Therapeutic applications of siRNA and microRNA
RNAi (RNA interference) still remains a recently discovered technology, leading the fields of diagnostics and therapeutics in genetic research and immunology. Being one of the greatest scientific achievements within the last decade, RNAi appears to originate from an ancient and widespread immune mechanism originating from genetic processes. Studies have shown RNAi to play a major role in a cell’s defense against several types of viruses and that it is involved in a number of regulatory mechanisms within the cell.
The significance of RNAi is proven by a recent Nobel Prize in physiology being awarded to the research duo of Craig Mello and Andrew Fire for their initial involvement in this field. Initially, RNAi was viewed as a tool utilized to study particular genes and their related functions by transfecting siRNA into cancer cell lines. Over a decade’s worth of research in this field has armed scientists with algorithms to predict mRNA targets and methods to quantify miRNA expression levels. Such methods include the use of qRT-PCR, arrays, protein expression level detection, and RNA-Seq. The use of siRNA and miRNA synthetic mimics in in vitro transfections or in vivo studies in combination with these methods has resulted in a larger understanding of the role of the RNAi machinery in endogenous gene regulation.
Many scientists recognized the importance of RNAi at its inception, especially its involvement in cancer, but there was intense skepticism in its potential as a therapeutic. With recent advances, interest in RNAi as a therapeutic active pharmaceutical ingredient (API) is rapidly growing and it is reasonable to expect significant results in the near future.
Some cells utilize the RNAi pathway for gene expression regulation. This process can be manipulated, and it is plausible that manipulating the RNAi cellular machinery to target viruses or control gene pathways in animals is a feasible application. In contrast to small molecule or antibody therapeutic approaches, the driving factor of RNAi therapeutics is the knowledge that miRNAs target multiple genes. A multi-target therapeutic precludes cells from becoming resistant to treatment by targeting multiple genes in a pathway, unlike single target therapies in which a cell can bypass a single gene in a pathway (e.g. EGFR resistance, etc).
Initial studies utilizing RNAi proved promising results, especially in the realm of viral infection. In 2002, scientists at MIT stated they had used the RNAi mechanism to inhibit the life cycle of the HIV virus at various stages. However, rapid mutations in the HIV virus makes it resistant to any therapy, including RNAi. Broader applications of in vivo RNAi therapy involving human patients is currently under clinical evaluation.
As with any therapeutic, building on the successful results of in vitro studies with in vivo efficacy is a giant hurdle. The major factor with oligonucleotide therapeutics is the delivery to target cells, as the delivery of hydrophilic synthetic RNA across a hydrophobic cell membrane remains troublesome. In order to address delivery issues, the oligo therapeutic can be encapsulated in a lipid nanoparticle, conjugated to an antibody or modified to overcome degradation. Along with delivery issues, another area to address in oligo therapeutic development is half-life. Since efficacy is most likely transient, gene silencing through the RNAi pathway needs to be sustained for as long as possible. By optimizing delivery conditions and adding modifications to limit degradation, the results will be an increase in bioavailability (i.e. amount of circulating drug) which increases the overall effects of the therapeutic.