Second Annual Meeting of the Oligonucleotide Therapeutics Society

Web Sites

Ambion, the RNA Interference Resource
This site, maintained by Ambion, Inc., publishes recent news about RNAi research.

Genome-Wide RNAi Global Initiative
A press release from Dharmacon, Inc. describing the formation of an international alliance of biomedical research centers to make use of recent advances in siRNA research.

Nature Focus RNAi Interference
Three animations, on gene expression, gene silencing, and RNAi amplification.

NOVA ScienceNow RNAi
Contains video of a segment on RNAi broadcast on NOVA, hosted by Robert Krulwich. The site also offers additional background information.

Oligonucleotide Therapeutics Society
A nonprofit forum created to foster academia- and industry-based research and development of oligonucleotide therapeutics.

The RNAi Consortium
A public-private consortium based at the Broad Institute in Cambridge, MA, created to develop and validate tools and methods for using RNAi to study the function of human and mouse genes.

RNAi.net
The "world of RNA interference"—including headlines, interviews, upcoming events, and information on suppliers.

The RNA World Website
Published by the Institute for Molecular Biology in Jena, Germany, this site features extensive links concerning research on RNA.

Companies conducting research in oligonucleotide therapeutics:

Acuity Pharmaceuticals
Ambion
Alnylam Pharmaceuticals
Archemix
AVI BioPharma
Calando Pharmaceuticals
Coley Pharmaceutical Group
Dharmacon RNA Technologies
Ester Neuroscience
Genta
Hybridon
Invitrogen
Isis Pharmaceuticals
OncoGeneX Technologies
OSI Pharmaceuticals
Sirna Therapeutics
Topigen Pharmaceuticals
VasGene Therapeutics

Books

Crooke ST, ed. 2001. Antisense Drug Technology: Principles, Strategies, and Applications. Marcel Dekker, New York.
Amazon | Barnes & Noble

Engelke D. 2004. RNA Interference (RNAi): The Nuts & Bolts of siRNA Technology (Nuts & Bolts Series). DNA Press, Eagleville, PA.
Amazon | Barnes & Noble

Fire A. 2005. RNA Interference Technology: From Basic Science to Drug Development. Cambridge University Press, Cambridge, UK.
Amazon | Barnes & Noble

Gibson I, ed. 2003. Antisense and Ribozyme Methodology: Laboratory Companion (Laboratory Companion Series). Wiley-VCH, Hoboken, NJ.
Amazon | Barnes & Noble

Hannon GJ, ed. 2003. RNAi: A Guide to Gene Silencing. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Amazon | Barnes & Noble

Hartmann RK, Bindereif A, Schön A, Westhof E. 2005. Handbook of RNA Biochemistry. John Wiley & Sons, New Jersey.
Amazon | Barnes & Noble

Herdewijn P. 2004. Oligonucleotide Synthesis: Methods and Applications (Methods in Molecular Biology). Humana Press, New Jersey.
Amazon

Schepers U. 2005. RNA Interference in Practice: Principles, Basics, and Methods for Gene Silencing in C.elegans, Drosophila, and Mammals. (Hardcover) John Wiley & Sons, New Jersey.
Amazon | Barnes & Noble

Journal Articles

General Background

Beal J. 2005. Silence is golden: can RNA interference therapeutics deliver? Drug Discov. Today 10: 169-172.

Session I

Glazer MJ, Fidanza A, McGall GH, et al. 2006. Kinetics of oligonucleotide hybridization to photolithographically patterned DNA arrays. Anal. Biochem. 358: 225-238.

Knauert MP, Kalish JM, Hegan DC, Glazer PM. 2006. Triplex-stimulated intermolecular recombination at a single-copy genomic target. Mol. Ther. 14: 392-400.

Koller E, Propp S, Murray H, et al. 2006. Competition for RISC binding predicts in vitro potency of siRNA. Nucleic Acids Res. 34: 4467-4476. FULL TEXT

Le Sage C, Agami R. 2006. Immense promises for tiny molecules: uncovering miRNA functions. Cell Cycle 5: 1415-1421. Epub 2006 Jul 1.

Prasanth KV, Prasanth SG, Xuan Z, et al. 2005. Regulating gene expression through RNA nuclear retention. Cell 123: 249-263.

Voorhoeve PM, le Sage C, Schrier M, et al. 2006. A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors. Cell 124: 1169-1181.

Session II

Dowler T, Bergeron D, Tedeschi AL, et al. 2006. Improvements in siRNA properties mediated by 2′-deoxy-2′-fluoro-beta-D-arabinonucleic acid (FANA). Nucleic Acids Res. 34: 1669-1675. FULL TEXT

Li L, Lin X, Staver M, et al. 2005. Evaluating hypoxia-inducible factor-1α as a cancer therapeutic target via inducible RNA interference in vivo. Cancer Res. 65: 7249-7258. FULL TEXT

Lin X, Ruan X, Anderson MG, et al. 2005. siRNA-mediated off-target gene silencing triggered by a 7 nt complementation. Nucleic Acids Res. 33: 4527-4535. FULL TEXT

McNamara JO 2nd, Andrechek ER, Wang Y, et al. 2006. Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras. Nat. Biotechnol. 24: 1005-1015.

Rusconi CP, Scardino E, Layzer J, et al. 2002. RNA aptamers as reversible antagonists of coagulation factor IXa. Nature 419: 90-94.

Sullenger BA, Gilboa E. 2002. Emerging clinical applications of RNA. Nature 418: 252-258.

Vester B, Hansen LH, Lundberg LB, et al. 2006. Locked nucleoside analogues expand the potential of DNAzymes to cleave structured RNA targets. BMC Mol. Biol. 7: 19. FULL TEXT

Xu D, Kang H, Fisher M, Juliano RL. 2004. Strategies for inhibition of MDR1 gene expression. Mol. Pharmacol. 66: 268-275. FULL TEXT

Xu D, McCarty D, Fernandes A, et al. 2005. Delivery of MDR1 small interfering RNA by self-complementary recombinant adeno-associated virus vector. Mol. Ther. 11: 523-530. FULL TEXT

Session III

Ali MM, Nagatsugi F, Sasaki S, et al. 2006. Application of 2-amino-6-vinylpurine as an efficient agent for conjugation of oligonucleotides. Nucleosides Nucleotides Nucleic Acids 25: 159-169.

Cohen JC, Boerwinkle E, Mosley TH Jr., et al. 2006. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N. Engl. J. Med. 354: 1264-1272. FULL TEXT

Geisbert TW, Hensley LE, Kagan E, et al. 2006. Postexposure protection of guinea pigs against a lethal ebola virus challenge is conferred by RNA interference. J. Infect. Dis. 193: 1650-1657.

Heidel JD. 2006. Linear cyclodextrin-containing polymers and their use as delivery agents. Expert Opin. Drug Deliv. 3: 641-646.

Hoyer D, Thakker DR, Natt F, et al. 2006. Global down-regulation of gene expression in the brain using RNA interference, with emphasis on monoamine transporters and GPCRs: implications for target characterization in psychiatric and neurological disorders. J. Recept. Signal Transduct. Res. 26: 527-547.

Hu-Lieskovan S, Heidel JD, Bartlett DW, et al. 2005. Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing's sarcoma. Cancer Res. 65: 8984-8992. FULL TEXT

Judge AD, Bola G, Lee AC, et al. 2006. Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo. Mol. Ther. 13: 494-505.

Judge A, McClintock K, Phelps JR, et al. 2006. Hypersensitivity and loss of disease site targeting caused by antibody responses to PEGylated liposomes. Mol. Ther. 13: 328-337.

Krutzfeldt J, Poy MN, Stoffel M. 2006. Strategies to determine the biological function of microRNAs. Nat. Genet. 38 Suppl: S14-19.

Krutzfeldt J, Rajewsky N, Braich R, et al. 2005. Silencing of microRNAs in vivo with 'antagomirs'. Nature 438: 685-689.

Krutzfeldt J, Stoffel M. 2006. MicroRNAs: a new class of regulatory genes affecting metabolism. Cell Metab. 4: 9-12.

Kwon BS, Jung HS, Song MS, et al. 2005. Specific regression of human cancer cells by ribozyme-mediated targeted replacement of tumor-specific transcript. Mol. Ther. 12: 824-834.

Mishra S, Heidel JD, Webster P, Davis ME. 2006. Imidazole groups on a linear, cyclodextrin-containing polycation produce enhanced gene delivery via multiple processes. J. Control Release Jun 27.

Morrissey DV, Lockridge JA, Shaw L, et al. 2006. Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nat. Biotechnol. 23: 1002-1007.

Sasaki S, Nagatsugi F. 2006. Application of unnatural oligonucleotides to chemical modification of gene expression. Curr. Opin. Chem. Biol. [Epub ahead of print]

Senn, JJ, Burel S, Henry SP. 2005. Non-CpG-containing antisense 2′-methoxyethyl oligonucleotides activate a proinflammatory response independent of Toll-like receptor 9 or myeloid differentiation factor 88. J. Pharmacol. Exp. Ther. 314: 972-979. FULL TEXT

Soutschek J, Akinc A, Bramlage B, et al. 2004. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432: 173-178.

Thakker DR, Hoyer D, Cryan JF. 2006. Interfering with the brain: use of RNA interference for understanding the pathophysiology of psychiatric and neurological disorders. Pharmacol. Ther. 109: 413-438.

Zimmermann TS, Lee AC, Akinc A, et al. 2006. RNAi-mediated gene silencing in non-human primates. Nature 441: 111-114.

Session IV

Colonna M. 2006. Toll-like receptors and IFN-α: partners in autoimmunity. J. Clin Invest. 116: 2319-2322. FULL TEXT

Hornung V, Ellegast J, Kim S, et al. 2006. 5′-triphosphate RNA is the ligand for RIG-I. Science 314: 994-997.

Ishii KJ, Akira S. 2005. TLR ignores methylated RNA? Immunity 23: 111-113.

Ishii KJ, Coban C, Kato H. 2006. A Toll-like receptor-independent antiviral response induced by double-stranded B-form DNA. Nat. Immunol. 7: 40-48.

Ishii KJ, Uematsu S, Akira S. 2006. 'Toll' gates for future immunotherapy. Curr. Pharm. Des. 12: 4135-4142.

Kariko K, Buckstein M, Ni H, et al. 2005. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23: 165-175.

Kariko K, Weissman D, Welsh FA. 2004. Inhibition of oll-like receptor and cytokine signaling—a unifying theme in ischemic tolerance. J. Cereb. Blood Flow Metab. 24: 1288-1304.

Lafyatis R, York M, Marshak-Rothstein A. 2006. Antimalarial agents: closing the gate on Toll-like receptors? Arthritis Rheum. 54: 3068-3070.

Marshak-Rothstein A. 2006. Tolling for autoimmunity-prime time for 7. Immunity 25: 397-399.

Marshak-Rothstein A. 2006. Toll-like receptors in systemic autoimmune disease. Nat. Rev. Immunol. 6: 823-835.

Schlee M, Hornung V, Hartmann G. 2006. siRNA and isRNA: two edges of one sword. Mol. Ther. 14: 463-470.

Smyth DJ, Cooper JD, Bailey R, et al. 2006. A genome-wide association study of nonsynonymous SNPs identifies a type 1 diabetes locus in the interferon-induced helicase (IFIH1) region. Nat. Genet. 38: 617-619.

Session V

Alter J, Lou F, Rabinowitz A, et al. 2006. Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology. Nat. Med. 12: 175-177.

Jackson DA, Juranek S, Lipps HJ. 2006. Designing nonviral vectors for efficient gene transfer and long-term gene expression. Mol. Ther. 14: 613-626.

Kim K, Liu F. 2004. In vitro selection of RNase P ribozymes that efficiently cleave a target mRNA. Methods Mol. Biol. 252: 399-412.

Manzini S, Vargiolu A, Stehle IM, et al. 2006. Genetically modified pigs produced with a nonviral episomal vector. Proc. Natl. Acad. Sci. USA 103: 17672-17677. FULL TEXT

Roberts J, Palma E, Sazani P, et al. 2006. Efficient and persistent splice switching by systemically delivered LNA oligonucleotides in mice. Mol. Ther. 14: 471-475.

Sazani P, Kole R. 2003. Therapeutic potential of antisense oligonucleotides as modulators of alternative splicing. J. Clin. Invest. 112: 481-486. FULL TEXT

Sazani PL, Larralde R, Szostak JW. 2004. A small aptamer with strong and specific recognition of the triphosphate of ATP. J. Am. Chem. Soc. 126: 8370-8371.

Shahid KA, Majumdar A, Alam R, et al. 2006. Targeted cross-linking of the human beta-globin gene in living cells mediated by a triple helix forming oligonucleotide. Biochemistry 45: 1970-1978.

Ting R, Thomas JM, Lermer L, et al. 2004. Substrate specificity and kinetic framework of a DNAzyme with an expanded chemical repertoire: a putative RNaseA mimic that catalyzes RNA hydrolysis independent of a divalent metal cation. Nucleic Acids Res. 32: 6660-6672. FULL TEXT

Trang P, Kim K, Liu F. 2004. Developing RNase P ribozymes for gene-targeting and antiviral therapy. Cell Microbiol. 6: 499-508.

Clinical Briefings (Industrial Satellite Meeting)

Bedikian AY, Millward M, Pehamberger H, et al. 2006. Bcl-2 antisense (oblimersen sodium) plus dacarbazine in patients with advanced melanoma: the Oblimersen Melanoma Study Group. J. Clin. Oncol. 24: 4738-4745.

DeVincenzo JP, Alvarez R, Tripp R, et al. 2006. Development of an antiviral for respiratory syncytial virus (RSV) utilizing RNA interference (RNAi). Euro. Acad. of Pediatrics, October 7-10, 2006, Barcelona, Spain.

Fortin M, Ferrari N, Higgins ME, et al. 2006. Effects of antisense oligodeoxynucleotides targeting CCR3 on the airway response to antigen in rats. Oligonucleotides 16: 203-212.

Higgins D, Rodriguez R, Milley R, et al. 2006. Modulation of immunogenicity and allergenicity by controlling the number of immunostimulatory oligonucleotides linked to Amb a 1. J. Allergy Clin. Immunol. 118: 504-510. Epub 2006 Jun 27.

Kastelein JJ, Wedel MK, Baker BF, et al. 2006. Potent reduction of apolipoprotein B and low-density lipoprotein cholesterol by short-term administration of an antisense inhibitor of apolipoprotein B. Circulation 114: 1729-1735.

Krieg AM. 2006. Therapeutic potential of Toll-like receptor 9 activation. Nat. Rev. Drug Discov. 5: 471-484.

Mahadevan B, Arora V, Schild LJ, et al. 2006. Reduction in tamoxifen-induced CYP3A2 expression and DNA adducts using antisense technology. Mol. Carcinog. 45: 118-125.

Sponsorship

Gold Sponsor

Silver Sponsors

Alnylam Pharmaceuticals

Coley Pharmaceutical Group

Isis Pharmaceuticals

Pfizer

Bronze Sponsors

Acuity Pharmaceuticals

Agilent Technologies - Boulder

Avecia

Biosearch Technologies, Inc.

Calando Pharmaceuticals

Dainippon Sumitomo Pharma Co., Ltd.

Dharmacon

Ercole Biotechnologies, Inc.

Gene Tools

Glen Research

Integrated DNA Technologies

Invitrogen

Koken Co., Ltd.

Merck Research Laboratories

Nature Publishing Group

Novartis

Oligonucleotide Therapeutics Society (OTS)

OSI Eyetech

Pachyonychia Congenita Project

Qiagen

RNAi.net

Rosetta Inpharmatics/Merck Research Laboratories

Sanofi Aventis

Sirna Therapeutics

Topigen Pharmaceuticals

U.S. Army

The field of oligonucleotide therapeutics has transitioned rapidly from bench to clinic. Several companies are developing therapies for unmet medical needs such as diabetes, advanced macular degeneration, and asthma. Some compounds are in Phase I and II safety trials, while others have moved into large-scale Phase III efficacy trials.

Antisense oligonucleotides

Isis Pharmaceuticals: Treating high cholesterol and type 2 diabetes

Isis Pharmaceuticals has two antisense oligonucleotide (ASO) compounds in Phase II clinical trials, reported Sanjay Bhanot. ISIS 301012 is being developed for hyperlipidemia (high cholesterol) and ISIS 113715 is a treatment for type 2 diabetes. As with most ASO therapies, these compounds work via enzyme-mediated destruction of the target mRNA. Each binds to its complementary mRNA sequence to form a DNA/mRNA duplex that is then cleaved by an endogenous enzyme, RNase-H.

To improve the pharmacological properties of these ASOs, investigators have introduced modifications to the phosphodiester DNA backbone. The first is a sulfur-substitution that converts the phosphodiester to a phosphorothioate. Another is the replacement of some of the nucleotides with 2′-O-(2-methoxyethyl)-modified oligonucleotides, known as 2′-O-MOEs, spaced with phosphorothioate gaps between the MOEs. The resulting "MOE-gapmers" retain the ability to induce cleavage by RNase-H but have longer retention time in the body. The result, says Bhanot, is "a simple saline injection that can be given as infrequently as once a week to once a month."

The first compound, ISIS 301012, reduces cholesterol in blood by inhibiting apoB-100, a protein involved in the synthesis and transport of low-density lipoprotein (LDL), the so-called "bad" cholesterol implicated in heart disease. A single agent Phase IIa study with 10 patients receiving injections every other week resulted in a 42% decline of LDL cholesterol and a 46% reduction of triglycerides at a dose of 200 mg/wk for 3 months. In another project addressing type 2 diabetes, ISIS 113715 inhibits PTP-1B, a protein that Bhanot called the "brake on insulin signaling." In preclinical studies, PTP-1B inhibition reduced blood glucose levels and boosted insulin sensitivity. Previous data from the Phase I trial indicate that the drug was well tolerated. In an ongoing single agent phase IIa trial in mildly diabetic patients, ISIS 113715 produced robust reduction in fasting and post-prandial glucose levels and was well tolerated.

Topigen Pharmaceuticals: ASM8 for the Therapy of Asthma

Topigen Pharmaceuticals is developing an ASO treatment for asthma, Paolo M. Renzi reported. The compound, TPI-ASM8, works by blocking multiple gene pathways involved in immune cell recruitment and chronic inflammation. TPI-ASM8 is a combination of two gene-silencing oligonucleotides, a 19-mer and a 21-mer, that inhibit two cytokines involved in inflammatory cell influx and airway hyper-responsiveness. In a Phase II study of the drug delivered via inhaler, TPI-ASM8 was able to stem the influx of eosinophils, neutrophils, and other inflammatory cells into the lungs and inhibit target gene expression. It is the first study in humans to demonstrate the application of an RNA-targeting drug in lung disease.

Genta Pharmaceuticals: Genasense for patients with melanoma and chronic lymphocytic leukemia

Genta Pharmaceuticals's Loretta M. Itri gave an update on Genasense (oblimersen sodium), an 18-mer phosphorothioate antisense oligodeoxynucleotide designed to inactivate BCL-2, a gene implicated in a number of cancers. This therapy could be combined with conventional chemotherapeutics such as dacarbazine (DTIC) to treat patients with melanoma and chronic lymphocytic leukemia (CLL). In Phase III trials of BCL-2 antisense combined with the chemotherapeutic drug fludarabine/cyclophosphamide for CLL and melanoma, the researchers found that the combination therapy enhanced patients' duration of survival, especially among individuals who enter the study with nonelevated levels of lactate dehydrogenase (LDH), a marker of tissue breakdown.

AVI BioPharma: Liver enzymes as an antisense target

Can we change how the liver metabolizes drugs? That is the question that Patrick Iversen (AVI BioPharma) wants to answer by using oligonucleotides to inhibit the liver enzymes responsible for breaking down drugs. When it comes to drugs, most people are either fast or slow metabolizers. Fast metabolizers have liver enzymes that break down a drug so quickly that it doesn't have much time to work. An oligotherapeutic that inhibits specific liver enzymes could prolong the activity of a drug in fast metabolizers.

AVI BioPharma has developed oligotherapeutics called phosphorodiamidate morpholino oligomers (PMOs), so-called because of the addition of a six member sugar and a neutral backbone. AVI-4557 is a PMO that inhibits expression of the liver enzyme cytochrome P-450 3A4 (CYP3A4), which metabolizes 50% of all prescription drugs. In human Phase I trials, AVI-4557 effectively inhibited expression of CYP3A4 following intravenous and oral administration.

siRNA

Sirna Therapeutics: A treatment for age-related macular degeneration (AMD)

A leading cause of blindness in people age 60, AMD is caused by neovascularization in the macula, a specialized area of the retina, resulting in a black and blurry obstruction in the center of the visual field. Sirna-027 blocks production of vascular endothelial growth factor receptor 1 (VEGFR1), a protein that fosters formation of new blood vessels in the eye, reported Roberto Guerciolini.

Visual acuity stabilized or improved in 92% of treated patients at 12 weeks, with no evidence that the intravitreal injections resulted in systemic exposure. No serious adverse events or dose limiting toxicities were reported. The drug's performance compares favorably with products on the market but could require fewer intra-eye injections, which can cause discomfort. The trial is the first human experience of a chemically-modified siRNA.

Alnylam Pharmaceuticals: ALN-RSV01 for respiratory syncytial virus

Akshay K. Vaishnaw of Alnylam Pharmaceuticals reported on the promising findings of a Phase I study of the siRNA therapeutic ALN-RSV01 as a treatment for respiratory syncytial virus (RSV) infection. RSV causes respiratory infection in young children and results in 120,000 hospitalizations per year in the United States alone. In the Phase I study of intranasal administration in adult male volunteers, ALN-RSV01 appeared safe and did not appear to result in systemic exposure.

Immunostimulatory Therapeutics

Coley Pharmaceutical Group: Treatments for hepatitis C and non-small cell lung cancer

Coley Pharmaceutical Group is developing a number of CpG-based therapies, said Arthur M. Krieg, including CpG 10101 (ACTILON) for chronic hepatitis C and PF-3512676 for non-small cell lung cancer. CPG 10101 is in Phase Ib clinical trials and early results show that the compound in combination with the antiviral drug ribavirin plus pegylated interferon provides a greater decrease in HCV RNA than does ribavirin and pegylated interferon alone.

Another CpG therapeutic targets non-small cell lung cancer (NSCLC). In collaboration with Pfizer, PF 3512676 (formerly CPG 7909) is now being tested in two Phase III trials with and without chemotherapy. Investigators found an 80% higher median survival time in patients randomized to PF-3512676 plus chemotherapy versus chemotherapy alone.

Dynavax: Enhancing vaccines with immunostimulatory oligonucleotides

Harnessing the power of the oligonucleotide-stimulated immune response could yield improved vaccines. Gary Van Nest of Dynavax described two 20-mer unmethylated, CpG-containing oligonucleotides that can enhance vaccine efficacy. In one example, the immunostimulatory oligonucleotide is mixed with vaccine antigens to create the HEPLISAV vaccine for hepatitis B. In a small human trial, HEPLISAV yielded a faster response, greater seroprotection and greater duration of antibody response with fewer doses.

The second compound, TOLAMBA, features an immunostimulatory oligonucleotide linked directly to a ragweed allergen known as Amb a 1. This combination facilitates dendritic cell uptake, promotes antigen presentation, stimulates Th2 to Th1 shift in ragweed-allergic patients, and essentially transforms the allergen into drug, said Van Nest. Dynavax is now working on applying this technology to the influenza virus, and is also working on advanced formulations of ISS mixed with vaccine antigens for the treatment of anthrax.

Are other RNAs being regulated in a similar way to Malat-1? Is there a class of RNAs kept in the nucleus ready for rapid response?

Is Bruce Sullenger's aptamer–siRNA delivery system generalizable to other siRNAs? Why does it work so well, and what governs how aptamers bind receptors?

Are MOE gapmers active in the liver and other tissues?

Will PCSK9 siRNA prove to be a viable therapeutic for the prevention of high cholesterol or heart disease?

What is the biodistribution of nanoparticle-encapsulated siRNA in mice?

Can siRNA molecules and targeting ligands be further optimized to improve the effect?

Are modified RNAs such as synthetic 5′ triphosphate RNA stable in cerebral spinal fluid? Are there other ways to inject them or administer them?

Why is RNA expression better without modification? Is it related to immune stimulation?

Does signaling involving the 5′ triphosphate RNA ligand/RIG-I system work in nonmammalian systems such as plants?

Could an S/MAR vector be used to make transgenic mice?

How can the number of S/MAR vectors that can be incorporated into a cell be increased?

How can we further optimize the ability of anti-HCMV ribozymes to block infection?

Can the R27 ribozyme block viral infection or reduce virulence of HCMV in humans?

Can LNA SSOs be administered orally?

Can LNA SSO human versions be made?

New perspectives on an old phenomenon

Early in the development of oligonucleotide therapeutics, researchers noticed that the compounds sometimes had immunostimulatory effects, causing an increase in the production of cytokines and other cell-signaling molecules. These immune responses probably developed to defend the cell against foreign viral DNA. While viewed as an undesirable side effect by some, other researchers are harnessing these immunostimulatory properties for therapeutics. Many aspects of the basic biology behind these effects remain to be explained, and several new discoveries form the basis of the talks below.

Researchers are harnessing previously undesirable effects for therapeutics.

So far, researchers have discovered that oligonucleotides stimulate immune reactions through two main pathways, the Toll-like receptors (TLRs) found in immune cells, and cytoplasm-based receptors MDA5 and RIG-I found in all cell types.

The Toll-like receptors are located in the endosomal membrane, and four have been identified. When a bit of foreign DNA or RNA enters the nucleus, enzymes degrade it and free the oligonucleotide strand, which can then be recognized by TLRs. TLR-3 recognizes double-stranded RNA, TLR-7 recognizes single-stranded or short double-stranded RNA, TLR-8 recognizes single-stranded RNA, and TLR-9 recognizes single-stranded DNA such as CpG DNA.

The second system is composed of two receptors, known as RIG-I and MDA5, located in the cytosol. Both recognize poly I:C, a synthetic double-stranded RNA, and researchers have recently discovered that RIG-I also recognizes single-stranded RNA if it contains additional modifications. This system is also capable of DNA recognition, although the DNA receptor has not been identified.

This session featured a number of new discoveries, including the revelation of the long-sought ligand for the RIG-I receptor, insights into how nucleoside modifications can reduce immunostimulatory effects without destroying the translatability of RNA, and how TLRs play a role in self-recognition of nucleic acids.

TLR-dependent and -independent mechanisms in host response to RNA viruses

When a virus enters the body, immune defenses get to work. One such response occurs in immune cells known as plasmacytoid dendritic cells (pDCs), a subset of dendritic cells from bone marrow that enter the blood. Viruses trigger the secretion of IFN-α (interferon-α) and IFN-β, which prime both innate and adaptive immune responses. pDCs sense viruses through TLRs -7 and -9. Classical dendritic cells also utilize TLR-7 and TLR-9 to detect viruses and induce proinflammatory cytokines, but oddly, these two receptors are not involved in the classical DC's induction of IFN-α. "This suggested to us that classical DCs can sense viruses and secrete IFN-α independently of TLR-7 and -9," said Marco Colonna (Washington University School of Medicine).

After testing a few possibilities, Colonna and his team turned their attention to the cytosolic double-stranded RNA receptors RIG-I and MDA5. These receptors bind to RNA, unwind it, and initiate production of IFN-α/β.

Turning first to MDA5, the team generated MDA5 knockout mice. They found that the MDA5-deficient dendritic cells could not produce IFN-α or certain other cytokines and were more susceptible to viral infection. These experiments led them to conclude, "MDA5 is a major receptor for double-stranded RNA," said Colonna.

When double-stranded RNA (poly I:C) is low, the cytosolic MDA5 receptor plays a major role in detection. When poly I:C is high, TLR-3 plays the more important role.

The team also explored the role of TLR-3, which detects double-stranded RNA but is only expressed in certain DC subsets and macrophages. Colonna's findings suggest that when double-stranded RNA (poly I:C) is low, the cytosolic MDA5 receptor plays a major role in detection, whereas when poly I:C is high, TLR-3 plays the more important role. Thus, pDC use the endosomal TLR-7/-9 pathway while classical DC use the cytosolic (MDA5) pathway and the TLR-3 pathway.

These discoveries may help researchers develop therapeutics for the treatment of autoimmune diseases such as systemic lupus erythematosus (SLE) and type 1 diabetes.

Nucleoside modification in RNA recognition

Researchers may be able to skirt immunogenicity by making nucleoside modifications to oligonucleotides. Katalin Karikó (University of Pennsylvania School of Medicine) and colleagues have created nucleoside-modified RNAs that are less immunogenic in dendritic cells (DCs).

The work began when the researchers noted that naturally produced mRNAs are not as immunogenic as synthetic ones. "We were surprised to find that natural RNAs had different levels of induction," said Karikó. "One thing came to mind: If we modified the in vitro transcribed RNA, would it become less immunogenic?"

Modification of the RNA can be done in numerous ways, but Karikó singled out three common modifications to see if these reduced the immunogencity of mRNA. These were 2′-O-methylation, RNA base methylation, and pseudouridylation (ψ), where an altered uridine is substituted into the RNA.

The researchers made all three modifications and tested them for reduction of the cytokines TNF-α and IL-12. The pseudouridylation worked best.

The investigators noted that the ψ-containing RNAs did not activate primary human DCs (so they were not immunogenic) while remaining translatable into proteins. When injected into mice intravenously at 1 and 3 µg RNA (0.05 and 0.15 mg/kg), the ψ-containing RNAs accumulate and translate in the spleen, and do not induce TNF-α or IFN-γ. When delivered to the lungs, the ψ-modified RNA translate and do not induce TNF-α, whereas unmodified RNAs do.

Deciphering the RNA ligand for RIG-I

As stated above, oligonucleotide immunogenicity is activated by both the TLR system and the cytosolic receptors MDA5 and RIG-I. It's long been clear that Poly I:C is the ligand for MDA5, but for years researchers have not known the ligand for RIG-I.

According to Veit Hornung (University of Munich, Germany), it appears this mystery is at least partially solved. In a paper published in Science the same week as the conference, Hornung and his colleagues describe the discovery of a ligand, 5′ triphosphate RNA, that binds to RIG-I.

The discovery began when the researchers observed that in vitro-transcribed single-stranded RNA (ssRNA) was very immunogenic in monocytes whereas synthetic ssRNA was not. However, in plasmacytoid dendritic cells (pDCs), both in vitro-transcribed and synthetic ssRNA were immunogenic.

This led the investigators to wonder, what is the difference between naturally occurring (in vitro-transcribed) ssRNAs and synthetic ssRNAs? The sequence is the same, said Hornung, but the difference is found at the 5′ end. In vitro-transcribed RNA is triphosphorylated at the 5′ end, but synthetic RNA, usually purchased from a company, is not.

The difference between naturally occurring ssRNAs and synthetic ssRNAs is in the triphosphorylation in the 5′ end of the naturally occurring ssRNA.

Investigating this discrepancy, they removed the triphosphate at the 5′ end from the in vitro-transcribed ssRNA and tested to see if it still triggered the immune response in the pDC and monocytes. The result was surprising: Without the 5′ triphosphate, the naturally occurring ssRNA induced an immune response in monocytes, mediated through the cytosolic receptors, but not in pDCs, which work via TLR-7. This led Hornung and his colleagues to conclude that "the 5′ triphosphate end is the immunogenic part."

Yet, if the 5′ triphosphorylated ssRNA is so immunogenic, how does the immune system ignore self-produced ssRNAs and only react to non-self ssRNAs, such as those produced by viruses? The cell has developed coping strategies to discriminate self from non-self ssRNAs, such as converting the triphosphate of a self-ssRNA to a monophosphate and capping the 5′ end with other substrates. The investigators were able to repeat these strategies and completely eliminate immunogenicity of the ssRNAs in both monocytes and pDCs. Hornung found that the body avoids mounting an immune response against its own 5′ triphosphate ssRNA by producing "common post-transcriptional modifications [that] hamper the recognition of 5′ triphosphate RNA oligonucleotides."

What receptors are involved in this immunogenicity? Using knockout experiments, the researchers ruled out the possibility that Toll-like receptors are involved. They then turned to MDA5 and RIG-I. By transfecting 5′ triphosphate ssRNA into cells, they saw complete loss of activity in RIG-I knockout mice. In short, mice that lacked RIG-I did not respond immunogenically to 5′-triphosphate ssRNA.

RIG-I recognition is a natural response to viral infection.

Hornung's team wondered whether RIG-I recognition of viruses could be a physiological mechanism by which the cells detect and respond to viruses, or if it was just a laboratory curiosity. They later found that rabies virus ssRNA elicits an immune response only if it retains the triphosphate on its 5′ end. In other words, RIG-I recognition is a natural response to viral infection.

They also discovered that RIG-I binds directly to the 5′ triphosphate RNA, and that this induces signal transduction. Or as Hornung explained, "5′ triphosphate RNA is the long-sought ligand for RIG-I." Common postranscriptional modifications reduce the immunogenicity of 5′ triphosphate RNA.

The investigators also found that viral replication was not needed for the RNA viruses to be recognized. "This is very important because this is a big dogma out there that you need replication of the virus it to be recognized," said Hornung. "At least for this protein and for this route of activation it is not the case."

What is more, the research sheds light on how the cell distinguishes self-made RNA from viral RNA. "Recognition of foreign RNA depends on a defined molecular structure and this is the 5′ triphosphorylated RNA," said Hornung. "There are few, if any, structures out there that can explain the non-self detection process as we can show here with the RIG-I protein."

More progress on immunomodulating agents

A number of additional presentations addressed the growing knowledge of immunostimulatory effects and how they can be harnessed as therapeutics.

• Ken Ishii discussed TLR-independent mechanisms for recognizing immunostimulatory RNA and DNA.
• Ann Marshak-Rothstein described DNA- and RNA-associated autoantigens that activate autoreactive B cells through a mechanism that depends on TLR-9 and/or TLR-7.
• Tao Lan presented novel RNAs that are potent agonists of TLR-8, or both TLR-7 and -8.
• Hendrick Poeck discussed the role of isRNA and siRNA in immunotherapy.
• Montserrat Puig described new targets for TLR-9 called thermolytic oligodeoxynucleotides.

The four-letter genetic code may seem simple, but it contains the potential for the creation of new therapeutics to fight cancer, viral infections, and other disorders that have resisted traditional pharmaceutical approaches. Scientists now know that novel drugs made from short nucleic acid segments, or oligonucleotides, can target DNA and RNA to inactivate genes involved in causing disease.

Discoveries about antisense activity and RNA interference have fueled the excitement that study of oligonucleotides will yield much-needed drugs.

This promising field has come a long way since oligonucleotides were first discovered in the late 1970s to be capable of inhibiting the expression of specific genes. These single strands of nucleic acids can bind complementarily to disease-related messenger RNA (mRNA), thereby inactivating the mRNA and preventing production of proteins associated with disease. Chemical modifications have improved the stability of these "antisense" oligonucleotide drugs, and one antisense drug, Isis Pharmaceuticals's Vitravene, was approved in 1998 for viral eye infections.

Some of these oligonucleotides have the ability to stimulate the immune system by interacting with Toll-like receptors localized to the cell surface or endosome of immune cells, and another receptor system located in the cytosol of all cell types. Researchers are actively investigating whether these immunostimulatory effects could be employed against cancer, viral diseases, or as immune system boosters for vaccines or chemotherapeutics.

Perhaps the most important discovery to affect the development of oligonucleotide therapeutics has been that of RNA interference (RNAi), a mechanism by which RNA inhibits gene expression. RNA interference refers to naturally occurring gene silencing mechanisms, including one that involves small 21- to 23-nucleotide double-stranded RNAs called short interfering RNAs (siRNA), which are capable of knocking down specific gene expression to a few percent of its original level.

One sign of the potential scientists see for RNA interference was the awarding of the 2006 Nobel Prize in Physiology or Medicine to two co-discoverers of the phenomenon, Andrew Fire and Craig Mello. Therapeutics based on siRNA are also being developed as treatments for age-related macular degeneration, respiratory syncytial virus infection, hepatitis C, and HIV infections. Delivery of siRNAs to their targets is an active area of research as well.

All of these developments have fueled the excitement that this promising field will soon yield much-needed new drugs. A greater understanding of how oligonucleotides cause immune responses is helping researchers design therapeutics that either avoid immunogenic effects or take maximum advantage of them. "It is an exciting time to be working in the field of oligonucleotides," said John J. Rossi, chairman of the Division of Molecular Biology at the Beckman Research Institute of City of Hope and a conference organizer.

The Second Annual Meeting of the Oligonucleotide Therapeutics Society, held October 19–21, 2006 at the Rockefeller University, presented an opportunity for the exchange of ideas among molecular biologists involved in designing oligonucleotide drugs, chemists who create and optimize the compounds for maximum efficacy, pharmacologists who study delivery and metabolism, and physicians who bring the compounds to the clinic. The conference was cosponsored by the New York Academy of Sciences and the Oligonucleotide Therapeutics Society (OTS), a nonprofit forum formed in 2003 to foster academic and industry-based research and development in the field. The First Annual Meeting of the Oligonucleotide Therapeutics Society, which was also cosponsored by the New York Academy of Sciences, was held in September 2005 and drew academic and industrial researchers from around the world.

Highlights of the 2006 conference included:

Cell biology and enzymology of mRNA metabolism and oligonucleotide function

• The paradigm "DNA makes RNA makes protein" is outdated since RNA has many roles in the cell.
• Short interfering RNAs (siRNAs) can compete with each other for ability to silence gene expression via the involvement of a protein called Argonaute2.
• Human Clp1 is the enzyme that facilitates the silencing ability of non-phosphorylated siRNAs.

Oligonucleotide synthesis and technologies

• Aptamer-siRNA chimeras targeted to prostate specific membrane antigen (PSMA) can reduce the growth of prostate tumors in mice.
• Arabinose-modified nucleic acids can increase the stability of oligonucleotides for both antisense and RNAi therapies.
• Novel siRNA-like constructs called sisiRNAs are strong and lasting, and have fewer off-target effects.

Progress in gene targeting oligonucleotide therapeutic development

• Oligonucleotide stability and delivery can be improved through novel chemical modifications, encapsulation in nanoparticles, and chemical crosslinking.
• SiRNA formulated in stabilized nucleic acid-lipid particles called SNALPs can prevent guinea pigs exposed to Ebola virus from developing hemorrhagic fever.
• Novel modified antisense oligonucleotides just 12 nucleotides in length can significantly lower glucose levels in a mouse model of type 2 diabetes.

Progress of immunomodulating oligonucleotide therapeutic development

• Antiviral responses include both Toll-like receptor- and RIG-I and MDA5-dependent mechanisms.
• The ligand for the RIG-I receptor was discovered this year to be a 5′ triphosphate-single-stranded RNA.
• Nucleoside modification of RNA can decrease recognition by Toll-like receptors, permitting therapeutic siRNA to avoid causing an immune response.

New and alternate gene targeting approaches

• Non-viral episomal vectors have been developed that are safer than current viral-based vectors. They can be used to transfer genes into sperm of large farm animals.
• Catalytic RNAs known as ribozymes can inactivate viruses such as human cytomegalovirus (HCMV) and were shown to block viral infection in immune-compromised mice.
• Oligonucleotide-based therapies can repair mutated genes that cause disorders such as sickle cell disease and Duchenne muscular dystrophy.

Clinical reports from ongoing human trials

Oligonucleotide-based treatments are in development for treatment of asthma, respiratory syncytial virus, type 2 diabetes, age-related macular degeneration and more.

• Isis Pharmaceuticals is conducting Phase II trials of two antisense compounds, one a treatment for hypercholesterolemia and the other for type 2 diabetes.
• Sirna Therapeutics is testing an siRNA therapeutic for the treatment of age-related macular degeneration (AMD) in a Phase I trial.
• Coley Pharmaceutical Group is testing the safety and tolerability of CpG therapeutics for viral diseases such as hepatitis C.
• Topigen Pharmaceuticals is evaluating ASM8, an antisense therapeutic delivered via nebulization to treat asthma, in a Phase II trial.
• Genta Pharmaceuticals is involved in an ongoing Phase III clinical trial of Genasense in combination with chemotherapy for the treatment of melanoma and chronic lymphocytic leukemia.
• Alnylam Pharmaceuticals has an ongoing Phase I trial of ALN-RSV01 siRNA to treat respiratory syncytial virus infection.
• AVI BioPharma is exploring compounds that can inactivate the liver enzymes responsible for metabolizing pharmaceuticals as a strategy for keeping these drugs active for longer.

The conference brought together key participants in the rapid transition of a benchside discovery into a viable therapeutic platform. Several advances in our understanding of the immunological effects of oligonucleotides are transforming the potential for use of these compounds as drugs. Enhancements in antisense oligonucleotide stability and potency are bringing these therapies closer to clinical use than ever. And rapid progress in the field of RNA interference is presenting new opportunities for therapeutics that can dramatically improve patient survival and quality of life.