Targeting gene pathways using microRNA therapeutics
More than 750 microRNAs have been identified to date, regulating an estimated one-third of all human genes (3). Using sophisticated
bioinformatics analyses and enhanced detection methodologies, scientists demonstrated that a single microRNA may be capable
of regulating hundreds of messenger RNAs that function in the same or related pathways. Because microRNAs have functions in
multiple biological pathways, a change in expression or function of microRNAs might give rise to diseases, such as cancer,
fibrosis, metabolic disorders and inflammatory disorders. The demonstration that several microRNAs are up-regulated in a particular
disease phenotype provides the rationale to use anti-miR technology to restore the balance of normal gene regulation inside
the cell (see Table I) (4).
Introduction to microRNAs
MicroRNAs are small noncoding RNAs that are approximately 20–25 nucleotides in length. They regulate expression of multiple
target genes through sequence-specific hybridization to the 3' untranslated region (UTR) of messenger RNAs and block either
translation or direct degradation of their target messenger RNAs (5). MicroRNA genes are expressed in the cell nucleus as
a precursor called the primary microRNA which, upon further processing by an enzyme called Drosha, lead to pre-microRNA (see
Figure 2). Once exported into the cytoplasm, the pre-microRNA is cleaved by the Dicer enzyme into a 20–25 nucleotide-long
double-stranded RNA that is then loaded into RISC. This process is followed by the unwinding of the two RNA strands, the degradation
of the passenger strand, and the retention of the mature microRNA. Through the RISC, the microRNA guides and targets messenger
RNAs through direct base pairing. The 5' region of microRNA, also known as the "seed" region (nucleotides 1 through 8 or 2
through 9), is the most critical sequence for targeting and function (6). The microRNA target sites, located in the 3' UTR
of messenger RNAs, are often imperfectly matched to the microRNA sequence.
MicroRNAs do not require perfect complementarity for target recognition, so a single microRNA is able to regulate multiple
messenger RNAs. Although microRNAs exert subtle effects on each individual messenger RNA target, the combined effect is significant
and produces measurable phenotypic results. The ability of microRNAs to influence an entire network of genes involved in a
common cellular process provides tremendous therapeutic potential and differs from the specificity of today's drugs, which
act on specific cellular targets. MicroRNAs play integral roles in several biological processes, including immune modulation,
metabolic control, neuronal development, cell cycle, muscle differentiation, and stem-cell differentiation. Most microRNAs
are conserved across multiple animal species, indicating the evolutionary importance of these molecules as modulators of critical
biological pathways and processes (3).
The association of microRNA dysfunction with disease has created enormous potential for selective modulation of microRNAs
using anti-miR oligonucleotides, which are rationally designed and chemically modified to enhance target affinity, stability,
and tissue uptake. Aberrantly expressed or mutated microRNAs that cause significant changes in critical biological pathways
represent potential targets whose selective modulation could alter the course of disease. From a mechanistic view, the inhibition
of the microRNA target is based on the specific annealing of the anti-miR (see Figure 3). A stable, high-affinity bond between
the anti-miR and the microRNA will compete with binding to the 3' UTR target region.
Figure 3: Single-stranded oligonucleotide anti-miRs pharmacologically modulate dysregulated microRNAs. The anti-miR oligonucleotide
(black) binds and hybridizes to the abnormally expressed microRNA (red), blocking its function within the cell. DGCR8 is DiGeorge
critical region, and miR is microRNA.
Studies by Regulus Therapeutics and others have demonstrated that modulating microRNAs through anti-miR oligonucleotides can
effectively regulate biological processes and produce therapeutically beneficial results in murine models of cardiac dysfunction;
reducing cancer metastases in murine tumor models; and reducing viral load in the chimpanzee model of hepatitis C virus infection
(7,8,9). Most recently, advances in oligonucleotide chemistry have improved potency and stability by modification with novel
2',4'-constrained 2'O-ethyl (cEt) nucleotides (10). The ability to achieve increased inhibitory potency with this next generation
of bicyclic nucleic acid chemistry could make a significant positive impact on the design of anti-miR inhibitors for a vast
array of microRNA disease targets.