RNA-based therapeutics hold significant potential as promising

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1 Supplement to the May 2011 Issue of pharmtech.com 2011 The Industry s Authoritative Source MicroRNA Therapeutics Perspectives in MicroRNA Therapeutics Kevin Steffy, Charles Allerson, and Balkrishen Bhat Decades of research and development have produced a rich, deep pipeline of preclinical and clinical programs based on oligonucleotide therapeutics. In particular, anti-mir therapeutics represent an exciting opportunity in the field of microrna drug discovery. The authors provide further insight into microrna biology, and the simplicity of anti-mir oligonucleotide drug delivery, which can restore balance and function to dysregulated microrna pathways of gene expression. Kevin Steffy, PhD,* is the global alliance manager, Charles Allerson, PhD, is the associate director of chemistry, and Balkrishen Bhat, PhD, is the senior director of chemistry, all at Regulus Therapeutics, 3545 John Hopkins Ct., San Diego, CA 92121, tel , ksteffy@regulusrx.com. *To whom all correspondence should be addressed. RNA-based therapeutics hold significant potential as promising treatment options for human disease. In the past 20 years, advances in the RNA field have identified several novel RNAbased therapies that are currently under clinical investigation, including antisense oligonucleotides, small interfering RNA (sirna), and microrna. By targeting RNA and modulating human biology at the molecular level, these new technologies have allowed drug-discovery efforts to focus on a broad range of disease targets once deemed to be undruggable. Leading RNA biotechnology companies have since expanded the target space and generated multiple clinical candidates characterized by improved target specificity, improved drug safety, and demonstrated efficacy in patients. These companies have traditionally focused on targeting specific genes relevant to the disease indication through the control 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 (doublestranded 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 specificity 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 reaching the ribosome (1). Antisense drugs are short (15 23mer) chemically modified nucleotide chains that hybridize to a specific complementary area of mrna. On hybridization, the mrna is recognized as a RNA- DNA hybrid and degraded through an RNase H cleavage mechanism and not translated by the ribosome into a functional protein (see Figure 2). By inhibiting the production of proteins involved in disease, antisense drugs can create pharmacologic benefit for patients.

2 MicroRNA Therapeutics Figure 1: The RNA therapeutics opportunity. MicroRNAs represent a new set of drug targets capable of regulating an entire network of related genes. Figure 2: MicroRNAs are key regulators of the genome. Hybridization of micrornas (red) to their target seed sequence in mrnas regulates and 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-dependent 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 mrna. This results in site-specific mrna cleavage and protein degradation (see Table I) (2). The presence of the RNAi cellular components, combined with silencing, specificity, and efficacy makes it an attractive mechanism for targeting dysregulated gene expression in human disease. 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 micror- NAs 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 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 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). Anti-miR therapeutics 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 poten- ALL FIGURES COURTESY OF THE AUTHORS 2 Pharmaceutical Technology BIOPROCESSING & STERILE MFG PharmTech.com

3 Table I: Overview of the current RNA-based drug-discovery platform. Technology Compound Target Delivery Mechanism Regulus microrna platform sirna ASO Single stranded anti-mirs (15-19nt) Double-stranded mir-mimics (21-23bp) Double-stranded RNAs (22bp) Single-stranded oligos (15-20nt, gapmer) micrornas No DDS for anti-mirs DDS required for mimics (single strand mimic in process) microrna targeting leads to pathway modulation messenger RNA DDS required Cleavage of a single mrna by RISC/AGO2 messenger RNA No DDS Cleavage of a single mrna in nucleus by RNase H tial 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. 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 (). 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. Anti-miR oligonucleotide drug delivery Up until nearly a decade ago, insufficient in vivo stability, limited methods of delivery and tissue distribution of oligonucleotides hampered successful clinical development for several promising oligonucleotide therapeutic agents. As high molecular weight, highly charged polyanionic molecules, oligonucleotides faced many hurdles in reaching their target organ or target cell type. First-generation antisense phosphorothiolated oligodeoxynucleotide clinical candidates administered into the bloodstream had a low affinity for their target, poor stability because of nuclease degradation, unfavorable immunostimulatory properties, and rapid excretion by renal clearance, resulting in shortened half-lives (11). To increase their metabolic stability and tissue half-life, antisense and anti-mir oligonucleotides from second-generation nucleoside chemistries were developed that dramatically altered the pharmacokinetic properties of these molecules (,12). The introduction of chemical modifications such as 2 methoxyethyl (MOE) and 2, 4 -constrained 2 O-ethyl (cet) into the ribose sugar ring significantly improved both the pharmacokinetic and safety profile of antisense oligonucleotides. Once delivered systemically, these second-generation compounds rapidly partition from the plasma and are taken up by cells of multiple tissues without the need of formulation or a delivery vehicle. Benefitting from nearly 20 years of oligonucleotide chemistry advances at companies such as Isis Pharmaceuticals, leading developers of anti-mir therapies have garnered a tremendous advantage in improved delivery strategies. The high water solubility of anti-mir oligonucleotides due to their polyanionic chemical structure has allowed anti-mir formulation in simple aqueous solutions such as buffered saline (13). The only limiting factor is the viscosity of the solution, which is generally concentration-dependent for single-stranded oligonucleotides (13). This simple anti-mir formulation is in contrast to the requirements for double-stranded sirna drug delivery, which must fully encapsulate the sirna in a lipid nanoparticle to systemically deliver its contents to a target tissue (14). Anti-miR route of administration and tissue distribution Bioavailability and tissue distribution of anti-mirs have been studied extensively in rodents and nonhuman primates. The preferred route of administration for most therapeutic anti-mir compounds is subcutaneous systemic delivery, because it provides efficient dissemination of the drug to different tissues including the liver, kidney and adipose tissue without the need of a drug delivery system. Additionally, the biodistribution of anti-mirs in multiple animal species following subcutaneous administration provides valuable information 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. Pharmaceutical Technology BIOPROCESSING & STERILE MFG

4 MicroRNA Therapeutics regarding organs that may be successfully treated, as well as those organs unlikely to be affected. Multiple studies were performed in mice and monkeys with second generation anti-mir 1 and anti-mir 2 compounds given subcutaneously once weekly over several weeks. A quantitative analysis of tissues demonstrated broad biodistribution of modified anti-mirs among multiple tissue types including the kidney, liver, lymph nodes, adipose tissue, and spleen, as demonstrated by mass spectrometry analysis (see Figure 4). These organs have been previously shown to be target sites for oligonucleotide distribution after parenteral administration (13). Additionally, the similar pharmacokinetics and correlated tissue distribution of each anti-mir in different preclinical animal models provide important guidance for selection of different disease indications and may assist in better clinical trial designs with anti-mir therapies (see Figure 5). Effective delivery of anti-mir oligonucleotides has also been demonstrated in different species through multiple routes of administration including: intravenous, intraperitoneal, intratracheal, intranasal, and intracerebral. A more detailed analysis of anti-mir tissue distribution using quantitative whole body autoradiography to provide additional quantitative information is in progress. Anti-miR delivery and function mrna expression profiling methods coupled with statistical techniques that can measure small changes in the expression of many genes have become powerful tools to further our understanding of the biological role and function of micrornas. Relying on the scientific findings that some micrornas are capable of regulating hundreds of messenger RNAs, studies were performed in mice to determine anti-mir delivery to different cell types. Mice were treated with a specific anti-mir (intraperitoneal injection) and multiple cell types were harvested to for mrna expression studies using Sylamer enrichment analysis (15). Anti-miR oligonucleotides are distributed to peritoneal macrophages as evidenced by flow cytometry analysis and target gene up-regulation (see Figure 6). An analysis identifying an overrepresented set of genes associated with a specific anti-mir biological effect was conducted and a data plot from the isolated macrophages was generated that demonstrated the most up-regulated sets of genes after anti-mir treatment. P values generated for this dataset suggest statistically significant preferential up-regulation of genes matched to their target sequence after anti-mir treatment. Conclusion Targeting pathways of human disease with microrna-based drugs represents a novel and potentially powerful therapeutic approach. Recent data demonstrate not only that dysregulated micrornas are associated with and can cause human disease, but that selective modulation through anti-mir intervention can provide therapeutic benefits. Anti-miR oligonucleotides can be easily administered through local or parenteral injection routes with sufficient uptake of the agent to achieve sustained target inhibition in tissues and organs without the need of formulation. Improvements in antimir chemical design and pharmacokinetic properties will allow further exploration of microrna biology and broaden the utility of microrna therapeutics. Figure 4: Similar pattern of tissue distribution for chemically modified anti-mirs in mouse and monkey. Anti-miR oligonucleotide quantitation was performed on tissues by either mass spectrometry analysis or capillary gel electrophoresis. IP is Intraperitoneal, SC is subcutaneous. ug Anti-miR/g tissue (μm) Mouse kidney Intestine Muscle Lymph 34 mg/kg/week delivered IP for 3 weeks References 1. S.T. Crooke et al., Mechanisms of Antisense Drug Action, an Introduction, in Antisense Drug Technology: Principles, Strategies, and Applications, S.T. Crooke, Ed. (CRC Press, Boca Raton, 2007), pp S.M. Elbashir et al., Nature 411 (6386), (2001). 3. A. Jackson and P.S. Linsley, Discovery Medicine 9 (47), (20). 4. J. Krutzfeldt et al., Nature 438 (7068), (2005). 5. D.P. Bartel, Cell 116 (2), (2004). 6. E.C. Lai, Nat. Rev. Genet 30 (4), (2002). 7. T. Thum et al., Nature 456 (7224), (2008). 8. L. Ma et al., Nature 28 (4), (20) Monkey kidney Lymph nodes Bone marrow 25 mg/kg/week delivered SC for 6 weeks Figure 5: The distributions of anti-mirs in mice and monkey are highly correlated. Quantitative analysis of drug concentration by mass spectrometry revealed a good correlation of drug tissue distribution across multiple species. Monkey drug levels (μg/g) Monkey Vs. Mouse Lymph Mouse drug levels (μg/g) Anti-mR 1 Anti-mR 2 Kidney Pharmaceutical Technology BIOPROCESSING & STERILE MFG PharmTech.com

5 Figure 6: Functional drug delivery of anti-mirs in mouse peritoneal macrophages. Flow-cytometry studies (a) and gene-regulation studies (b) demonstrate the internalization of anti-mir and target engagement in macrophages (Sylamer analysis). Potential targets containing heptamer 1-7 (GCATTAA) or heptamer 2-8 (AGCATTA) are enriched. X-axis is ranked genes by fold change. Y axis is -log (P-value enrichment). PBS is Phosphatebuffered saline. (a) PBS Anti-miR-Cy (b) 3 - UGGGGAUAGUGUUAAUCGUAAUU-5 CD11b MFI Cy log Enrichment P-value GCATTAA AGCATTA Target cell Sorted sequences (most up- to most down-regulated) Messenger RNA 9. R.E. Lanford et al., Science 327 (5962), (20).. P.P. Seth et al., J. Med. Chem. 52 (1), 13 (2009). 11. A.A. Levin, R. Zu, and R.S. Geary, Basic Principles of the Pharmacokinetics of Antisense Oligonucleotide Drugs, in Antisense Drug Technology: Principles, Strategies, and Applications, S.T. Crooke, Ed. (CRC Press, Boca Raton, 2007), pp B. Monia et al. J. Biol. Chem. 268 (19), (1993). 13. R.S. Geary, R. Zu, and A.A. Levin, Pharmacokinetic/Pharmacodynamic Properties of Phosphorothioate 2 -O-(2-Methoxyethyl)-Modified Antisense Oligonucleotides in Animals and Man, in Antisense Drug Technology: Principles, Strategies, and Applications, S.T. Crooke, Ed. (CRC Press, Boca Raton, 2007), pp S.C. Semple et al., Nature Biotech. 28 (2), (20). 15. S.V. Dongen et al., Nature Methods 5 (12) (2008). PT Posted with permission from the May 2011 Supplement to Pharmaceutical Technology. Copyright 2011, an Advanstar publication. All rights reserved. # Managed by The YGS Group, For more information visit