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Perspectives in Microrna Therapeutics

Perspectives in Microrna Therapeutics

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 includingR 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 by improved target specificity, improved drug safety, and demonstrated expression. efficacy in patients. These companies have traditionally focused on tar- geting specific 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 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 . The antisense approach targets a specific gene and interrupts the 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 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 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 . 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 through expression in human disease. direct base pairing. The 5’ region of microRNA, also known as the “seed” region ( 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- 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 , 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 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; reducing cally deliver its contents to a target tissue (14). cancer metastases in murine tumor models; and reducing viral load in the chimpanzee model of hepatitis C virus infection (7,8,9). Most Anti-miR route of administration and tissue distribution recently, advances in oligonucleotide chemistry have improved potency Bioavailability and tissue distribution of anti-miRs have been stud- and stability by modification with novel 2’,4’-constrained 2’O-ethyl (cEt) ied extensively in rodents and nonhuman primates. The preferred nucleotides (10). The ability to achieve increased inhibitory potency with route of administration for most therapeutic anti-miR compounds this next generation of bicyclic chemistry could make a is subcutaneous systemic delivery, because it provides efficient dis- significant positive impact on the design of anti-miR inhibitors for a semination of the drug to different tissues including the liver, kidney vast array of microRNA disease targets. and adipose tissue without the need of a drug delivery system. Addi- tionally, the biodistribution of anti-miRs in multiple animal species Anti-miR oligonucleotide drug delivery following subcutaneous administration provides valuable information Up until nearly a decade ago, insufficient in vivo stability, limited meth- ods of delivery and tissue distribution of oligonucleotides hampered Figure 3: Single-stranded oligonucleotide anti-miRs pharmacologically successful clinical development for several promising oligonucleotide modulate dysregulated microRNAs. The anti-miR oligonucleotide (black) therapeutic agents. As high molecular weight, highly charged polyan- binds and hybridizes to the abnormally expressed microRNA (red), blocking ionic molecules, oligonucleotides faced many hurdles in reaching their its function within the cell. DGCR8 is DiGeorge critical region, and miR is target organ or target cell type. First-generation antisense phosphoro- microRNA. thiolated oligodeoxynucleotide clinical candidates administered into the bloodstream had a low affinity for their target, poor stability because of 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 chem- istries were developed that dramatically altered the pharmacokinetic properties of these molecules (10,12). 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 sec- ond-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 oligonucle- otide chemistry advances at companies such as Isis Pharmaceuticals, leading developers of anti-miR therapies have garnered a tremendous

Pharmaceutical Technology BIOPROCESSING & STERILE MFG. 2011 3 MicroRNA Therapeutics

regarding organs that may be successfully treated, as well as those Figure 4: Similar pattern of tissue distribution for chemically modified organs unlikely to be affected. Multiple studies were performed in anti-miRs in mouse and monkey. Anti-miR oligonucleotide quantitation mice and monkeys with second generation anti-miR 1 and anti-miR 2 was performed on tissues by either mass spectrometry analysis or compounds given subcutaneously once weekly over several weeks. A capillary . IP is Intraperitoneal, SC is subcutaneous. quantitative analysis of tissues demonstrated broad biodistribution of Mouse Monkey modified anti-miRs among multiple tissue types including the kidney, 4000 400 liver, lymph nodes, adipose tissue, and spleen, as demonstrated by 300 3000 m) mass spectrometry analysis (see Figure 4). These organs have been pre- 200 2000 viously shown to be target sites for oligonucleotide distribution after 100 parenteral administration (13). Additionally, the similar pharmacoki- 1000 netics and correlated tissue distribution of each anti-miR in different 15 preclinical animal models provide important guidance for selection 10 ug Anti-miR/g tissue ( μ of different disease indications and may assist in better 5 0 designs with anti-miR therapies (see Figure 5). Effective delivery of Liver Lung Heart Liver Lymph Lung kidney Spleen Muscle Heart kidney Spleen anti-miR oligonucleotides has also been demonstrated in different Intestine 34 mg/kg/week delivered IP Lymph nodes Lymph species through multiple routes of administration including: intrave- for 3 weeks Bone marrow 25 mg/kg/week delivered SC nous, intraperitoneal, intratracheal, intranasal, and intracerebral. A for 6 weeks more detailed analysis of anti-miR tissue distribution using quantita- tive whole body autoradiography to provide additional quantitative information is in progress. Figure 5: The distributions of anti-miRs in mice and monkey are Anti-miR delivery and function highly correlated. Quantitative analysis of drug concentration by mass spectrometry revealed a good correlation of drug tissue distribution mRNA expression profiling methods coupled with statistical tech- across multiple species. niques that can measure small changes in the expression of many genes have become powerful tools to further our understanding Monkey Vs. Mouse 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 10000 to determine anti-miR delivery to different cell types. Mice were Kidney treated with a specific anti-miR (intraperitoneal injection) and multiple cell types were harvested to for mRNA expression stud- 1000 ies using Sylamer enrichment analysis (15). Anti-miR oligonucle- Liver otides are distributed to peritoneal macrophages as evidenced by g/g) Lymph Heart Spleen

flow cytometry analysis and target gene up-regulation (see Figure ( μ 6). An analysis identifying an overrepresented set of genes asso- 100 ciated with a specific anti-miR biological effect was conducted Lung and a data plot from the isolated macrophages was generated that Monkey drug levels demonstrated the most up-regulated sets of genes after anti-miR 10 treatment. P values generated for this dataset suggest statistically 110 100 1000 significant preferential up-regulation of genes matched to their Mouse drug levels target sequence after anti-miR treatment. (μg/g) Conclusion Anti-mR 1 Targeting pathways of human disease with microRNA-based drugs Anti-mR 2 represents a novel and potentially powerful therapeutic approach. Recent data demonstrate not only that dysregulated microRNAs References are associated with and can cause human disease, but that selective 1. S.T. Crooke et al., “Mechanisms of Antisense Drug Action, an Introduc- modulation through anti-miR intervention can provide therapeu- tion,” in Antisense Drug Technology: Principles, Strategies, and Applica- tic benefits. Anti-miR oligonucleotides can be easily administered tions, S.T. Crooke, Ed. (CRC Press, Boca Raton, 2007), pp. 3–47. through local or parenteral injection routes with sufficient uptake 2. S.M. Elbashir et al., 411 (6386), 494–498 (2001). of the agent to achieve sustained target inhibition in tissues and 3. A. Jackson and P.S. Linsley, Discovery Medicine 9 (47), 311–318 (2010). 4. J. Krutzfeldt et al., Nature 438 (7068), 685–689 (2005). organs without the need of formulation. Improvements in anti- 5. D.P. Bartel, Cell 116 (2), 281–297 (2004). miR chemical design and pharmacokinetic properties will allow 6. E.C. Lai, Nat. Rev. Genet 30 (4), 363–364 (2002). further exploration of microRNA biology and broaden the utility 7. T. Thum et al., Nature 456 (7224), 980–984 (2008). of microRNA therapeutics. 8. L. Ma et al., Nature 28 (4), 341–347 (2010).

4 Pharmaceutical Technology BIOPROCESSING & STERILE MFG. 2011 PharmTech.com 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 Phosphate- buffered saline. (a) (b) PBS Anti-miR-Cy3 3’ - UGGGGAUAGUGUUAAUCGUAAUU-5’ 78.17 2.67 30.98 54.47

20 103 103 GCATTAA AGCATTA 15 102 102

CD11b 101 101 10

100 19.14 0.02 100 13.24 1.31 5 100 101 102 103 100 101 102 103 MFI 0

Cy3 -5 -log 10 Enrichment P-value

Target cell -10 0 5000 10000 15000 20000 Sorted sequences (most up- to most down-regulated) Messenger RNA

9. R.E. Lanford et al., Science 327 (5962), 198–201 (2010). 13. R.S. Geary, R. Zu, and A.A. Levin, “Pharmacokinetic/Pharmacodynamic 10. P.P. Seth et al., J. Med. Chem. 52 (1), 10–13 (2009). Properties of Phosphorothioate 2’-O-(2-Methoxyethyl)-Modified Anti- 11. A.A. Levin, R. Zu, and R.S. Geary, “Basic Principles of the Pharmacoki- sense Oligonucleotides in Animals and Man,” in Antisense Drug Technol- netics of Antisense Oligonucleotide Drugs,” in Antisense Drug Technol- ogy: Principles, Strategies, and Applications, S.T. Crooke, Ed. (CRC Press, ogy: Principles, Strategies, and Applications, S.T. Crooke, Ed. (CRC Press, Boca Raton, 2007), pp. 306–326. Boca Raton, 2007), pp. 183–215. 14. S.C. Semple et al., Nature Biotech. 28 (2), 172–176 (2010). 12. B. Monia et al. J. Biol. Chem. 268 (19), 14514–14522 (1993). 15. S.V. Dongen et al., Nature Methods 5 (12) 1023–1025 (2008). PT

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