miRNA

Therapeutic Targeting of MicroRNAs

Speaker: Peter S. Linsley, PhD
Regulus Therapeutics

MicroRNA are endogenous non-coding RNAs that post-transcriptionally regulate gene expression. microRNAs can act as master regulators in biological pathways central to many areas of biology, including development, cancer, metabolism, and immunity. Each microRNA may regulate many genes by binding to target sites in 3′UTR regions of their mRNAs. Their ability to modulate disease pathways makes targeting or augmenting microRNAs an exciting new approach for drug discovery. What distinguishes microRNAs from more traditional therapeutic approaches is their ability to induce phenotypic changes by simultaneously influencing multiple targets in a single pathway.

One approach to targeting microRNAs is through the use of antisense molecules (antagomirs or anti-miRs) to inhibit their function. Previous studies showed that miR-122, the predominant microRNA in mammalian liver, could be inhibited using antisense approaches without causing undue toxicities. Moreover, antisense inhibition of miR-122 in high fat-fed mice resulted in reduced plasma cholesterol and hepatic steatosis.

While these results were encouraging, the ability of microRNAs to target transcript networks complicates our understanding of how best to optimize their beneficial effects, and how to avoid unwanted toxicities. In this talk, I will present current information from ongoing studies into the development of microRNA therapeutics. These studies include the improvement of potency and specificity of anti-miRs and understanding links between microRNA levels and disease pathways.

Mechanisms of microRNA-mediated Gene Regulation in Animal Cells

Speaker: Timothy W. Nilsen, PhD
Case Western Reserve University School of Medicine

Several lines of evidence suggest that microRNAs (miRNAs) regulate the expression of the majority of genes in animals. In almost all cases, miRNAs appear to function by binding to target mRNAs and repressing protein synthesis. The molecular mechanism(s) by which repression is achieved remains controversial and evidence for many seemingly contradictory modes of action have been proposed. In this presentation, the evidence for multiple mechanisms of miRNA function will be evaluated and discussed. In addition, evidence will be presented that the magnitude of miRNA-mediated regulation can vary dramatically depending upon cell type.

Enhancement of Hepatitis C Virus Replication by Liver-specific microRNA miR-122

Speaker: Peter Sarnow, PhD*
Stanford University School of Medicine

MicroRNAs interact generally with sites residing in 3′ noncoding regions in target mRNAs, leading to posttranscriptional downregulation of mRNA expression. In contrast, liver-specific microRNA miR-122 is known to bind to a site close to the 5′ end of the hepatitis C virus (HCV) RNA genome, resulting in upregulation of viral RNA abundance. Studies with replication-defective viral RNAs demonstrated that miR-122 did not affect mRNA translation or mRNA stability, suggesting that miR-122 affects mRNA abundance likely by increasing HCV RNA replication. We found that the location of the miR-122 binding site in the viral genome dictates it's effect on gene regulation, because insertion of the miR-122 binding site into the 3′ noncoding region of a reporter mRNA leads to downregulation of mRNA expression. Furthermore, we discovered a juxtaposed, second miR-122 binding site in HCV, which separates the two seed match sequence elements by a nine nucleotide sequence element which is highly conserved among all HCV genotypes. Results obtained with mutated HCV genomes argue that both sites are likely to be occupied in the same molecule for efficient regulation to occur. Subcellular analyses in cells continuously replicating HCV RNA, i.e. replicon cells, have shown that HCV replication proteins at least partly co-localize with components of P-bodies, which are cytoplasmic structures that harbor microRNA-bound mRNAs. Curiuosly, P-bodies are dispersed in cells infected with infectious HCV, suggesting that P-bodies may play roles in innate immune responses to HCV or that components of P-bodies are subverted to aid in viral replication. These findings set a paradigm for dual functions of a tandem microRNA binding site in a position-dependent manner. Furthermore, a concerted binding of two miR-122 molecules to the HCV genome offers a potential new antiviral intervention by targeting an oligomeric microRNA-HCV complex.

*Coauthors: Catherine L. Jopling^1,2, Cara Pager¹, and Sylvia Schütz¹.

¹ Stanford University School of Medicine
² University of Cambridge

Studying micro-RNA Mediated Gene Regulation by Ablating One Specific Target Site
at a Time

Speaker: F. Nina Papavasiliou, PhD
The Rockefeller University

B lymphocytes perform somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin locus to generate an antibody repertoire diverse in both affinity and function. These somatic diversification processes are catalyzed by activation-induced cytidine deaminase (AID), a potent DNA mutator whose expression and function are highly regulated. miR-155 is one of a number of microRNAs with expression patterns that are suggestive of a function in the regulation of these reactions. Indeed, target prediction algorithms suggested that AID itself could be a target of miR-155. To study the role of miR-155 in AID regulation we specifically mutated the presumed target site in the 3′UTR of AID. Here we show that point mutations in the portion of the target site predicted to anneal with the miR seed region, lead to both quantitative and temporal deregulation of AID levels which is accompanied by specific functional consequences for CSR and affinity maturation. Thus, miR-155, which has recently been shown to play important roles in regulating the germinal center reaction, does so in part by directly downmodulating AID expression.

*Coauthors: G. Teng, P. Hakimpour, P. Landgraf, A. Rice, T. Tuschl, and R. Casellas.

Delivery Strategies

High Throughput Approaches for Creating Nanosystems for Delivering DNA and siRNA

Speaker: Robert S. Langer, PhD
Massachusetts Institute of Technology

Nanoparticles provide a unique opportunity for oligonucleotide delivery because their small size enables them to be taken up by cells. We have developed a novel high throughput approach for synthesizing polymers or lipids whereby tens of thousands of new materials can be synthesized, combined with DNA or siRNA, and tested for safety and efficacy. We discuss these approaches in the context of different animal models, including primates, of various diseases.

Ligand-Targeted Delivery of siRNAs to Pathologic Cells and Tissues

Speaker: Philip S. Low, PhD*
Purdue Univeersity

We have developed methods to deliver both low molecular weight drugs and macromolecules specifically to pathologic cells, thereby avoiding the collateral toxicity associated with drug uptake by healthy cells. In the case of cancer, we have exploited the strong upregulation of the folate receptor on malignant cells to target: i) siRNAs, ii) chemotherapeutic agents, iii) gene therapy vectors, iv) protein toxins, v) radioimaging agents, vi) nanoparticles, vii) liposomes with entrapped drugs, viii) immunotherapeutic agents, and ix) numerous types of imaging agents to tumor tissues by linking the various agents to the vitamin folic acid. Current clinical trials of five distinct folate-linked drugs demonstrate that the folate-targeting strategy holds significant promise for improving the magnitude and specificity of drug uptake by solid tumors.

Folate-linked drugs must obviously extravasate and penetrate the tumor tissue before they can bind to FR-expressing cancer cells. Consequently, size, shape, and water-solubility influence a folate conjugate's tumor targeting ability. Data showing the real time penetration, binding and internalization of various fluorescent folate conjugates by tumor cells in live mice, as monitored by multiphoton intravital microscopy, will be presented. These studies will illustrate the impact that particle size and shape have on the rate and magnitude of tumor penetration. Included in these analyses will be studies describing the targeting specificity, tumor penetration and uptake kinetics, and target gene suppression of folate-conjugated siRNA molecules in vitro and in vivo.

We are also developing drug targeting strategies for the imaging and therapy of rheumatoid arthritis, Crohn's disease, atherosclerosis, lupus, psoriasis, glomerulonephritis, atherosclerosis, and multiple sclerosis. Finally, we have discovered a number of novel targeting ligands for drug delivery selectively to various infectious pathogens. Methods associated with these technologies will also be discussed.

*Coauthors: Erina Vlashi, Mini Thomas, Ligia Sega, Purdue University; Yingqun Huang, Yale University School of Medicine; and Muthiah Manoharan, Alnylam Pharmaceuticals.

Delivery of siRNA for Tumor Therapy

Speaker: Leaf Huang, PhD
University of North Carolina at Chapel Hill

We have developed a surface-modified LPD (liposome-polycation-DNA) nanoparticle formulation. This self-assembled nanoparticle formulation was around 120 nm in diameter with 90% encapsulation efficiency for siRNA. siRNA was first mixed with a high molecular weight polyanion carrier (either DNA or hyaluronic acid) and then complexed with the protamine into a compact core. The core was subsequently coated with two cationic lipid bilayers. The inner lipid bilayer was stabilized by the charge-charge interaction between the cationic lipids and the compact core. Upon addition of a PEGylated lipid, DSPE-PEG, the outer lipid bilayer was stripped off and the DSPE-PEG was inserted into the outer leaflet of the inner bilayer, resulting in approximately 10.6 mol% modification of DSPE-PEG on the surface of the nanoparticles. The high degree of PEGylation completely shielded the charge of the nanoparticles with the zeta potential close to neutral (−5.6 ± 4.5 mV) and abolished the reticuloendothelial uptake in the isolated liver perfusion. When i.v. injected into tumor bearing mice (s.c. human lung cancer xenograft model in the nude mice), the nanoparticles delivered 70–80% injected siRNA/g into the tumor, while the normal organs only showed a moderate uptake (10–20% injected siRNA/g). After the conjugation of a targeting ligand, anisamide, at the distal end of the PEG, the intracellular delivery of siRNA into the sigma receptor expressing tumor was significantly enhanced. This led to efficient EGFR silencing, significant apoptosis induction and tumor growth inhibition at the dose of 1.2 mg siRNA/kg for three consecutive injections. An experimental murine lung metastasis model was established by i.v. injecting mouse melanoma cells, which were stably transduced with a luciferase gene by retrovirus, into the mice. An improved metastatic tumor delivery of siRNA was discovered by using the nanoparticles. Approximately 70–80% luciferase activity in the lung metastasis was reduced after a single injection of the nanoparticles containing siRNA against luciferase at the dose of 0.15 mg/kg. When a combination of three siRNA sequences (MDM2, c-myc and VEGF) were delivered, the oncogenes in the lung metastasis were silenced simultaneously, leading to 70–80% tumor load reduction and 30% prolongation in animal lifespan. The nanoparticle formulation showed minimal or no immunotoxicity in both animal models with little organ damage at the therapeutic dose. The results promise the potential use of this formulation clinically.

Work supported by NIH grant CA129835.

Delivery of RNA-Based Therapeutics: Advances and Challenges

Speaker: Laura Sepp-Lorenzino, PhD
RNA Therapeutics, Merck Research Laboratories

Oligonucleotide-based technologies offer great potential for enabling the development of therapeutic antagonists for targets previously considered un-druggable using traditional therapeutic modalities, with superior specificity profiles and with shorter timelines than the current norm for small molecules. However, despite steady advances in the field, significant issues remain that must be overcome for successful development of safe and efficacious dosage forms for treating systemic disease in humans. This requires selection of specific siRNAs with high intrinsic potency and duration of silencing, as well as low affinity for oligonucleotide innate immune receptors, together with an effective and safe systemic delivery approach. One strategy for overcoming some of these barriers is through chemical modification. Chemical modification protects siRNAs from nuclease degradation, increases duration of effect and reduces off-target gene silencing thereby enhancing selectivity for the intended target. In addition, siRNA chemical modification significantly diminishes activation of toll like receptors, and associated downstream responses. To address the challenges associated with delivery, we are evaluating a number of delivery strategies internally as well as part of our extensive external research and licensing efforts in this area. The application of these approaches will be described using case studies, in proof-of-concept studies aimed at validating our platforms. Specifically, we will provide data on systemic delivery of siRNAs targeting ApoB and benchmarking genes being delivered in cationic lipid nanoparticles (LNP), in multiple species including non-human primates.

siRNA Delivery by Peptide Transduction – dsRNA Binding Domain Fusions

Speaker: Steven F. Dowdy, PhD
Howard Hughes Medical Institute and UCSD School of Medicine

Induction of RNA interference (RNAi) by siRNAs to degrade specific mRNAs has great potential to treat human disease, especially cancer with the myriad of genetic alterations that distinguish the cancer cells from the surrounding normal cells. However, due to their size (~14,000 Dalton) and charge, siRNAs have no bioavailability to enter unperturbed cells. Current siRNA delivery approaches fail to deliver siRNAs into a high percentage of cells in a non-cytotoxic fashion, especially primary cells. Thus, siRNA delivery remains the rate-limiting step for RNAi therapeutic development. To address the siRNA delivery problem, we developed a Peptide Transduction Domain – dsRNA Binding Domain (PTD–DRBD) fusion protein siRNA delivery approach. DRBDs bind siRNAs with high avidity independent of sequence, mask the negative charge and allow for PTD-mediated siRNA delivery. PTD–DRBD delivered siRNAs induced RNAi responses in the entire cell population of 20+ cell types assayed in a non-cytotoxic fashion, including primary human umbilical vein endothelial cells (HUVEC), primary T cells, primary human fibroblasts, keratinocytes, neuronal cells, hematopoietic lineages and human embryonic stem cells.

We tested the in vivo ability of PTD–DRBD mediated siRNA delivery to induce a synthetic lethal response to kill glioblastoma cells in vivo by selectively targeting intersecting oncogenic pathways. Glioblastoma multiforme remains the most deadly and intractable human malignancy. Activation of multiple oncogenic pathways in glioblastoma leads to increased cell growth, proliferation and tumor cell survival. In vivo PTD–DRBD mediated delivery of combination therapeutic siRNAs targeting the EGF-Receptor and Akt2 synergized to induce apoptosis throughout the tumor and significantly increased survival in intracerebral glioblastoma mouse models, whereas delivery of irrelevant control siRNAs did not alter longevity. These observations demonstrate the exquisite in vivo synergistic potential of RNAi to treat cancer by simultaneously selecting and degrading multiple specific oncogenic mRNA targets to induce synthetic lethal responses.

Drug and Therapeutic Development

A Phase II Trial of ALN-RSV01, an RNAi Therapeutic for Respiratory Syncytial Virus

Speaker: Akshay K. Vaishnaw, MD, PhD*
Alnylam Pharmaceuticals, Inc.

Confirmation of RNAi as a widespread physiologic phenomenon in mammalian systems has raised the possibility of pharmacologic intervention using synthetic siRNAs. Before RNAi therapeutics can be evaluated in clinical trials however, they must be designed so that these simple dsRNA species acquire 'rug-like' properties. We have made significant progress towards this goal, our most advanced program being ALN-RSV01 which targets the N gene transcript of respiratory syncytial virus (RSV). RSV is a major lower respiratory tract pathogen of young children and accounts for approximately 80% of childhood bronchiolitis cases and 50% of infant pneumonias. Currently, there are no viable treatment options for this infection. The primary site of RSV infection is in the epithelium lining the respiratory tract, suggesting accessibility to an inhaled RNAi therapeutic. In pre-clinical studies ALN-RSV01 substantially inhibited in vitro RSV replication in plaque assays at sub-nM concentrations, and, after direct pulmonary delivery, attenuated lung RSV burden by several log order in both prophylactic and therapeutic mouse models of disease. Rat and monkey toxicology studies have been completed, as have multiple intranasal and inhalational single- and multi-dose Phase 1 human clinical studies. To date, the ALN-RSV01 safety, tolerability and PK profiles look encouraging. The development of anti-virals against respiratory pathogens is further assisted by the availability of experimental infection models. Recently, we have completed a randomized, double-blind, placebo-controlled study evaluating the safety and anti-viral efficacy of ALN-RSV01 in subjects experimentally infected with RSV. The data demonstrate clear evidence of a strong anti-viral effect with good safety and tolerability. In the presentation, I will review the data from prior Phase I safety studies, the current Phase II data and upcoming development plans.

*Coauthors: Jared Gollob, Rachel Meyers, and Rene Alvarez; Alnylam Pharmaceuticals, Inc.; John DeVincenzo, University of Tennessee School of Medicine.

Clinical Application of Systemically-Administered siRNA-Containing Nanoparticles
for Cancer

Speaker: Jeremy Heidel, PhD*
Calando Pharmaceuticals

Calando has advanced CALAA-01, a polymer-based, transferrin-targeted, siRNA-containing nanoparticle formulation targeting ribonucleotide reductase subunit M2 (RRM2), into Phase I clinical investigation in patients with solid tumors. CALAA-01 is the first formulated, targeted, systemic siRNA to reach the clinic; it is also the first siRNA in the clinic for oncology. Pre-clinical safety and efficacy of CALAA-01 will be discussed; aspects of characterization and formulation; as well as clinical protocol and status, will be described.

*Coauthors: Yi-Ching (Joanna) Liu, Shyam Rele, and Yongchao Liang, Calando Pharmaceuticals; and Mark Davis, California Institute of Technology.

Clinical Development of Antisense Drugs for Type 2 Diabetes: A Novel
Therapeutic Strategy

Speaker: Sanjay Bhanot, MD, PhD
Isis Pharmaceuticals

Type 2 Diabetes (T2D) is a growing epidemic with over 240 million adult cases worldwide. The International Diabetes Foundation estimates that by the year 2025, over 330 million adults will be diagnosed with diabetes. Recent findings indicate that the only class of approved insulin sensitizers, the thiazolidinediones, may increase cardiovascular risk. These findings have resulted in black box warnings for these compounds and have increased the urgency to develop drugs with novel mechanisms and improved safety profiles for the treatment of T2D. Isis Pharmaceuticals currently has four such antisense compounds in development, each of which has a unique mechanism of action and the potential of becoming first-in-class therapeutic for the treatment of T2D. ISIS 113715, a specific antisense inhibitor of protein tyrosine phosphatase 1B (PTP-1B) is one such insulin sensitizer that is under development for T2D. In Phase 2 studies, ISIS 113715 treatment for 6–12 weeks resulted in reductions in fasting and post-prandial blood glucose levels and HbA1c in treatment naïve type 2 diabetic patients. In addition, by the end of treatment, patients treated with ISIS 113715 achieved significant reductions in total and LDL cholesterol levels as compared to the placebo group. ISIS 113715 was safe, well tolerated and did not cause hypoglycemia or body weight gain. Phase 2 studies are in progress to evaluate the effects of ISIS 113715 in T2D patients uncontrolled with oral anti-diabetic agents. The presentation will highlight Phase 2 data and the anticipated product profile of ISIS 113715 and will summarize the unique attributes of other antisense drugs under development, especially their potential to provide therapeutic benefit that extends beyond glucose control, including lipid-lowering and anti-obesity effects.

A Phase II Randomized Study of Custirsen (OGX-011) Combination Therapy in Patients with Poor-Risk Hormone Refractory Prostate Cancer (HRPC) Who Relapsed on or Within Six Months of 1st-line Docetaxel Therapy

Speaker: Martin Gleave, MD*
OncoGenex Technologies, Vancouver, and the University of British Columbia

Secretory clusterin (sCLU-2) is a stress-induced, cytoprotective chaperone up-regulated by androgen ablation and chemotherapy to inhibit cell death and confer broad-spectrum treatment resistance. sCLU-2 is a potent inhibitor of protein aggregation and proteotoxic stress. We recently elucidated sCLU-2 as a ubiquitin binding protein that enhances NF-kB transcriptional activity and cell survival by facilitating COMMD1 and I-kB ubiquitination and proteasome degradation. Additional cytoprotective mechanisms include inhibition of activated Bax and Bad which inhibit cytochrome C release and caspase activation. These data indicate that knockdown of sCLU can sensitize cancer cells to treatment stress via several distinct survival pathways.

The 2nd generation antisense drug, custirsen (OncoGenex, OGX-011), decreases cytoprotective sCLU, while increasing pro-apoptotic alternative spliced nuclear (nCLU), levels to enhance hormone- and chemo-therapy in many preclinical xenograft models. A novel, dose escalating Phase I pre-prostatectomy trial defined the optimal biologically dose and usual toxicity parameters of OGX-011, with maximal dose-dependent knockdown of clusterin in prostate and lymph node tissues at the 640 mg dose level. Phase I combination studies confirmed that 640 mg OGX-011 can be combined with standard doses of the chemotherapy agents. The current status of randomized phase II trials of chemotherapy +/− OGX-011 in lung and mHRPC will be updated.

A recently completed Phase II trial in docetaxel-recurrent mHRPC evaluated the safety and efficacy of custirsen in combination with either docetaxel or mitoxantrone as 2nd-line treatment. Pts were eligible if they had progressed while receiving or within 6 mo of 1st-line docetaxel. Following 3 loading doses, all pts received 640 mg of weekly IV custirsen. Pts were randomized to standard doses of docetaxel/prednisone (DPC) or mitoxantrone/prednisone (MPC) on a 21-day cycle for up to 9 cycles. Protocol defined PD was based on RECIST, pain and performance score but not solely on PSA. As of Jan07, median follow-up was 13.3 mo. in 42 pts. Median # of cycles delivered was 7.5 for DPC and 6.0 for MPC, with 1/3 completing 9 cycles. Best PSA response (≥90, ≥50 ≥30%): DPC-20/40/55%, MPC-0/27/32%. Pain response occurred in 67% for DPC and 50% for MPC, with a median duration of 6 mos in both arms. KM estimate of PFS was 4.7 mo. for DPC and 2.6 mo. for MPC, with similar deaths (40% and 36%) in both arms. The data indicate both 2nd line custirsen combination treatments were well tolerated for up to 9 cycles and associated with higher-than predicted PSA and pain responses. Custirsen+docetaxel appeared superior to custirsen+mitoxantrone in both efficacy and safety. A Phase III study is planned utilizing docetaxel +/− custirsen as 2nd-line Rx in patients progressing after 1st-line docetaxel.

*Coauthors: Kim Chi and Fred Saad, OncoGenex Technologies, Vancouver, and the University of British Columbia.

TPI 1100: A Novel Inhaled FANA-containing Antisense Oligonucleotide Product Against Phosphodiesterase 4 and 7 for the Treatment of COPD

Speaker: Nicolay Ferrari, PhD
Topigen Pharmaceuticals Inc., Montreal

Chronic Obstructive Pulmonary Disease (COPD) is largely irreversible disorder in which neutrophils are believed to play a key role in the development and maintenance of the underlying inflammation and tissue matrix breakdown. These inflammatory processes can be downregulated by an increase in cAMP, whose accumulation in cells can be modulated by phosphodiesterases (PDEs), in particular PDE4, the predominant PDE isoenzyme in pro-inflammatory cells. PDE4 has become a lead target for the treatment of COPD, however, the development of PDE4 inhibitors has been limited by a low therapeutic ratio, the lack of specificity and side effects related to the oral route of administration.

TPI 1100 is a novel inhaled RNA-targeting compound comprised of two AON containing 2′-deoxy-2′-Fluoro-β-D-Arabinonucleic Acid (FANA) designed against the human PDE subtypes 4B, 4D and 7A.

In vitro, we have demonstrated that TPI 1100 was very effective at specifically inhibiting all three PDE subtypes in both lung epithelial and immune (PBMC) cells. Moreover, in stimulated cells, TPI 1100 prevented induction of mRNA expression and protein secretion of key inflammatory markers (including IL-8, MCP-1, GM-CSF, TNF-α and IL-2). Studies performed in animal models of COPD indicated that lung delivery of TPI 1100 exerted marked protective effects against cigarette smoke-induced inflammation. TPI 1100 demonstrated potent anti-neutrophilic effects as well as inhibition of the induction of pro-inflammatory cytokines/chemokines (including KC, MIP-1α and MIP-2) and metalloproteases (MMP-9). In our model, TPI 1100, at a dose 25-fold lower, was shown to be more effective than orally administered Roflumilast, a small molecule inhibitor of PDE4 currently in Phase 3 trials for COPD.

The toxicity of TPI 1100 was assessed in a standard battery of in vitro and in vivo genetic toxicity assays and in 14-day inhalation toxicology studies in mice and monkeys. Results from these studies demonstrated that this new AON-based product is well tolerated and that delivery via the inhaled route achieves localized deposition in the pulmonary tract with very limited systemic exposure and reduced toxicity compared to other routes of AON administration. In conclusion, TPI 1100 is a novel inhaled FANA-containing anti-PDE AON drug which demonstrates the potential for unique protective effects on biomarkers that are believed to be important in the prognosis of COPD.

*Coauthors: Hélène D'Anjou, Marylène Fortin, Marie-ève Higgins, Paméla Aubé, Serge Séguin, Jasmine Gougeon, Antonia Balassy, Elisabeth Viau, Rosanne Séguin, Alain Guimond, and Kamel Monktefi; Topigen Pharmaceuticals Inc., Montreal.

Clinical Development of an Anti-VWF Aptamer, ARC1779

Speaker: James C. Gilbert, MD
Archemix Corp

ARC1779 is an aptamer which blocks the binding of the A1 domain of VWF to GPIb on platelets with high affinity and specificity. The VWF-platelet interaction is a potentially therapeutically relevant target in atherothrombosis and in hematologic disorders such as TMA or VWD-2B. ARC1779 has demonstrated proof of mechanism in healthy volunteers and is now being tested in Phase 2 clinical trials in patients with TMA, VWD-2B, and carotid endarterectomy. The experience with ARC1779 illustrates some of the advantages and challenges that typify clinical development of systemically administered aptamer therapeutics.

Antagonizing microRNAs for Therapeutics

Speaker: Sakari Kauppinen, PhD
University of Copenhagen and Santaris Pharma

MicroRNAs (miRNAs) are an abundant class of short endogenous non-coding RNAs that act as important post-transcriptional regulators of gene expression by base-pairing to their target mRNAs, thereby mediating mRNA cleavage or translational repression. Recent data suggest that miRNAs are aberrantly expressed in many human cancers and that they may play significant roles as oncogenes or tumour suppressors. Apart from cancer, miRNAs have also been implicated in viral infections, heart disease and neurological disorders. Hence, disease-associated miRNAs represent a potential new class of targets for antisense-based therapeutics, which may yield patient benefits unobtainable by other therapeutic approaches.

LNA (locked nucleic acid) is the first true conformational analogue of RNA, in which the furanose ring in the sugar-phosphate backbone is locked in an RNA-like, C3′-endo conformation. This conformational restriction results in unprecedented binding affinity between single-stranded LNA oligonucleotides and their complementary RNA targets and high stability in blood and tissues in vivo. LNA-modified oligonucleotides have proven outstanding in microRNA recognition and detection due to their high specificity and affinity. Here we report that short, unconjugated LNA-antimiR oligonucleotides can be used as potent molecules for sequence-specific antagonism of disease-associated miRNAs in vitro and in vivo in rodents and non-human primates.

Restoration of Dystrophin by a Peptide Conjugated Morpholino Oligomer Improved Skeletal and Cardiac Functions in Dystrophin-Deficient MDX Mice

Speaker: Hong M. Moulton, PhD*
AVI BioPharma

Duchenne muscular dystrophy (DMD) is a serious X-linked disease caused by mutations in the human dystrophin gene, most commonly resulting in premature termination of translation so that no functional dystrophin is produced. Splice-switching oligonucleotides have been shown to correct out-of-frame mutations in DMD and restore truncated yet functional dystrophin. However, systemic application of the oligos has been limited due to their insufficient delivery to nuclei of muscle cells and failure to restore dystrophin in heart. Here, we show that a cell-penetrating peptide-conjugated morpholino oligomer (PPMO) targeted to the mutated exon restored dystrophin to >80% and 50% of normal level in skeletal and cardiac muscles, respectively, with a single intravenous injection in mdx mice at 30 mg/kg. Three months treatment with a total of six injections at 30 mg/kg each restored not only dystrophin to nearly normal levels in all muscles but also dystrophin-associated proteins. The PPMO treatment with four daily injections at 12 mg/kg each produced a persistent exon-skipping effect where the exon-skipped transcript was still detectable in the heart, diaphragm, and quadriceps of the mdx mice 9 weeks after the last injection, indicating that PPMO has prolonged tissue retention. Animal health was improved by the treatments, reflected by improvement of the pathology, strength and function of skeletal and cardiac muscles as well as by reduction in levels of serum creatine kinase. No toxicity or immunogenicity was detected at the dose regimens used. Our study demonstrated that PPMO are very promising therapeutics for treatment of DMD.

*Coauthors: Peter Sazani and Ryszard Kole, AVI BioPharma; Bo Wu and Qi Long Lu, Carolinas Medical Center, Charlotte; and Natee Jearawiriyapaisarn, University of North Carolina.

Preclinical Models and Clinical Programs PART I: Gene Silencing

The Sarcoma Genome Project: Molecular Subtyping and the Development of Selective Therapeutic Strategies

Speaker: Samuel Singer, MD
Memorial Sloan-Kettering Cancer Institute

Soft tissue sarcomas are an uncommon and heterogeneous group of mesenchymal cancers that are a challenge to diagnose and treat. Despite aggressive therapy, 50% of newly diagnosed sarcoma patients will die from disease highlighting a pressing need to develop new targeted therapies. Therefore we initiated the Sarcoma Genome Project to characterize the spectrum of genetic aberrations in high-grade soft tissue sarcoma. We performed systematic genomic profiling of 207 patients across seven high-grade soft tissue sarcoma types that included high-throughput re-sequencing for somatic mutations, high-resolution single-nucleotide polymorphism (SNP) arrays to characterize copy-number aberrations and loss-of-heterozygosity (LOH), and paired gene expression profiling. This compendium of somatic alterations is then used to inform the molecular classification of soft tissue sarcoma types, to discover novel subtypes, and to identify driver genes in a loss-of-function (RNAi) screen of all significantly amplified genes in dedifferentiated liposarcoma. Genome-wide copy-number changes, combined with expression analysis, identified known sarcoma genes along with potentially novel ones in regions of significant amplification and deletion. Moreover, distinct patterns of copy-number alterations were associated with clinicopathological features, and point to novel molecular sub-classifications. Finally, for functional target discovery, we performed a genomics-driven RNA interference screen in dedifferentiated liposarcoma, knocking down its entire amplicome in genotype-matched cell lines. We identified a signature of essential genes that reveal genotype-specific sensitivities, and a novel set of synergistically acting candidate oncogenes. Together, we present an integrative genetic and functional analysis that informs not only the genetic abnormalities contributing to sarcomagenesis, but also an initial signature of type-specific essential genes representing potential therapeutic targets.

*Coauthors: Jordi Barretina, Barry S. Taylor, and Matthew Meyerson, Memorial Sloan-Kettering Cancer Institute.

Therapeutic RNAi targeting PCSK9

Speaker: Kevin Fitzgerald, PhD
Alnylam Pharmaceuticals, Inc.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates the level of low density lipoprotein receptor (LDLR) protein and its function. Loss of PCSK9 protein increases LDLR levels in liver and reduces plasma LDL cholesterol (LDLc). PCSK9 activity decreases liver LDLR levels and increases plasma LDLc. We have developed active cross-species small interfering RNAs (siRNAs) capable of targeting murine, rat, non-human primate (NHP), and human PCSK9. PCSK9 and control siRNAs were formulated in a lipidoid nanoparticle (LNP) and delivered to several different in vivo models. Liver specific siRNA silencing of PCSK9 in mice and rats reduced PCSK9 mRNA levels by 50–70%. The reduction in PCSK9 transcript resulted in up to a 60% reduction in plasma cholesterol. These effects were shown to be mediated by an RNAi mechanism using 5′-RACE. In NHP, a single dose of siRNA targeting PCSK9 resulted in a rapid, durable, and reversible lowering of plasma PCSK9, apolipoprotein B and LDLc, without measurable effects on HDL cholesterol (HDLc) or trigycerides (TGs). The duration of PCSK9 silencing lasted for three weeks after a single intravenous dose. These results validate PCSK9 targeting with RNAi therapeutics as an approach to specifically lower LDLc.

*Coauthors: M. Frank-Kamenetsky¹, T. Racie¹, B. Bramlage², A. Akinc¹, A. Grefhorst³, N. Anderson³, K. Charisse¹, R. Dorkin¹, C. Gamba-Vitalo¹, P. Hadwiger², M. John², M. Maier¹, L. Nechev¹, T. Read¹, P. Tan², J. Wong¹, T. Zimmermann¹, D. Anderson⁴, H. Vornlocher², M. Manoharan¹, V. Koteliansky¹, and J. Horton³.

¹ Alnylam Pharmaceuticals, Inc.
² Roche Kulmbach GmbH, Germany.
³ University of Texas Southwestern Medical Center at Dallas. ⁴ Massachusetts Institute of Technology.

Oblimersen Yields Long-term Benefit in Advanced Melanoma and CLL: Extended Analysis and Future Directions in Clinical Development

Speaker: Loretta M. Itri, MD*
Genta Incorporated

The antiapoptotic protein Bcl-2 provides cancer cells with a survival advantage and is associated with chemoresistance. Oblimersen (Genasense®) is a DNA phosphorothioate oligonucleotide complementary to the first 6 codons of an open reading frame in bcl-2 mRNA that decreases bcl-2 mRNA and Bcl-2 protein. Additional mechanisms may contribute to its antitumor effect. Oblimersen enhances antitumor activity when combined with various anticancer therapeutics.

In advanced melanoma (N=771), oblimersen by continuous IV (CIV) infusion plus dacarbazine significantly increased overall response rate (p=0.007), durable response rate (p=0.027), and progression-free survival (p=0.0007); a favorable trend in overall survival also was observed (p=0.077) (Bedikian et al, J Clin Oncol, 2006). Treatment effect was highly correlated with the ratio of baseline LDH to the upper limit of normal range (ULN; Keilholz et al, ASCO, 2007). In patients with baseline LDH ≤ 0.8 × ULN (the lowest LDH category studied [N=274]), median overall survival was 12.3 vs 9.9 months (HR=0.64; p=0.0009). A confirmatory study (AGENDA) is ongoing in patients with low baseline LDH. In relapsed/refractory CLL, oblimersen by CIV infusion plus fludarabine/cyclophosphamide significantly increased complete response rate (p=0.025) and duration of complete response (p=0.03) (O'Brien et al, J Clin Oncol, 2007). Moreover, 5-year follow-up showed a significant survival benefit (45% vs. 24% alive) in patients who achieved a complete or partial response with oblimersen (median: 56.0 vs. 37.8 mos; HR=0.60; p=0.038). Patients sensitive to fludarabine at baseline had the greatest survival benefit (median: 45.8 vs. 32.6 mos; HR=0.50; p=0.004).

Oblimersen research is continuing in the clinical setting with administration in combination with novel, multidrug regimens and as a short (1- to 2-hour) IV infusion.

*Coauthors: Steven Novick and Jane Wu, Genta Incorporated.

ATL/TV1102, an Oligonucleotide Targeting VLA-4 mRNA, Significantly Reduces New Active Lesions in Patients with Relapsing Remitting Multiple Sclerosis

Speaker: Christopher J. Wraight, PhD*
Antisense Therapeutics Limited

ATL/TV1102 is a 2nd generation antisense inhibitor of CD49d, a subunit of Very Late Antigen 4 (VLA-4) which plays a key role in cell adhesion to vessel walls. VLA-4 blockade, as shown by monoclonal antibodies such as natalizumab, prevents activated lymphocytes from migrating into the CNS and significantly reduces disease activity in MS.

Objective: To evaluate VLA-4 Antisense (ATL/TV1102) in the treatment of RR-MS.

Clinical Trial Design: Randomized, double-blind, placebo-controlled multicenter Phase-IIa trial. 77 patients with RR-MS were treated for 8 weeks with either 200mg of ATL/TV1102 or placebo subcutaneously twice weekly and evaluated for 16 weeks. MRI scans were performed at screening, and then monthly over 16 weeks. Primary efficacy variable: cumulative number of new active lesions (CNNAL; new gadolinium enhancing T1 lesions (T1-Gd), new or enlarging T2 lesions) on MRIs taken at weeks 4, 8 and 12. Secondary efficacy variable: cumulative volume of T1-Gd lesions (CVT1L) on MRIs taken at weeks 4, 8 and 12.

ITT population: 74 patients with a valid baseline MRI and at least one postbaseline MRI scan after first injection of study medication (n=39 placebo, n=35 ATL/TV1102).

Outcomes: ATL/TV1102 showed a significant reduction, 54.4%, in CNNAL (6.2 placebo, 3.0 ATL/TV1102; p=0.01). A reduction of 66.7% (p=0.002) was observed in the cumulative number (weeks 4,8,12) of new T1-Gd lesions with ATL/TV1102. A reduction in CVT1L was also observed under ATL/TV1102 but did not reach significance (589.4 mm³ placebo, 358.0 mm³ ATL/TV1102; p=0.1068). Adverse events that were more frequent under ATL/TV1102 included mild to moderate injection site reactions and a tendency for decreased platelet counts which were reversible after treatment interruption.

This proof-of-concept study of a drug designed to inhibit VLA-4 mRNA showed a significant reduction of the cumulative number of new active lesions in RR-MS patients following 8 weeks of treatment. These promising results warrant further investigation.

*Coauthors: V. Limmroth, Cologne City Hospitals, University of Cologne; F. Barkhof, VU University Medical Center, Amsterdam; N. Desem, Antisense Therapeutics Ltd, Melbourne.

Antisense-mediated Exon Skipping for Duchenne Muscular Dystrophy

Speaker: Annemieke Aartsma-Rus, PhD*
Leiden University Medical Center, the Netherlands

Antisense-mediated reading frame restoration is presently the most promising therapeutic approach for Duchenne muscular dystrophy (DMD). In this approach, antisense oligoribonucleotides (AONs) induce specific exon skipping during pre-mRNA splicing of mutated dystrophin transcripts. This is aimed to restore the disrupted open reading frame and allow synthesis of internally deleted, partly functional Becker-like dystrophin proteins. The approach is theoretically applicable to over 70% of all patients. Proof of concept has been achieved in cultured muscle cells from patients carrying different mutation types, as well as in the mdx mouse model. Recently we achieved the first proof of applicability to humans in vivo in a local-administration clinical trial in 4 patients, in which exon 51 skipping and dystrophin restoration was confirmed after a single intramuscular dose of AON, while no adverse effects were seen. Current research focuses on optimization of bioavailability, biodistribution, exon skipping efficiencies and functional benefit after systemic delivery of AONs. We find that after intravenous or subcutaneous injections, AONs are more easily taken up by dystrophic fibers when compared to healthy fibers. In the mdx mouse model, we are able to induce exon skipping and dystrophin restoration in all muscles, including the heart, after short term treatment with high AON doses and long term treatment with lower doses of AON. This was accompanied by improvement of muscle integrity and function. In parallel, we are assessing the specificity and bio-distribution of different AON chemistries after local and systemic treatment in various mouse models. Our hDMD model, which contains a complete copy of the human DMD gene stably integrated into the mouse genome, allows us to test human-specific AONs in vivo. The first results indicate that morpholino and 2′-O-methyl phosphorothioate AONs are equally efficient for most human target sequences, but that morpholinos may be less sequence-specific, probably due to their higher binding strength.

*Coauthors: Hans Heemskerk¹, Christa de Winter¹, Maaike van Putten¹, Anneke Janson², Jan Verschuuren³, Johan den Dunnen¹, Judith van Deutekom2, and Gert-Jan van Ommen¹.

¹ Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands.
² Prosensa BV, Leiden, the Netherlands.
³ Department of Neurology, Leiden University Medical Center, Leiden, the Netherlands.