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Jul 1, 2009 (Vol. 29, No. 13)

Creating Therapies via the RNAi Route

New Transfection Systems Tackle Delivery Issues and Off-Target Effects

  • Electroporation in a Pipette Tip

    Click Image To Enlarge +
    Standard preplated transfection vs. reverse transfection with siPORT™ NeoFX™ transfection agent
    (Ambion/Life Technologies)

    Christofer Cunning, Ph.D., senior marketing manager at Life Technologies, says that reproducibility, toxicity concerns, and the ability to successfully transfect primary cells and difficult-to-transfect cells, including immune cells and stem cells, remain challenging in siRNA experiments. “In general, people have used vector approaches with viral delivery systems for difficult transfections,” he says. “Use of viral vectors, however, remains time-consuming, requires cloning steps, is relatively expensive initially, and may require significant safety precautions.” In addition, while electroporation has been widely applied, “it requires strict adherence to already set protocols and a lot of siRNA.”

    To address these issues, Invitrogen launched its Neon™ transfection system earlier this year. In contrast to other systems, Neon allows transfection to take place in a pipette tip, using as few as 2x104 cells. “This technology is amenable to changes in protocols that can be defined by the user,” Dr. Cunning asserts.

    Unlike standard cuvette-based electroporation chambers, the Neon system uses a biologically compatible pipette tip chamber that generates a more uniform electric field. The company says this design allows better maintenance of physiological conditions resulting in high cell survival compared to conventional electroporation. A universal reagent kit allows use with all cell types.

    Invitrogen recently launched Invivofectamine™ for in vivo siRNA transfection. The company claims that in contrast to other marketed products, Invivofectamine can be injected in small volumes (microliters as opposed to milliliters) and without high pressure, thereby minimizing the potential of inconsistent results and gross harm to the subject animal.

    This reagent also reportedly provides stability to siRNA so that it remains intact and ready to perform the selected knockdown. Invitrogen has demonstrated successful delivery of intact siRNA molecules via direct injection into established tumors, tail vein injection to the liver, kidney, lungs, spleen, and pancreas, as well as effective and specific knockdown in these tissues.

  • Dynamic PolyConjugate Transfection

    Roche Madison was formed following Roche’s purchase of Mirus Bio in 2008. It has developed Dynamic PolyConjugate  (DPC) technology specifically to transfect and deliver siRNA in vivo. David Lewis, Ph.D., program director for RNAi, says the special properties that enable in vivo siRNA delivery include a positively charged membrane-active polymer that can act as a transfection agent once inside a cell. 

    “We shield, or mask, the positive charge for in vivo use and covalently bind siRNA to the polyconjugate via a disulfide bond,” Dr. Lewis explains. “The polymers are directed to specific locations through targeting ligands, for example, n-acetyl galactosamine, that targets the polymer to liver hepatocytes. Through the targeting ligand, the polymer can engage asialo-glycoprotein receptors and be taken up into liver cells via receptor-mediated endocytosis.” 

    In the endosome, the polymer is unmasked and becomes cationic, he says, allowing it to act as a transfection agent. The polymer lyses the endosome, “exposing it to the cytoplasm, a reducing environment that reduces the disulfide bond tethering the siRNA to the polymer and frees it to engage with RISC complexes.

    “To date, we have been able to silence apolipoprotein B as well as other target genes in rodents and nonhuman primates, and we envision this technology as a potential delivery system for therapeutic siRNAs,” Dr. Lewis adds. “Following apoB siRNA delivery, we have seen a decrease in LDL and serum cholesterol in the animals.” In addition, DPC can be delivered by simple intravenous injection in small volumes and does not require injection in potentially harmful large hydrodynamic volumes.


Readers' Comments

Posted 07/02/2009 by Professor

Very useful for for me! I needed to update on various currently available options.

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