ahead of print.
  5. Agrawal, A. Min, D. Singh, N. et al.  Functional delivery of siRNA in mice using dendriworms.  ACS Nano (2009) 3:  2495-2504
  6. Zhou, Y. Drummond, DV. Zou, H.  et al.  Impact of single-chain Fv antibody fragment affinity on nanoparticle targeting of epidermal growth factor receptor-expressing tumor cells.  J Mol Bio (2007) 371: 934-47
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Scientist & Co-founder, Manus Biosynthesis";s:15:"profile_teambio";s:432:"Dr. Pirie is a scientist and co-founder at Cambridge, MA based Manus Biosynthesis. Chris earned his PhD in Biological Engineering from MIT in 2011 where his thesis research was focused on therapeutic protein engineering and he was a recipient of the NSF Graduate Research Fellowship. Also while at MIT he served on the Institute Committee on Intellectual Property and served as a writer and editor for the Entrepreneurship Review.";s:11:"profile_bio";s:1019:"He received his Bachelors degree, cum laude with college honors, in Bioengineering from the University of Washington where he studied intracellular drug delivery. In addition to his academic research he has served as a student representative to the Institute Committee on Intellectual Property where he helps guide MIT policy on important IP issues like open source publishing and patent reform. While at Washington he served on the Bioengineering Curriculum Committee pushing for improved course work in molecular transport and mathematical modeling. Born in the San Francisco Bay Area, he grew up in Seattle and now lives in Cambridge. As a life sciences editor and writer for MITER he sources primarily research focused articles with tangential, intellectual excursions into ideas on venture capital and technology translation. Chris enjoys sailing the Charles River, reading classic literature and astrophysics books, traveling to places other people only read about, and BASE jumping off the Green Building.