Supplement to the May 2011 Issue of 2011 pharmtech.com The Industry’s Authoritative Source MicroRNA Therapeutics Perspectives in MicroRNA Therapeutics Kevin Steffy, Charles Allerson, and Balkrishen Bhat Decades of research and development have NA-based therapeutics hold significant potential as promis- produced a rich, deep pipeline of preclinical ing treatment options for human disease. In the past 20 years, and clinical programs based on oligonucleotide advances in the RNA field have identified several novel RNA- based therapies that are currently under clinical investigation, therapeutics. In particular, anti-miR therapeutics Rincluding antisense oligonucleotides, small interfering RNA (siRNA), represent an exciting opportunity in the field of and microRNA. By targeting RNA and modulating human biology at microRNA drug discovery. The authors provide the molecular level, these new technologies have allowed drug-discovery further insight into microRNA biology, and the efforts to focus on a broad range of disease targets once deemed to be simplicity of anti-miR oligonucleotide drug “undruggable.” delivery, which can restore balance and function Leading RNA biotechnology companies have since expanded the target space and generated multiple clinical candidates characterized to dysregulated microRNA pathways of gene by improved target specificity, improved drug safety, and demonstrated expression. efficacy in patients. These companies have traditionally focused on tar- geting specific genes relevant to the disease indication through the con- trol of protein synthesis at the RNA level. More recently, drug discovery researchers are attempting to regulate entire networks of genes through the modulation of a single microRNA. Targeting microRNAs with either oligonucleotide inhibitors, namely anti-miRs, or miR-mimics (double- stranded oligonucleotides that replace microRNA function), provides a novel class of therapeutics and a unique approach to treating disease by modulating entire biological pathways (see Figure 1). Targeting specific genes using antisense oligonucleotides and siRNA Antisense oligonucleotides and siRNA have great potential to become mainstream therapeutic entities. This is due, in part, to their high spec- ificity and wide therapeutic target space in the genome. The antisense approach targets a specific gene and interrupts the translation phase of the protein production process by preventing the mRNA from reach- Kevin Steffy, PhD,* is the global alliance manager, ing the ribosome (1). Antisense drugs are short (15–23mer) chemically Charles Allerson, PhD, is the associate director modified nucleotide chains that hybridize to a specific complementary of chemistry, and Balkrishen Bhat, PhD, is the area of mRNA. On hybridization, the mRNA is recognized as a RNA- senior director of chemistry, all at Regulus Therapeutics, 3545 John Hopkins Ct., San Diego, CA 92121, tel. DNA hybrid and degraded through an RNase H cleavage mechanism 858.202.6321, [email protected]. and not translated by the ribosome into a functional protein (see Figure 2). By inhibiting the production of proteins involved in disease, anti- *To whom all correspondence should be addressed. sense drugs can create pharmacologic benefit for patients. MicroRNA Therapeutics Figure 1: The RNA therapeutics opportunity. MicroRNAs represent Figure 2: MicroRNAs are key regulators of the genome. Hybridization of a new set of drug targets capable of regulating an entire network of microRNAs (red) to their target seed sequence in mRNAs regulates and related genes. directs the expression of an entire network of genes. AGO is Argonaute protein, DGCR8 is DiGeorge critical region 8, miR is microRNA, RISC is RNA induced silencing complex. RNA interference (RNAi) is a highly conserved sequence-de- pendent eukaryotic process for regulating gene expression. Small stretches of double-stranded RNA ranging from 19 to 25 base pairs, and known as siRNA, utilize the RNA induced silencing complex (RISC) pathway to target a specific gene and bind to its homologous by the Dicer enzyme into a 20–25 nucleotide-long double-stranded mRNA. This results in site-specific mRNA cleavage and protein RNA that is then loaded into RISC. This process is followed by the degradation (see Table I) (2). The presence of the RNAi cellular unwinding of the two RNA strands, the degradation of the passenger components, combined with silencing, specificity, and efficacy strand, and the retention of the mature microRNA. Through the makes it an attractive mechanism for targeting dysregulated gene RISC, the microRNA guides and targets messenger RNAs through expression in human disease. 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 Targeting gene pathways using critical sequence for targeting and function (6). The microRNA microRNA therapeutics target sites, located in the 3’ UTR of messenger RNAs, are often More than 750 microRNAs have been identified to date, regulating imperfectly matched to the microRNA sequence. an estimated one-third of all human genes (3). Using sophisticated MicroRNAs do not require perfect complementarity for target rec- bioinformatics analyses and enhanced detection methodologies, ognition, so a single microRNA is able to regulate multiple messenger scientists demonstrated that a single microRNA may be capable of RNAs. Although microRNAs exert subtle effects on each individual regulating hundreds of messenger RNAs that function in the same messenger RNA target, the combined effect is significant and pro- or related pathways. Because microRNAs have functions in multiple duces measurable phenotypic results. The ability of microRNAs to biological pathways, a change in expression or function of microR- influence an entire network of genes involved in a common cellular NAs might give rise to diseases, such as cancer, fibrosis, metabolic process provides tremendous therapeutic potential and differs from disorders and inflammatory disorders. The demonstration that sev- the specificity of today’s drugs, which act on specific cellular targets. eral microRNAs are up-regulated in a particular disease phenotype MicroRNAs play integral roles in several biological processes, includ- provides the rationale to use anti-miR technology to restore the bal- ing immune modulation, metabolic control, neuronal development, ance of normal gene regulation inside the cell (see Table I) (4). cell cycle, muscle differentiation, and stem-cell differentiation. Most microRNAs are conserved across multiple animal species, indicating Introduction to microRNAs the evolutionary importance of these molecules as modulators of criti- MicroRNAs are small noncoding RNAs that are approximately cal biological pathways and processes (3). 20–25 nucleotides in length. They regulate expression of multiple target genes through sequence-specific hybridization to the 3’ Anti-miR therapeutics untranslated region (UTR) of messenger RNAs and block either The association of microRNA dysfunction with disease has cre- translation or direct degradation of their target messenger RNAs ated enormous potential for selective modulation of microRNAs (5). MicroRNA genes are expressed in the cell nucleus as a precur- using anti-miR oligonucleotides, which are rationally designed and sor called the primary microRNA which, upon further processing chemically modified to enhance target affinity, stability, and tissue by an enzyme called Drosha, lead to pre-microRNA (see Figure 2). uptake. Aberrantly expressed or mutated microRNAs that cause Once exported into the cytoplasm, the pre-microRNA is cleaved significant changes in critical biological pathways represent poten- AUTHORS THE OF COURTESY FIGURES ALL 2 Pharmaceutical Technology BIOPROCESSING & STERILE MFG. 2011 PharmTech.com Table I: Overview of the current RNA-based drug-discovery platform. Technology Compound Target Delivery Mechanism Regulus microRNA • Single stranded microRNAs • No DDS for anti-miRs microRNA targeting leads to platform anti-miRs (15-19nt) • DDS required for mimics pathway modulation • Double-stranded (single strand mimic in miR-mimics (21-23bp) process) siRNA Double-stranded RNAs messenger RNA DDS required Cleavage of a single mRNA by (22bp) RISC/AGO2 ASO Single-stranded oligos messenger RNA No DDS Cleavage of a single mRNA in (15-20nt, gapmer) nucleus by RNase H tial targets whose selective modulation could alter the course of dis- advantage in improved delivery strategies. The high water solubility of ease. From a mechanistic view, the inhibition of the microRNA tar- anti-miR oligonucleotides due to their polyanionic chemical structure get is based on the specific annealing of the anti-miR (see Figure 3). has allowed anti-miR formulation in simple aqueous solutions such as A stable, high-affinity bond between the anti-miR and the mi- buffered saline (13). The only limiting factor is the viscosity of the solu- croRNA will compete with binding to the 3’ UTR target region. tion, which is generally concentration-dependent for single-stranded Studies by Regulus Therapeutics and others have demonstrated oligonucleotides (13). This simple anti-miR formulation is in contrast that modulating microRNAs through anti-miR oligonucleotides can to the requirements for double-stranded siRNA drug delivery, which effectively regulate biological processes and produce therapeutically must fully encapsulate the siRNA in a lipid nanoparticle to systemi- beneficial results in murine models of cardiac dysfunction;