4. Biological challenges for gene inactivation

There are certain challenges exist, such as off-target effects, cytotoxicity, need for efficient delivery methods, their clinical implementation need efficient delivery vehicles and siRNA activity, itself, non-specific gene silencing, activation of innate immune system, the lack of efficient in vivo delivery systems still remain to be handled [80]. The effective delivery of RNAi therapeutics in vivo is one of the important challenge and have to consider several parameters for an efficient silencing, particle sizing, duration of the RNAi effect, its stability and modification, the delivery system and clearing off-target effects [81]. Apart from these challenges, the development of efficient tissue-specific and differentiation dependent expression of siRNA is essential for transgenic and therapeutic approaches. Bioactive drugs have been shown to perturb the naturally running system as these can clog/saturate the biochemical pathways. Since siRNA/shRNA relies on the endogenous microRNA machinery, thereby high doses of ectopic RNA have the risk of saturating all component of the miRNA pathway components. This was observed in the work by Grimm et al. [82] observed fatality association with high doses of liver-directed AAV-encoded shRNAs in mice, where high doses killed the recipient mice within 2 months. The length threshold of siRNAs seems to vary among cell types and it is an important consideration as dsRNA would induce innate immune responses that would eventually lead to cell death in mammalian. However, dsRNA less than 30 nucleotides have been shown good enough for no induction of cellular toxicity in mammalian and longer dsRNA is known to rapidly induce interferon responses. This suggests the careful risk assessment strategies when using longer and more potent Dicer substrates siRNAs. Moreover, correct RNAi targets are must, though ideal specificity of RNAi targets has not been shown. However if RNAi is going to silence off-targets, it can alter the gene function, which is clearly undesirable, therefore, care should be taken before-hand not to suppress the off-targets. If one third of siRNA are chosen randomly that it results in a toxic phenotype [83]. Comparison of siRNA and miRNA is described Table 4. However, there are successful in vitro and in vivo experiments for raising hopes in treating human disease with RNAi. The epigenetic network is one of the complex regulatory networks where epigenetic mechanisms such as DNA


Table 4. Comparison of siRNA and miRNA.


Table 5. Disadvantages, advantages of RNAi and possible therapeutic strategies.

methylation and modifications to histone proteins regulate gene expression and high-order DNA structure [84]. Epigenetics is basically a study of heritable changes in phenotypes where the DNA sequences are not changed anymore. DNA methylation [85] is an epigenetic factor that represents the inclusion of a methyl group (–CH3) to the fifth position of a cytosine pyrimidine ring or to the sixth nitrogen position of an adenine purine ring in genomic DNA. DNA methylation generally decreases belong to the gene expression level. In this connection, copy number variation (CNV) [86] is another latest domain of research in genomics. It is basically an event where the repetition of different portions of the genome continuously happens, and an alteration on the number of repeats in the genome is recognized between individual to individual in the human population. Copy number variation is a category of structural changes, especially, it is a type of either duplication or deletion event which generally influences a reasonable number of base pairs. It has been realized from recent researches that around two-thirds of the total human genome is made up of repeats. In the case of mammals, copy number alteration provides a significant contribution on producing the necessary deviation in both the population and disease phenotype. Cancer forms by various types of somatic genetic changes including copy-number alternations which affect the activity of the critical genes regulating the growth of the cell. Disadvantages and advantages of RNAi, and possible overcome strategies are demonstrated briefly in Table 5.
