While RNA molecules were historically considered to function as intermediates in the flow of genetic information between DNA and proteins, RNA is now known to play many additional roles. It can fold into diverse shapes with the potential to bind to targets ranging from small organic molecules to large proteins. Such high-affinity RNA molecules – called aptamers – have binding properties analogous to antibodies. Certain other RNA sequences, called ribozymes, have catalytic abilities, mimicking enzymes. Small noncoding RNAs such as small interfering RNAs (siRNAs) and microRNAs (miRNAs) can regulate gene expression. SomaGenics develops both therapeutic small RNAs and tools for analyzing small RNAs as biomarkers.
RNA interference (RNAi) is a naturally occurring, highly specific mode of gene regulation mediated by short double-stranded RNA (dsRNA) of approximately 20-22 base pairs. One strand of the dsRNA, called the guide strand, shuttles into a cytoplasmic multiprotein complex called the RNA-induced silencing complex (RISC). The guide strand bound to RISC searches the resident population of messenger RNAs (mRNAs) for complementary sequences. Base pairing between the guide and the target mRNA sequence leads to the down-regulation of the expression of the targeted mRNA by any of several pathways. When the complementarity between the guide and the target RNAs is high, the mechanism of down regulation involves the cleavage of the mRNA by an Argonaute protein within the RISC. These short dsRNAs that are capable of inducing RNAi-mediated down regulation of gene expression can be either produced by the cell or introduced externally into the cell using chemically synthesized or in vitro transcribed RNA sequences. These exogenous RNAi molecules can be either siRNAs or shRNAs.
The mechanism of RNA interference
In humans, microRNAs (miRNAs) generally represent the natural RNAi pathway involved in gene regulation. There are over 1000 miRNAs encoded by the human genome, which regulate almost all biological pathways. As a result, the miRNA expression profiles of pathological cells are different from the normal healthy cells, making them attractive as diagnostic markers. Genes encoding microRNAs are transcribed into long transcripts containing primary miRNAs (pri-miRNAs). Two RNAse III-type endonuclease enzymes, Drosha and Dicer, sequentially process these pri-miRNAs. Drosha processes pri-miRNAs to generate ~70-80-nt precursor-miRNAs (pre-miRNAs), which are then cleaved by Dicer to produce mature miRNAs. In most cases, only one strand within the mature miRNA duplex serves as the guide to enter into RISC for gene silencing. A single miRNA has the potential to silence hundreds of genes.
Small interfering RNAs (siRNAs) are chemically synthesized and introduced into cells to down regulate the expression of specific genes for the purpose of gene target validation and also towards the development of target-specific RNAi-based therapeutics. Most biopharmaceutical companies that develop RNAi-based drugs use siRNAs as their therapeutic platform.
Short hairpin RNAs (shRNAs) are generally produced by intracellular expression using shRNA-expression cassettes, which are introduced into cells using viral vectors, plasmids or DNA constructs. The expressed shRNAs that resemble pre-miRNAs are processed by Dicer to generate siRNAs from which the guide strands are loaded into RISC to initiate the RNAi process. The expressed-shRNA platform is predominantly being used in a gene therapy approach.
Beyond their usefulness in the context of expressed RNAi, shRNAs have great therapeutic potential as synthetic triggers of RNAi. SomaGenics has established a proprietary design of short synthetic shRNAs (sshRNAs) that are highly potent, effectively silencing target genes in cultured cells at low picomolar concentrations (IC50).