RNAi technology - promises and pitfalls

RNA interference (RNAi) technology is making revolutionary changes in therapeutics, diagnosis and health technology. Messenger RNA (mRNA) being the key component of communication between the ribosomes and DNA, are responsible for programmed protein synthesis. The RNAi interferes with protein synthesis and thus prevents prognosis of the diseases. RNAi have been found useful in many therapeutic segments like diabetes, hypertension and cancer, thus defective protein synthesis is implicated in disease process. RNAi, is a biological process, in which RNA molecule inhibit gene expression, typically by inhibiting or destruction of specific mRNA. In the 1990’s, it was discovered that a gene responsible for petal pigmentation in petunia, was tried to be over expressed, instead it produces complete blockage in petal pigmentation. Thus, this discovery not only reveals a naturally occurring process, but also opened a vast opportunities for genome technology.

RNAi is an RNA dependent gene silencing process that is controlled by the RNA-induced silencing complex (RISC) and is initiated by short double stranded RNA (dsRNA) molecules in a cell’s cytoplasm. Endogenous dsRNA initiates RNAi by activating the ribonuclease protein dicer, which binds and cleaves double stranded RNAs (dsRNA) to produce double stranded fragments of 20 to 25 base pairs with a 2 nucleotide overhang at the 3 end, these short double stranded fragments are called small interfering RNAs (siRNAs). These siRNAs are then separated into single strands and integrated into an active RISC complex. After integration into the RISC, siRNA base pair to their target m-RNA and cleave it, thereby preventing it from being used as a translation template.

RNAi screens have been conducted in the past to identify synthetic lethality that underscore the tumour dependencies required for sustained proliferation of cancer cells, either within the same pathway of the oncogenic pathway or in parallel pathways. This provided an opportunity to target novel avenues for therapeutic intervention and to reverse resistance selectively in cells harbouring oncogenic mutations.

A promising application of RNAi screening in cancer therapeutics would be identified in novel targets that sensitize the cells to the effect of the administered drugs. Several RNAi screens have been performed in the past to search or genes who are down regulation would act synergistically with drugs like bortezomib in myelomas, taxol in breast cancer, erlotinib in lung cancer and so on.

Another example would be that of cancers with altered notch signalling pathway where “gamma-sectretase” has been viewed as a putative target for therapeutic intervention, however all small molecules failing to target it in clinical trial research. Thus, clearly RNAi could play a role in this alcove to identify sensitizers for cancers that are problematic to manage.

RNAi and oncology
Currently not used so much RNAi technology is still in its premature stage to be used as treatment for cancer. The information yet, suggest that it shows a strong potential in terms of treating cancer by silencing genes data differentially up regulated in tumour cells or genes involved in cell division. Development of therapy for cancer is a complex task and the complexity gets worse due the fact that the oncogenic pathway mutations initiating tumourigenesis might not even play a role in its maintenance, mutations including chemotherapy resistant mutations accumulate and sustain therapy and hence there is uninhibited proliferation of cancer cells.

Although time of its inception was believed to be the key of genomics, it is a trouble today. But, all this tells us development in genomics is used that guide to substantial therapy development. The research on RNAi not so speedy these days by the big pharmaceuticals, still is under great consideration as being a non-viral and safe delivery method. RNAi has already shown the great potential held by genomics, its great use in combination therapy with anti-cancer drugs was a great future if explored in detail since changing or interfering with synthesis of proteins can change a lot, in terms of diseased condition, or understanding and developing a therapy.

Developments, however slow are still going on and approximately 600 RNAi screens have already been published so far.  

RNAi have diversified applications not only in therapeutics also in vital areas of signal transduction, post pathogenic interaction and cancer research. The availability of RNAi screening has increased the expectations of the researches of the biological sciences. The integration of RNAi with high throughput technologies has several unprecedented opportunities of research and diagnosis in the study of pathogenesis of many critical diseases.

Application of RNAi in treating human diseases

Disease	Mechanism involved
Lymphoblastic leukaemia	Using siRNAs specific for the BCR–ABL transcript to silence the oncogenes
Pancreatic and colon carcinomas	The use of retroviral vectors to introduce interfering RNAs specific for an oncogenic variant of K-RAS
Colonic adenocarcinoma	Downregulation by miRNAs miR-143 and miR-145
Bladder cancer	Treatment by miRNAs as biomarkers
HIV	Down regulation of the cellular cofactors required for HIV infection by RNAi
Viral hepatitis	Inhibition of Fas expression by siRNA
Cardiovascular and cerebral vascular diseases	Using RNAi to intervene in the process of atherosclerosis or to reduce the damage to heart tissue and brain cells
Ocular diseases	Shutting down production of VEGF by siRNA
Malaria	RNAi can identify the genetic factors shape the vector parasite relationship may be crucial to identifying new genetic means of controlling mosquito-borne diseases
Metabolic disease and neurodegenerative disorders	Treatment of these diseases with miRNAs as potential therapeutic targets

Latest applications of RNAi cell technology
RNAi and stem cells: Recent studies have begun to investigate the role of miRNA during normal stem cell development in mammalian and non-mammalian systems. It was identi?ed miRNAs in undi?erentiated and di?erentiated mouse embryonic stem (ES) cells. Some of the miRNAs seemed to be ES-speci?c, and these ES-speci?c miRNAs were repressed as embryonic stem cells di?erentiated into embryoid bodies and were undetectable in adult mouse organs and tissues. Finally, miRNAs isolated from adult hippocampal neural stem cells played a critical role in mediating neuronal di?erentiation. miRNAs are also involved in the regulation of stem cell development in non-mammalian organisms. It is recently identi?ed that maintenance of primordial germ cell (PGC) self-renewal and inhibition of PGC di?erentiation towards somatic cell fate in Drosophila requires the non-coding PGC miRNA. RNA-polymerase-typeII-dependent transcription is normally repressed in PGCs of many animals during early development and this phenomenon might be important for the maintenance of germ line fate by preventing somatic cell di?erentiation. Germ cells lacking PGC miRNA, expressed a number of genes important for di?erentiation of nearby somatic cells.

RNAi applications to research
Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of the 3.5-day-old mouse blastocyst. These cells are an attractive model to study the molecular regulation of cell lineage commitment and cellular di?erentiation because embryonic stem cells can give rise to cells derived from all three primary germ layers: endoderm, mesoderm and ectoderm. Therefore, the ability to selectively knockdown speci?c target genes would aid in the understanding of multiple aspects of early development. Yang demonstrated the ability to diminish expression of a target gene in undi?erentiated embryonic stem cells by in situ production of long dsRNA from a transient transfection of a plasmid harbouring a 547 bp inverted repeat or by direct transfection of a 740-bp dsRNA made by in vitro transcription. However, these long dsRNAs could only mediate RNAi in undi?erentiated ES cells but not di?erentiated embryonic stem cells. As an alternative approach, transfected well-di?erentiated embryonic stem cells with siRNA, and found that these oligo nucleotides were e?ective in diminishing the expression of such genes as PU1 and c-EBPa.

Oct4 is a transcription factor which has been characterized as a key regulator of embryonic stem cell pluripotency. Genetic studies have indicated that the expression level of Oct4 is important in early lineage commitment of ES cells. Oct4-de?cient embryos fail to form an inner cell mass (ICM) but remaining cells commit to the trophoblast lineage. It was examined whether suppression of Oct4 expression via RNAi would alter ES cell lineage commitment decision. In their study, embryonic stem cells were transfected with plasmids containing an independently expressed reporter gene and an RNA polymerase type III promoter to constitutively express small stem-loop RNA transcripts corresponding to Oct4 mRNA. Cells transfected with Oct4 shRNA demonstrated reduced levels of Oct4 mRNA and exhibited characteristics of trophectodermal di?erentiation. More recently, Oct4 siRNAs delivered by transfection were e?ective in both human and mouse ES cells in diminishing Oct4 expression.

Scientific developments in RNAi tech with application of shRNA
In vivo delivery of RNAi also offers great promise for the future. Since current in vivo gene function studies involve the time-consuming development of transgenic mouse gene knockouts and double knockouts, a successful in vivo RNAi protocol would represent a tremendous step forward in terms of time allocation and likely lead to an explosion of knowledge obtained from such studies. Most current approaches to in vivo RNAi involve the systemic delivery of “naked” siRNAs. These so-called naked siRNAs are only moderately effective in the in vivo knockdown of a gene of interest and mostly are limited to genes expressed within the liver and kidney. In addition, since naked siRNAs exhibit poor pharmacokinetics, they are delivered at high concentrations, adding to their expense and putative off-target effects. However, there exists at least one in vivo siRNA delivery transfection reagent (Invivofectamine, Invitrogen, Inc.), which has shown promise in the delivery of much lower concentrations of siRNA to a mouse; however, this reagent remains cost prohibitive to most laboratories and these data remain to be reproduced readily outside of its commercial source. In addition, viral vectors encoding shRNAs have shown promise for in vivo delivery; however, most of these studies have utilized adenoviral delivery of shRNA, which has well-known toxic effects in the animal. Recently, adeno-associated viral vectors (AAV) have been designed with less toxicity and adequate shRNA delivery. Finally, there are numerous ongoing studies focused on virally mediated delivery of shRNA to hematopoietic stem cells (HSC) isolated from a mouse and re-implanted into an irradiated recipient mouse. These HSCs have been shown to give rise to cells with stable shRNA; however, the recipient mouse still retains gene expression within stromal cells. Nonetheless, this may prove an effective strategy for in vivo studies of gene expression in cells of an immune origin. Notwithstanding these advances in in vivo RNAi, there are still numerous challenges to methodology and application; however, with every new publication comes the exciting possibility of another breakthrough in RNAi technology which will likely advance this field far beyond what is conceivable today.

Drawbacks of RNAi technology
Along with several applications and advantages, RNAi technology has some prominent disadvantages. Researchers have demonstrated that RNAi sequences do not only bind to one target. These changes in the gene expression pattern of the cell and potentially of the phenotype give rise to an off-target signature. The unidentified effects of such a signature bear a high risk to create false-positive outcomes that can bring a complete project into jeopardy. Furthermore, even most up to date algorithm-based sequence designs show a knockdown efficiency that is generally at 80 per cent or less. The effects of such specific but low knockdown can be masked by the off-target signature with phenotypic changes being undetectable. These issues can make RNAi unpredictable, slow, and risky, in particular in drug discovery, where speed and reliability of results are crucial factors

Key issues are RNA interference is variable in nature. Incompleteness of slicing and knockdowns may cause genetic errors, potentially non-specific to reagents, prevalent and latest techniques are needed not a cost effective method and highly trained professionals with deep knowledge of genetics are required.