An evolutionary old mechanism of regulation of gene expression called RNA interference (RNAi), was discovered 10 years ago by Craig Mello and Andrew Fire [1]. Both of them were awarded with the Nobel Prize in medicine and physiology in 2006 to admire a deep impact made by their discovery on the molecular biology and potential application in therapy. RNAi is an RNA-dependent posttranscriptional gene silencing process induced in lower organisms by a long double stranded RNA, and in mammals by exogenously delivered short interfering RNAs (siRNAs) or by micro RNAs (miRNAs) coded in genome [2]. The mechanism employing endogenous miRNAs, operates in a cell as a powerful regulatory mechanism for genes switching on and off during various cellular processes, including cell development and carcinogenesis. The siRNA molecules are double-stranded 19-base pairs long RNA fragments with two-nucleotide overhangs at each 3’-end. Such duplexes are able to initiate gene silencing process as components of nucleoprotein RNA-induced silencing complexes (RISC) [3]. One of the siRNA strands, acting as a guide strand, hybridizes to the complementary sequence of mRNA and after RISC-performed cleavage excludes it from the translation process. Since their discovery, siRNA's have been widely used as convenient tools in functional genomics and as potential agents for therapeutic applications [4,5,]). Their silencing activity and specificity are determined by structural features, including thermodynamic stability of the duplex ends and their helical structure ,[6,]). The siRNA silencing activity can be modulated by chemical modifications in the functionally important regions (terminal and central domains of the duplex) [7]. RNAi can be effectively induced by exogenously delivered synthetic siRNAs or by siRNAs generated intracellulary from plasmid- or vector-coded shRNAs. After a long period of very limited success in the field of antisense/ribozyme approach (till now only one drug Vitravene has been approved by FDA), the RNAi technology kindled a new hope for revitalization of therapeutic strategies based on synthetic oligonucleotides. However, the RNAi technology still suffers from many limitations including siRNA intracellular instability, a problem of cell- or tissue-specific delivery and observed side effects. At present, there are a few siRNAs (for example, SiRNA027 and Cand5 designed for therapeutic treatment of the age-related macular degeneration (AMD)) in phase II and III of the clinical trials.

For several years, our research group interests have been focused on development of anti-amyloid strategy for Alzhemier’s disease (AD) based on sequence specific silencing of genes of enzymes involved in secretion of beta-amyloid peptides (Abeta). According to the amyloid cascade hypothesis, accumulation of Abeta is the primary factor driving neural degeneration. Abeta is the product of the proteolytic cleavage of the APP (amyloid precursor protein) substrate protein by beta- and gamma-secretases. It was demonstrated that beta-secretase (aspartyl protease Asp2), also called beta-site APP cleaving enzyme (BACE1), is an excellent target for anti-amyloid therapeutic drug design [8]. Till now, several approaches have been evaluated to find an effective inhibitor for human beta-secretase, mostly in the field of peptidomimetic, non-cleavable substrate analogues. Our approach is based on targeting of the BACE mRNA and its down-regulation with small inhibitory nucleic acids (siNAs) [9]. Those include catalytic nucleic acids (ribozymes and deoxyribozymes) as well as siRNAs. Plasmid-coded hammerhead ribozymes and synthetic deoxyribozymes 10-23 were the first investigated as efficient inhibitors of BACE1 gene expression in a sequence-specific manner, measured, both, at the mRNA and protein levels [10,11]. Later on, synthetic siRNA as well as vector-coded shRNAs proved to be useful to down-regulate BACE1 mRNA in human cell lines as well as in adult mice hippocampal neural stem cells and animal model [11,12].

[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):806-11.

[2] Hutvágner G, Zamore PD. A microRNA in a multiple-turnover RNAi enzyme complex. Science. 2002 Sep 20;297(5589):2056-60.

[3] Schwarz DS, Hutvágner G, Du T, Xu Z, Aronin N, Zamore PD. Asymmetry in the assembly of the RNAi enzyme complex. Cell. 2003, 115(2):199-208.

[4] Kim DH, Rossi JJ. Strategies for silencing human disease using RNA interference. Nat Rev Genet. 2007; 8(3):173-84.

[5] Aagaard L, Rossi JJ. RNAi therapeutics: principles, prospects and challenges. Adv Drug Deliv Rev. 2007 Mar 30;59(2-3):75-86.

[6] Nawrot B., Sipa K. Chemical and structural diversity of siRNA molecules, Curr. Top. Med. Chem. 2006, 6, 913-925.

[7] Sipa K, Sochacka E, Kazmierczak-Baranska J, Maszewska M, Janicka M, Nowak G, Nawrot B. Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA. RNA 2007, 13(8):1301-16.

[8] Citron M. Beta-secretase as a target for the treatment of Alzheimer's disease. J. Neurosci. Res. 2002, 70: 373-379.

[9]Nawrot B. Targeting BACE with small inhibitory nucleic acids – a future for Alzheimer’s disease therapy? Acta Biochim. Pol. 2004, 51 (2), 431-444.

[10] Nawrot B, Antoszczyk S, Maszewska M, Kuwabara T, Warashina M, Taira K, Stec WJ. Efficient Inhibition of b-Secretase (BACE) Gene Expression in HEK293T Cells by tRNAVal/CTE-Driven Hammerhead Ribozymes. Eur. J. Biochem. 2003, 270, 3962-3970.

[11]Nawrot B, Sipa K, Widera K, Antoszczyk S, Sierant M, Wojcik M, Maszewska M, Use of small inhibitory nucleic acids for down-regulation of genes involved in Alzheimer’s Disease, Proceedings of JMMC, Ed. Medimond s.r.l., Vienna, 2005, 49-54.

[12]Sierant M, Kubiak K, Warashina T, Kazmierczak-Baranska J, Nawrot B, In preparation

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1. Thermodynamic characterization and functional studies of chemically modified siRNAs
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