Chemically Modified siRNA Molecules - Term Paper Example

Chemically modified siRNA Introduction Gene regulation by RNA interference (RNAi), the process of modulating RNA level by small interfering RNA (siRNA), has provided investigators a tool to turn off genes at will. Cellular genes involved in human diseases, thus, can be targeted and silenced by exogenous introduction of siRNA or by introduction of gene constructs expressing short hairpin RNA (shRNA) that are converted into siRNA by the RNAi machinery (Tiemann and Rossi, 2009). However, like natural RNA in the human cells, siRNA is fragile and has a short life span if it is not protected from harsh environments in the body system. Delivery and nuclease resistance are two major challenges for the therapeutic application of siRNA. For example, siRNA itself cannot reach its mRNA targets inside cells since siRNA has large net negative charges and its size is too large for cell membrane crossing (Tiemann and Rossi, 2009). Various delivery tools for siRNA include lipids, cholesterol, virus, nanoparticles, and peptides (Tiemann and Rossi, 2009). Nuclease resistance of siRNA is another problem that needs to be addressed for the therapeutic application of siRNA. There are two major causes known to induce the siRNA degradation. One is nucleases present inside cells and the other is 2’ OH ribose groups innate to siRNA structure which, depending on its chemical environment, could initiate self-degradation of siRNA. The first step of siRNA degradation by various nucleases is the siRNA recognition by the nuclease through three dimensional fits. Chemical modifications on siRNA alter the three dimensional structures of siRNA and devoid the recognitions by nucleases and/or its neighboring 2’ OH destructive groups. Each chemical modification on siRNA, thus, could alleviate the degradation problem caused by nucleases and/or innate destructive nature of 2’ OH groups on siRNA. A very general form of well-tolerated modification concerns the use of 2’ F (2’-F-2’-deoxy-nucleoside residues) or 2’ OMe (2’-O-methyl-nucleoside residues) groups (Allerson et al., 2005). Chemically destructive innate 2’ OH groups on siRNA are displaced with either by 2’F or 2’ OMe groups in the study of Allerson et al. (2005). The displacements at 2’ OH groups have additional advantageous effect on the stability of siRNA by canceling its dimensional recognition by various nucleases. Additional present day approach has involved use of accurate placement of residues that contain 6-carbon sugars in place of ribose can result in siRNA with better stability and more enduring pharmacological actions (Fisher et al., 2007). In this paper, RNAi efficacies of chemically modified siRNA of 2’ F, 2’ OMe or altritol ribose (ANA) and the positional effects of partially modified siRNA with 2’ F, 2’ OMe or altritol ribose will be evaluated. Fully 2’-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA (Allerson et al., 2005). Allerson et al. (2005) of Isis Pharmaceuticals reported the effect of fully modified siRNA with 2’ F and/or 2’ OMe groups. Previously, 2’ F and 2’ OMe were known to increase the thermal stability of duplexes (Chiu and Rana, 2003). In this study, Allerson et al. have modified the entire sequence of siRNA with either 2’ F or 2’ OMe. RNAi efficacies of the modified siRNAs were then evaluated. The siRNA duplexes were designed to target one of two sites within the coding region of human PTEN mRNA, both previously reported as valid target sites for siRNA. Modified and unmodified duplexes were introduced into HeLa cells using a cationic lipid transfection reagent (lipofectin). In vitro activity was calculated by doing RT-PCR on PTEN mRNA and then making comparison to mRNA levels of untreated cells. The lower mRNA level than untreated cells would be expected if siRNA functions as designed. The efficacies of various siRNAs can be analyzed based on the mRNA levels on cells treated with each siRNA. Among the most challenging designs that came out from their studies was a completely modified duplex consisting of alternating 2’ OMe and 2’ F nucleotides. Duplexes resulting from this substitution demonstrated increased in vitro potency of 500 fold as compared to unmodified duplexes (Allerson et al., 2005). This study employed target sites coding regions of human PTEN mRNA which already had been established as valid siRNA targets. Thus it is easy to compare the RNAi effects of their chemically modified siRNA with previously published ones. This paper contributes to the research field that siRNAs with alternating 2’ F and 2’ OMe modifications enhance the efficacy of the siRNA. However, in vivo study is needed for this alternating modification to be adopted widely in RNAi-based drug development. Also, it may involve the extra steps in preparing the siRNA with alternating modifications. There should be an mechanistic explanation why the alternating modification is better than other types of modifications. Positional effect of chemical modifications on short interference RNA activity in mammalian cells (Prakash et al., 2005). Another Isis group (Prakash et al., 2005) evaluated positional effects of partially modified siRNA in mammalian cells. In this study, systematic investigation on the effect of 2’-sugar modifications (2’ F, 2’ OMe and 2’ O-MOE (2’-O-(2-methoxyethyl) nucleoside residues)) of three consecutive bases on the antisense and sense strands of siRNA was performed in HeLa cells. The research method employed here was using the same target Allerson et al. have used in their study on fully modified siRNA. Inhibition of PTEN expression by modified siRNA duplex was measured by real time quantitative RT-PCR as described earlier. The study of siRNA antisense strand revealed that activity is dependent on the sequence position where modification has taken place. The siRNAs with modified ribonucleotides at 5’ end of antisense strand were less active relative to the 3’ modified ones. The 2’ F sugar was generally well-tolerated on the antisense strand, whereas the 2’ OMe showed significant shift in activity depending on the position of modification. The 2’ O-MOE modification on the antisense strand leads to less active siRNA constructs in spite of placement position in the construct. The incorporation of the modified residues of 2’ OMe or 2’ O-MOE in the sense strand of siRNA did not show a strong positional preference (Prakash et al., 2005). This paper contributes to the research fields that chemical modifications of siRNA with 2’ F, 2’ OMe or 2’ O-MOE are showing strong positional effects. The results provide guidelines to design effective and stable siRNA for RNAi mediated therapeutic application. The positional effects of modifications are important tools towards the mechanistic investigation of RNAi as well as new drug developments. However, as evident limitation of the possible combinatorial placements, this investigation of positional effects will never be complete. For example, Prakash et al. have employed to modify three consecutive bases on siRNA strands in this 2006 investigation. But, it could be argued that less than (or more than) three base modification would be required to cause significant difference. Or could there be siRNA with higher efficacy with partial modification? Chemical modification of siRNAs to improve serum stability without loss of efficacy (Choung et al., 2006). In this 2006 study, Choung et al. at Bioneer and Korea Research Institute of Bioscience and Biotechnology (KRIBB) have used systematic modifications of 2’ F and 2’ OMe on Sur10058, a hyperfunctional siRNA which targets survivin mRNA. As compared to the unmodified Sur10058 which was frequently degraded in human serum, various modified structures of Sur10058 showed significantly enhanced serum stability. The RNAi effects of siRNAs were evaluated by the reduction of survivin protein. Here, it should be noted that Choung et al. employed protein quantitations, not an mRNA levels determination in order to determine the efficacies of their modified siRNA. End modification of 3-terminal nucleotides by 2’ OMe resulted in average increase in serum stability, with 3’ end modification being more effectual. Alternating modification by 2’ OMe substitution significantly stabilized Sur10058 with highest RNAi effect (Choung et al., 2006). Choung et al., in this study, provide the improved siRNA derivatives over unmodified siRNA with Sur10058 sequence. Also, the importance of terminal region on siRNA was illustrated by this study. This study also requires further in vivo investigation for future drug development. Unlike studies by Allerson et al. (2005) and Prakash et al. (2005) which showed superior siRNA effects by 2’ F and 2’ OMe alternating sequence, however, Choung et al. (2006) reported that alternating modifications of 2’ OMe and 2’ OH on full sequence yielded the highest level of siRNA action. It would be interesting to compare Isis formula of alternating 2’ F and 2’ OMe and KRIBB formula of alternating 2’ OMe and 2’ OH towards mRNA targets other than Sur10058 target. Inhibition of MDR1 expression with altritol-modified siRNAs (Fisher et al., 2007). Ribose moiety on siRNA was replaced with 6 carbon ring, altritol, by Fisher et al. of University of North Carolina and Rega Institute for Medical Research of Belgium in their 2007 study of MDR1 gene expression inhibition study. Altritol-modified nucleic acids (ANAs) assist RNA-like A-form structures when accommodated in oligonucleotide duplexes. Here Fisher et al. employed both RNA quantitation using RT-PCR and protein quantitation using Flow Cytometry, and revealed that ANA modified siRNAs pointing the MDR1 gene could display improved effectiveness in comparison to unmodified controls. Also, their positional study is much more extensive compared to previous results from Isis and KRIBB groups (Fisher et al., 2007). According to Fisher et al. (2007), only fully ANA modified siRNA was nuclease resistant while partially ANA modified siRNA was not nuclease resistant. SiRNA activity was retained through extensive ANA modification on the central part of the sense strand. ANA modifications on both strands result in effectiveness, on the other hand, when the number ANA residues increase on one strand, it does not alter anything . The greatest siRNA effects were attained with duplexes having 3’ ANA modifications in both strands. A decrease in MDR1 protein level observed subsequential to siRNA treatment were kept same by decrease in MDR1 mRNA levels. Substantially increased period of action of some of the ANA modified siRNAs was also observed (Fisher et al., 2007). The most important contribution by Fisher et al. (2007) is that partial ANA modification, especially at 3’ regions, enhances siRNA efficacy with increased duration of siRNA action. The increased duration provides significant advantage for therapeutic application of siRNA, especially when siRNA is acting as catalysts inside cells. The result that partially ANA modified siRNA is better than fully modified one is opposite of previous modifications at 2’ positions. It may be due to the fact that extensive modifications at ribose frame cause drastic conformational changes so that fully ANA modified siRNA cannot undergo RNAi cycle inside cells, while 2’ modifications can be tolerated more easily. It would be interesting to see results from in vivo study of this ANA modified siRNA. Comparison of ANA modification with other 2’ modifications in vivo will be particularly interesting, especially given the result of prolonged duration of ANA modified siRNA. Conclusions Developing nuclease resistant siRNA is one of the two major huddles in RNAi-based drug development. 2’ F and 2’ OMe are leading 2’ modifications while altritol is the leading ribose modification currently explored and, thus, on route to clinical trials. However, at this time, there does not seem to be one form of chemical modification that is better in every condition. For example, fully modified siRNA with alternating 2’ F and 2’ OMe was superior in one investigation while fully modified siRNA with alternating 2’ OMe and 2’ OH exhibited a better RNAi efficacy in another case. Also, full ribose modification is not tolerated by RNAi machinery. It may be that diverse forms of siRNA work excellent in different tissues. The prolonged duration of ANA modified siRNA is promising aspect for drug development. Only Fisher et al. have utilized both mRNA and protein quantitations and exhibited their corelations. It would be interesting to compare different types of modified siRNA in one biological system. In this case, we could eliminate variations like different cellular uptake. What about siRNA with both 2’ and ANA modifications at 3’ end? References Allerson C., Sioufi N., Jarres R., Prakash T., Naik N., Berdeja A., Wanders L., Griffey R., Swayze E. and Bhat B. (2005). Fully 2’-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified samm interfering RNA. J. Med. Chem.,48, 901-904. Chiu Y. and Rana T. (2003). SiRNA function in RNAi: A chemical modification analysis. RNA, 9, 1034-1048. Choung S., Kim Y., Kim S., Park H. and Choi Y. (2006). Chemical modification of siRNAs to improve serum stability without loss of efficacy. Biochemical and Biophysical Communications, 342, 919-927. Fisher M., Abramov M., Aerschot A., Xu D., Juliano R. and Herdewijn P. (2007). Inhibition of MDR1 expression with altritol-modified siRNAs. Nucleic Acids Research, 35(4), 1064-1074. Juliano R., Bauman J., Kang H. and Ming X. (2009). Biological barriers to therapy with antisense and siRNA oligonucleotides. Molecular Pharmaceutics, 6(3), 686-695. Prakash T., Allerson C., Dande P., Vickers T., Siofi N., Jarres R., Baker B., Swayze E., Griffey R. and Bhat B. (2005). Positional effect of chemical modifications on short interference RNA activity in mammalian cells. J. Med. Chem., 48, 4247-4253. Tiemann K. and Rossi J. (2009). RNAi-based therapeutics-current status, challenges and prospects. EMBO Molecular Medicine, 1, 142-151. Read More