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RNA Interference Technology-Development and Applications


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RNA Interference Technology-Development and Applications

RNA interference (RNAi) is a conserved gene-regulation mechanism in all eukaryotic cells, where small RNAs including small hairpin RNA (shRNA), small interfering RNA (siRNA), and micro-RNA (miRNA) interact with message RNAs (mRNAs) in a sequence-specific manner and cause the cleavage or translational blockage of a gene. Since its specificity and efficiency, RNAi has been widely utilized as a routine tool for gene function studies in biology laboratories worldwide.

Over the past decade, RNAi has been demonstrated in many eukaryotes including humans as well as some prokaryotic life forms and has been recognized to form an integral part of many gene regulatory networks during development. Currently, it is a routine laboratory practice to introduce the desired gene-specific dsRNA inducers into cells and selectively, systematically, and robustly silence the targeted sequence signature revealing its cellular function. In addition, RNAi has become an effective tool for agricultural biotechnology to improve production and combat disease pests as well as for medicine and molecular pharmacology to cure inflammatory, complex infectious and hereditary diseases.

The principle and mechanism of RNAi

RNAi is induced by the introduction of specific exogenous dsRNA either by virus genome RNAs, injection of synthetic dsRNAs. And in plants, RNA is mediated by Agrobacterium. RNAi is also the part of the normal development and dsRNAs are produced by endogenous genes encoding miRNA precursors or other long dsRNA molecules. In either case, dsRNAs are recognized by the enzyme Dicer and cleaved into short, double-stranded fragments of ~19-25 base pair long siRNAs. These siRNAs are divided into two single-stranded RNAs (ssRNAs), which are referred to as the “passenger” and the “guide” strands. The passenger strand is degraded, while the guide strand is picked up by the RNA-induced silencing complex (RISC) which has enzymatic digestion activity and contains the key components of Argonaute (AGO) and P-element induced wimpy testis (PIWI) proteins. The RISC proteins perform the unwinding of the guide and passenger strands in ATP-independent manner. Nevertheless, ATP is required to unwind and remove the cleaved mRNA strand from the RISC complex after catalysis. There are effector proteins such as RDE-4 (nematodes) and R2D2 (insects) which recognize exogenous dsRNAs and stimulate dicer activity.

Figure 1. Biogenesis of miRNA and gene silencing pathway

RNAi as a functional genomics tool

RNAi technology is applicable for gene silencing in many species. RNAi has been used extensively in C. elegans for functional genomics. High-throughput investigation of most of the ~19,000 genes has been accomplished. Ahringer has produced an RNAi library, representing ~86% of the genes of C. elegans. The strategy has also been successfully attempted in multiple other model organisms, including human.

In addition, RNAi has been utilized successfully in mammalian cells. A series of methods have been employed for siRNA knockdown of specific genes in mammalian cells. DNA-vector-mediated RNAi can silence genes transiently in mammalian cells, while other expression systems are used for stable silencing. The promoters of RNA polymerase (pol) II and III (U6 and H1) have been used for stable silencing. And tRNA promoter-based systems have been used for this purpose. There are two classes of retrovirus vectors that have been employed: (1) HIV-1-derived lentivirus vectors and (2) Oncoretrovirus-based vectors, such as Moloney murine leukemia virus (MoMuLV) and Murine stem cell virus (MSCV). Transgenic mice have been established with germline transmission of a shRNA expression cassette for silencing of genes not targeted by homologous recombination-based approaches. Desirable applications of this technique include inducible and cell type-specific expression patterns.

The applications of RNAi for therapy

Since RNAi blockage is very specific and at the transcriptional level, RNAi-based gene therapy (RNAi therapy) is thought to hold an enormous potential for treating many diseases, especially genetic disorders and viral infections. In fact, synthesized siRNA is regarded as a specialized drug for gene therapies. So far, promising results have been obtained with RNAi therapy in various diseases and many are being tested in clinical trials, including cancers, viral infections, and genetic or inflammatory disorders.

  • Reduce the risk of rejection in cell-based therapies

The high variability of the human leukocyte antigen (HLA) constitutes a main hurdle in allogeneic transplantation and to the application of off-the-shelf cell products in regenerative medicine. In studies, researches genetically modify the graft to silence its HLA expression to prevent the recognition of the allogeneic graft as non-self by the recipient’s immune system. In vitro assays have shown that HLA class I-silenced cells were protected against the antibody-mediated complement-dependent cytotoxicity. In addition, in T-cell cytotoxicity assays, significantly lower cell lysis rates were observed when HLA silenced cells were used as targets in comparison to fully HLA-expressing cells. Furthermore, HLA-silenced cells demonstrated to induce significantly lower T-cell proliferation, pro-inflammatory cytokine secretion, and degranulation. The residual HLA class I expression showed to be sufficient to prevent NK cell cytotoxicity. Altogether, HLA silenced cells showed a protective effect against the cellular and humoral allogeneic immune response.

  • Immune modulatory microRNAs in tumors

A close relationship between miRNAs and human cancer has been developed during the past years. High throughput analyses allow the comparison of miRNA expression pattern in normal and tumor tissues demonstrating global changes within the miRNA expression in different malignancies. Interestingly, the miRNA genes are frequently located at fragile sites and cancer-associated chromosomal regions. The deregulation of the biogenesis and expression of miRNAs is involved in the initiation as well as progression of tumors, metastasis formation, and therapy resistance. Some studies have indicated that miRNAs could be as putative targets for immune therapy (Figure 2).

Figure 2. miRNAs as putative targets for immune therapy

  • RNAi therapeutic potentials in CNS disease

Particularly, one attractive group of candidate diseases for RNAi therapy is the dominantly inherited neurodegenerative diseases, including polyglutamine disorders such as Huntington’s disease (HD), familial Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia caused by tau mutations. HD has been approaching with the animal model which mimics the human disease to provide some therapeutic clues in various ways. In the recently preclinical study, single injection of a cholesterol conjugated-siRNA was targeting mutant Huntingtin (mhtt), subsequently, the pathologic symptoms containing behavioral dysfunction were improved.

Future prospective

Fast progress in RNAi technology has shown promise for use in reverse genetics and therapy. While mechanistic complexities of the technology still need to be determined. Currently, RNAi has been established as a revolutionary tool for functional genomics in organisms. Multiple studies have defined the role of RNAi in plant and mammalian defense systems. A large number of studies have utilized RNAi technology to modulate gene expression. RNAi-based full genomic screens have allowed identification of specific genes, controlling a given trait with high accuracy. Further studies will continue to unravel the unlimited potential of RNAi to serve humankind.

References:

  1. Abdurakhmonov I Y. RNA Interference – A Hallmark of Cellular Function and Gene Manipulation // RNA Interference. 2016.
  2. Singh D, Chaudhary S, et al. RNA Interference Technology — Applications and Limitations // RNA Interference. 2016.
  3. Seliger B, Meinhardt A, Falke D. The Role of Immune Modulatory MicroRNAs in Tumors // RNA Interference. 2016.
  4. Jaimes Y, Seltsam A, et al. Regulation of HLA class II expression prevents allogeneic T-cell responses. Tissue Antigens, 2015, 77(1):36-44.
  5. Liu, Zhonghua, et al. Targeting beta-secretase with RNAi in neural stem cells for Alzheimer's; disease therapy. Neural Regeneration Research, 2013, 8(33):3095-3106.

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