Another popular option is the use of transfection reagent. Several companies offer specialized siRNA-delivery reagents. Careful optimization of variable factors should be ensured for all initial transfection experiment. It is based on this and further optimization that reproducible gene knock out results will be obtained.
Usually RNAi effect is seen within 4 hours and the maximum down regulation observed in hrs. The effect lasts several cell generations and from days depending on cell culture type. RNAi is a sequence specific chain of events. The yield and purity depends on the coupling efficiency. Initial experiments should be done at varying concentrations from nM. Some reports have used as high as 25nM concentrations.
This is another variable that has to be optimized and then maintained. Time points should be taken after transfection to determine the maximum inhibition.
Start at 4hrs and end at 72hrs initially. RNAi down regulates a gene function without actually interacting with the gene. The subtle action is by mRNA degradation. Thus the degree of RNA interference achieved is directly proportional to the level of mature mRNA and the translated proteins. The options are:. Measurements of target protein enzyme activity. This option is suitable if a robust assay is available or has been in prior use.
The assay would vary by the nature of the protein product. The main advantage of siRNA expression vectors is that they are suitable for long-term applications. Depending on the target tissue, siRNA therapeutics can be administered either locally or systemically via intravenous injection.
However, unprotected siRNAs are prone to rapid degradation by ubiquitous endo- and exonucleases and they are undetectable in the blood already 10 min after administration DeVincenzo et al. Due to a strong anionic charge of the phosphate backbone, siRNAs cannot passively diffuse through negatively charged cellular membranes. Several approaches have been developed to enhance siRNA stability and promote its cellular uptake.
The most widely used approach involves introduction of chemical modifications to ribose sugar, phosphate linkage, or base of nucleotide Kaczmarek et al.
RNA can be easily modified during chemical synthesis. Furthermore, bacteriophage polymerases e. Moreover, these modification render siRNAs unrecognizable for immune system Morrissey et al. RNA delivery to the target cells can be achieved by viral and non-viral vectors.
For the delivery to animals and humans, adeno-associated virus AAV vector is the first choice since it has been proved to be non-toxic, non-pathogenic, easy to produce, and it does not integrate into human genome Naso et al.
Of the non-viral vectors, nanoparticles comprised of cationic polymers poly-L-lysine, polyamidoamine, polyethyleneimine, chitosan or lipids are the best studied delivery vehicles Kaczmarek et al. In addition to nanoparticles, direct conjugates of bioactive ligands to siRNA molecules can facilitate their entry to the cell.
Once siRNA is complexed with positively charged polymer or lipid molecules, it can approach cell membrane close enough to be internalized via micropinocytosis or clathrin-dependent pathway Pozzi et al. If siRNA is conjugated with specific ligands e. In this case endosome escape agents must be applied to facilitate siRNA transport to cytosol, where it can be used by RNAi machinery Dominska and Dykxhoorn, In clinical studies, human respiratory tract can be easily achieved by inhalation of an aerosol indicating a plausible administration route for antivirals against respiratory viruses.
For a detailed review on the approaches to siRNA delivery please refer to Musacchio and Torchilin, ; Kaczmarek et al. Furthermore, ALN-RSV01 has been shown to reduce the risk of bronchiolitis obliterans syndrome in RSV-infected lung transplant patients in Phase 2 randomized, double-blind, placebo-controlled trials Zamora et al. However, in clinical trials were suspended for undisclosed reason. In our opinion, this could be related to the emergence of drug resistant viruses, which are easily generated if only a single-site siRNA molecule is used.
Nevertheless the results from these trials are mostly reported at different scientific meetings instead of publications in peer-reviewed journals and, therefore, the detailed information is scarce. In this paragraph we will focus on siRNAs developed by Arrowhead Pharmaceuticals due to the abundance of the published data available for analysis. The siRNAs were conjugated to cholesterol, which facilitates the cellular uptake and protects from degradation by serum RNAses Schroeder et al.
These conjugates were intravenously co-injected with polymer-based system Rozema et al. ARC tolerability and pharmacokinetics has been studied in healthy volunteers with no indicated adverse effects Schluep et al. However, the data from a phase II clinical trials Wooddell et al.
Although siRNAs themselves were well tolerated in humans, endosome escape agent caused some toxicity in experimental animals. This HIV-infected person developed an acute myeloid leukemia and was subjected to hematopoietic stem cell transplantation in A pool of three chemically modified siRNAs preventing synthesis of Zaire ebolavirus ZEBOV polymerase, viral proteins 24, and 35 completely protected rhesus macaques from lethal infection Geisbert et al.
However, TKM-Ebola did not improve survival, which might be connected to a poor design of the clinical trials and inclusion of only terminally sick patients with high viral loads Cross et al. Two clinical trials have been initiated to assess the efficacy and safety of shRNA-based TT therapeutics, which was created for chronic hepatitis C treatment and delivered to hepatocytes by AAV vector.
TT was shown to be safe and well-tolerated. However, in February Benitec Biopharma decided to discontinue hepatitis C program due to low commercial opportunities 3. In conclusion, the development of RNAi-based therapeutics is still in its early stage and has experienced numerous pitfalls.
Nonetheless, it has already been demonstrated that siRNAs can effectively inhibit the replication of various viruses despite different mechanisms they evolved to resist the pressure imposed by immune system and antiviral drugs. However, the possibility of generation of escape mutants, recently discovered inhibitors of RNAi in human viruses Fabozzi et al.
Furthermore, there are still unresolved issues with safe and efficient delivery of siRNAs to the target tissues and cells, which can be unraveled by fundamental research in the area. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Aalto, A.
RNA 13, — Alkhatib, G. Alvarez, R. RNA interference-mediated silencing of the respiratory syncytial virus nucleocapsid defines a potent antiviral strategy. Agents Chemother. Anderson, E. Identifying siRNA-induced off-targets by microarray analysis.
Methods Mol. Beaucage, S. Solid-phase synthesis of siRNA oligonucleotides. Drug Discov. PubMed Abstract Google Scholar. Bitko, V. Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses.
BMC Microbiol. Inhibition of respiratory viruses by nasally administered siRNA. Bobbin, M. Genome Med. Brummelkamp, T. A system for stable expression of short interfering RNAs in mammalian cells.
This natural mechanism for sequence-specific gene silencing promises to revolutionize experimental biology and may have important practical applications in functional genomics, therapeutic intervention, agriculture and other areas. A possible mechanism underlying the regulation of endogenous genes by the RNAi machinery was suggested from studies of C. A simplified model for the RNAi pathway is based on two steps, each involving ribonuclease enzyme. The benefit of shRNA is that they can be incorporated into plasmid vectors and integrated into genomic DNA for longer-term or stable expression, and thus longer knockdown of the target mRNA.
Lentiviral delivery of the shRNA enables stable expression and permanent knockdown of the target gene. The shRNA becomes stably integrated into the host cell genome. As cells divide, the shRNA is passed on to daughter cells.
0コメント