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Possible therapeutic applications and challenges[edit]

Since many disease states are caused by the overexpression of a gene responsible for a certain biochemical pathway, siRNA has been gathered support as a therapeutic agent. In a therapeutic application, an siRNA antisense to the gene that is overexpressed is introduced to the cell and binds to the mRNA transcript to cause the biochemical pathway that the gene was responsible for to stop functioning. For example, it has been shown that depleting polo-like kinase 1 can lead to apoptosis in cancer cells, but not normal cells.[1][2] Therefore, introduction of an siRNA that is antisense to the gene coding polo-like kinase 1 will lead to down regulation of the kinase, which causes the death of only tumor cells and therefore can be used to treat cancer.[3] Introducing siRNA into the body, however, is not easy. The siRNA molecules are too small to stay in circulation, but are too large to enter cells passively. Moreover, the immune response induced by exogenous siRNA has a large effect on the therapeutic value of the treatment.[4] Research on the use of siRNA as a therapuetic agent is largely focused on the safe and effective delivery of the siRNA molecules.

Phase I results of the first two therapeutic RNAi trials (indicated for age-related macular degeneration, aka AMD) reported at the end of 2005 demonstrated that siRNAs are well tolerated and have suitable pharmacokinetic properties.[5] siRNAs and related RNAi induction methods therefore stand to become an important new class of drugs in the foreseeable future.

In 2008, a team of researchers from Texas Tech University and Harvard University announced the development of a siRNA-based treatment that may ultimately counteract the Human Immunodeficiency Virus (HIV). Human cells infected with HIV, injected into rats, have been cured by the experimental treatment. Clinical trials on humans are expected to begin by 2010.[6][7][8]

In 2008 a novel DNA-siRNA delivery system that could lead to more efficient and more disease-specific vaccines against infectious diseases was developed by researchers at The University of Texas at Austin. Biomaterials based micron size particles carrying both the DNA vaccine and the siRNA to immune cells show potential to divert immune response in desirable directions.[9]

siRNA Delivery[edit]

Contemporary efforts to deliver siRNA focus on finding ways to successfully shuttle the siRNA molecules to cells while reducing the negative effects of foreign siRNA.

Lipid Nanoparticle (LNP) Systems[edit]

The most mature technology is the Lipid Nanoparticle (LNP) delivery system. In this system, a positively-charged lipid is formulated in a way that it is protonated at physiological pH, and can therefore bind to the negative charges on the siRNA backbone. This forms a micelle around the siRNA molecule which acts as a charge-shielding envelope.[10] Because of this, the micelle is significantly more stable in circulation, and can be taken into the cell through endocytosis. Once inside the cell, the change in pH across the cell membrane can protonate the phosphate backbone of the siRNA, making the siRNA neutral. The loss of negative charge causes the lipids to detach from the siRNA and the micelle begins to degrade. This allows the siRNA payload to be delivered to intracellular environments exclusively.

Recent Advancements[edit]

Further, the LNP can be “doped” with various polymers and other lipophilic components to allow the attachment of targeting molecules[11] that allow the LNP to deliver only to specific tissue types. This is important because, without some form of targeting, the LNPs affect any cell that takes them up.[12] Therefore, the introduction of targeting groups reduces off-target effects in pathways that exist in both healthy and diseased cells. Other molecules such as fusogenic peptides have been attached to the surfaces of the nanoparticles, which improve cellular uptake of the nanoparticles.[13] These peptides help the LNP penetrate the cell membrane by interacting with the charged surface of the phospholipid bilayer and inserting themselves into the membrane.[14] This helps the LNP initiate endocytosis, increasing the efficiency of the drug delivery.

Problems and Further Study[edit]

One issue that affects LNPs is that they activate a part of the immune system known as complement system.[15][16] In complement, the immune system identifies a foreign particle, here an LNP, and initiates pre-programmed cell death. However, since the LNPs are taken up into cells and deliver siRNA faster than complement is activated, the therapeutic index is significantly greater than that for unformulated siRNA.[17] Current research focuses on lessening the immunostimulatory nature of these particles; as the immune response to the particles decreases, the therapeutic index increases.

Bifunctional Gold Nanoparticles[edit]

Small clusters of neutral gold atoms known as gold nanoparticles have also been effective in siRNA delivery.[18] In these systems, polymers are attached to the surface of the nanoparticle by gold-sulfur bonds, followed by the annealing of the siRNA molecule to the polymer by disulfide bonds.[19] These particles are very stable to circulation until taken up into the cell, where enzymes within the cell cut the siRNA away from the nanoparticle.

Recent Advancements[edit]

The benefit of using gold nanoparticles is that in addition to modifying the surface of the particle, the core can also be modified. Some recent research has focused on adding a magnetic component to the particles.[20] In this way, the particle reacts to magnetic fields, allowing the particle to be manipulated in living systems using a magnet to attract or repel the particles. Other efforts have added imaging components to the particles.[21] By incorporating MRI contrast agents, such as amphiphol, into the nanoparticle, the particles can be visualized as they move through the body.[21][22] This allows the researcher or physician to be sure the particle is delivering the siRNA to the appropriate parts of the body during treatment.

Problems and Further Study[edit]

The particles, however, face problems with excretion, and can cause liver failure due to buildup of the particles.[22] Because there is no dedicated method for breaking down gold in the body, the nanoparticles tend to collect in cells until they are finally passed to the liver, where they still cannot be broken down.[19][22] While the particles are generally not immunostimulatory, they can cause a response if they remain in the liver long enough,[22][23] and as large quantities build up, toxicity leading to liver failure can be observed.[22] Current research is focused on finding ways to maximize efficacy to limit the amount of gold introduced to the body, and to find ways to increase excretion of the particles to limit accumulation,[24] which will significantly improve the therapeutic index of the gold nanoparticle drugs.


References[edit]

  1. ^ Chopra, P.; Sethi, G.; Dastidar, S. G.; Ray, A., Polo-like kinase inhibitors: an emerging opportunity for cancer therapeutics. Expert Opin. Investig. Drugs 19 (1), 27-43.
  2. ^ Liu, X. Q.; Lei, M.; Erikson, R. L., Normal cells, but not cancer cells, survive severe Plk1 depletion. Mol. Cell. Biol. 2006, 26 (6), 2093-2108.
  3. ^ Hu, K. J.; Lee, C.; Qiu, D. X.; Fotovati, A.; Davies, A.; Abu-Ali, S.; Wai, D.; Lawlor, E. R.; Triche, T. J.; Pallen, C. J.; Dunn, S. E., Small interfering RNA library screen of human kinases and phosphatases identifies polo-like kinase 1 as a promising new target for the treatment of pediatric rhabdomyosarcomas. Mol. Cancer Ther. 2009, 8 (11), 3024-3035.
  4. ^ Frank Y. Xie, M. C. W., Patrick Y. Lu, Harnessing in vivo siRNA delivery for drug discovery and therapeutic development. Drug Discovery Today 2006, 11 (1-2), 67-73.
  5. ^ Tansey B (11 August 2006). "Macular degeneration treatment interferes with RNA messages". San Francisco Chronicle. http://www.sfgate.com/cgi-bin/article.cgi?f=/c/a/2006/08/11/BUGPNKFU6T1.DTL."
  6. ^ "Texas Tech Researchers May Have Found AIDS Cure". KCBD. 2008-08-07. http://www.kcbd.com/Global/story.asp?S=8808748. Retrieved 2008-08-16.
  7. ^ Swaminathan, Nikhil (2008-08-07). "Researchers Silence HIV in Mice Engineered to Be Like Humans". Scientific American. http://www.sciam.com/article.cfm?id=researchers-silence-hiv-in-human-like-mice-rnai. Retrieved 2008-08-16.
  8. ^ "Researchers halt spread of HIV with RNAi". Harvard Medical School. http://web.med.harvard.edu/sites/RELEASES/html/080708_shankar.html. Retrieved 2008-08-16.
  9. ^ http://www.bme.utexas.edu/news/2008_roy_VDS.cfm.
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  12. ^ Singh, S. K.; Hajeri, P. B., siRNAs: their potential as therapeutic agents - Part II. Methods of delivery. Drug Discovery Today 2009, 14 (17-18), 859-865.
  13. ^ Hatakeyama, H.; Ito, E.; Akita, H.; Oishi, M.; Nagasaki, Y.; Futaki, S.; Harashima, H., A pH-sensitive fusogenic peptide facilitates endosomal escape and greatly enhances the gene silencing of siRNA-containing nanoparticles in vitro and in vivo. J. Control. Release 2009, 139 (2), 127-132.
  14. ^ Martin, I.; Ruysschaert, J. M., Common properties of fusion peptides from diverse systems. Biosci. Rep. 2000, 20 (6), 483-500.
  15. ^ Nakanishi, T.; Kunisawa, J.; Hayashi, A.; Tsutsumi, Y.; Kubo, K.; Nakagawa, S.; Nakanishi, M.; Tanaka, K.; Mayumi, T., Positively charged liposome functions as an efficient immunoadjuvant in inducing cell-mediated immune response to soluble proteins. J. Control. Release 1999, 61 (1-2), 233-240.
  16. ^ Scholer, N.; Hahn, H.; Muller, R. H.; Liesenfeld, O., Effect of lipid matrix and size of solid lipid nanoparticles (SLN) on the viability and cytokine production of macrophages. Int. J. Pharm. 2002, 231 (2), 167-176.
  17. ^ Christian Wolfrum, S. S., K Narayanannair Jayaprakash, Muthusamy Jayaraman, Gang Wang, Rajendra K Pandey, Kallanthottathil G Rajeev, Tomoko Nakayama, Klaus Charrise, Esther M Ndungo, Tracy Zimmermann, Victor Koteliansky, Muthiah Manoharan,Markus Stoffel, Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nature Biotechnology 2007, 25, 1149-1157.
  18. ^ Elbakry, A.; Zaky, A.; Liebkl, R.; Rachel, R.; Goepferich, A.; Breunig, M., Layer-by-Layer Assembled Gold Nanoparticles for siRNA Delivery. Nano Lett. 2009, 9 (5), 2059-2064.
  19. ^ a b DeLong, R. K.; Akhtar, U.; Sallee, M.; Parker, B.; Barber, S.; Zhang, J.; Craig, M.; Garrad, R.; Hickey, A. J.; Engstrom, E., Characterization and performance of nucleic acid nanoparticles combined with protamine and gold. Biomaterials 2009, 30 (32), 6451-6459.
  20. ^ Boyer, C.; Priyanto, P.; Davis, T. P.; Pissuwan, D.; Bulmus, V.; Kavallaris, M.; Teoh, W. Y.; Amal, R.; Carroll, M.; Woodward, R.; St Pierre, T., Anti-fouling magnetic nanoparticles for siRNA delivery. J. Mater. Chem. 2010, 20 (2), 255-265.
  21. ^ a b Qi, L. F.; Gao, X. H., Quantum dot-amphipol nanocomplex for intracellular delivery and real-time imaging of siRNA. ACS Nano 2008, 2 (7), 1403-1410.
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