Fourth Annual Meeting of the Oligonucleotide Therapeutics Society

Web Sites and Books

2008 Albert Lasker Foundation Prize in Basic Biomedical Research
The Lasker Foundation gave its 2008 Award to Victor Ambros, Gary Ruvkun, and David Baulcombe "for discoveries that revealed an unanticipated world of tiny RNAs that regulate gene function in plants and animals."

2006 Nobel Prize in Physiology or Medicine
Andrew Fire and Craig Mellow were awarded the Nobel Prize for their discovery of RNA interference. This Web site contains an illustrated presentation, advanced information containing the relevant background, and more.

Nanomedicine – on Science Daily
Nanomedicine is the medical application of nanotechnology and related research. It covers areas such as nanoparticle drug delivery and possible future applications of molecular nanotechnology (MNT) and nanovaccinology.

Nanomedicine Research
A free newsletter on nanomedicine research.

NOVA Science Now RNAi
This Web site accompanied a TV episode that introduced RNAi to the layperson.

Oligonucleotide Therapeutics Society
OTS is an open, non-profit forum to foster academia and industry-based research and development of oligonucleotide therapeutics (RNAi, CpG, antisense, and others).

PicTar
PicTar is an algorithm for the identification of microRNA targets.

siRNA at Whitehead
Software to help researchers select siRNA sequence to knock down expression of their gene of interest.

TargetScan
TargetScanS predicts biological targets of miRNAs by searching for the presence of conserved 8mer and 7mer sites that match the seed region of each miRNA.

Crooke ST, ed. 2008. Antisense Drug Technology: Principles, Strategies, and Applications, Second Edition. CRC Press, Boca Raton, FL.

Journal Articles

The Basics of MicroRNAs and the Targets They Regulate

Baek D, Villén J, Shin C, et al. 2008. The impact of microRNAs on protein output. Nature 455: 64-71.

Friedman RC, Farh KK, Burge CB, Bartel DP. 2008. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. Dec 2. [Epub ahead of print]

Lewis BP, Burge CB, Bartel DP. 2005. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120: 15-20.

Antisense and siRNA Development and Delivery

Chen Y, Huang L. 2008. Tumor-targeted delivery of siRNA by non-viral vector: safe and effective cancer therapy. Expert Opin. Drug Deliv. 5: 1301-1311.

Frank-Kamenetsky M, Grefhorst A, Anderson NN, et al. 2008. Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates. Proc. Natl. Acad. Sci. USA 105: 11915-11920. Full Text

Guimond A, Viau E, Aubé P, et al. 2008. Advantageous toxicity profile of inhaled antisense oligonucleotides following chronic dosing in non-human primates. Pulm. Pharmacol. Ther. 21: 845-854.

Li SD, Huang L. 2008. Targeted delivery of siRNA by nonviral vectors: Lessons learned from recent advances. Curr. Opin. Investig. Drugs 9: 1317-1323.

Meade BR, Dowdy SF. 2008. Enhancing the cellular uptake of siRNA duplexes following noncovalent packaging with protein transduction domain peptides. Adv. Drug Deliv. Rev. 60: 530-536.

Meade BR, Dowdy SF. 2007. Exogenous siRNA delivery using peptide transduction domains/cell penetrating peptides. Adv. Drug Deliv. Rev. 59: 134-140.

Oligonucleotide Therapeutics in the Clinic

Chi KN, Zoubeidi A, Gleave ME. 2008. Custirsen (OGX-011): a second-generation antisense inhibitor of clusterin for the treatment of cancer. Expert Opin. Investig. Drugs 17: 1955-1962.

Heidel JD, Liu JY, Yen Y, et al. 2007. Potent siRNA inhibitors of ribonucleotide reductase subunit RRM2 reduce cell proliferation in vitro and in vivo. Clin. Cancer Res. 13: 2207-2215.

Heidel JD, Yu Z, Liu JY, et al. 2007. Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNA. Proc. Natl. Acad. Sci. USA 104: 5715-5721.

Yu RZ, Lemonidis KM, Graham MJ, et al. 2008. Cross-species comparison of in vivo PK/PD relationships for second-generation antisense oligonucleotides targeting apolipoprotein B-100. Biochem. Pharmacol. Nov 14. [Epub ahead of print]

Oligonucleotide Immunoreactives

Agrawal S, Kandimalla ER. 2007. Synthetic agonists of Toll-like receptors 7, 8 and 9. Biochem. Soc. Trans. 35: 1461-1467.

Robbins M, Judge A, Ambegia E, et al. 2008. Misinterpreting the therapeutic effects of siRNA caused by immune stimulation. Hum. Gene Ther. Aug 19. [Epub ahead of print]

Robbins M, Judge A, Liang L, et al. 2007. 2′-O-methyl-modified RNAs act as TLR7 antagonists. Mol. Ther. 15: 1663-1669.

Redford TW, Yi AK, Ward CT, Krieg AM. 1998. Cyclosporin A enhances IL-12 production by CpG motifs in bacterial DNA and synthetic oligodeoxynucleotides. J. Immunol. 161: 3930-3935. Full Text

Additional Resources

Session I

Ebert BL, Pretz J, Bosco J, et al. 2008. Identification of RPS14 as a 5q- syndrome gene by RNA interference screen. Nature 451: 335-339.

Haasnoot J, Berkhout B. 2009. Nucleic acids-based therapeutics in the battle against pathogenic viruses. Handb. Exp. Pharmacol. 189: 243-263.

Rudnick SI, Swaminathan J, Sumaroka M, et al. 2008. Effects of local mRNA structure on posttranscriptional gene silencing. Proc. Natl. Acad. Sci. USA 105: 13787-13792.

Sandberg R, Neilson JR, Sarma A, et al. 2008. Proliferating cells express mRNAs with shortened 3′ untranslated regions and fewer microRNA target sites. Science 320: 1643-1647.

Ter Brake O, Legrand N, von Eije KJ, et al. 2008. Evaluation of safety and efficacy of RNAi against HIV-1 in the human immune system (Rag-2(-/-)(c)(-/-)) mouse model. Gene Ther. Jul. 31. [Epub ahead of print.]

Zhao H, Kalota A, Jin S, Gewirtz AM. 2008. The c-myb Protooncogene and microRNA (miR)-15a comprise an active autoregulatory feedback loop in human hematopoietic cells. Blood Sep 25. [Epub ahead of print]

Session II

Forsbach A, Nemorin JG, Montino C, et al. 2008. Identification of RNA sequence motifs stimulating sequence-specific TLR8-dependent immune responses. J. Immunol. 180: 3729-3738.

Georges SA, Biery MC, Kim SY, et al. 2008. Coordinated regulation of cell cycle transcripts by p53-Inducible microRNAs, miR-192 and miR-215. Cancer Res. 68: 10105-10112.

Jopling CL, Schutz S, Sarnow P. 2008. Position-dependent function for a tandem microRNA miR-122-binding site located in the hepatitis C virus RNA genome. Cell Host Microbe 4: 77-85.

Landthaler M, Gaidatzis D, Rothballer A, et al. 2008. Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs. RNA 14: 2580-2596.

Nilsen TW. 2008. Endo-siRNAs: yet another layer of complexity in RNA silencing. Nat. Struct. Mol. Biol. 15: 546-548.

Prakash TP, Kawasaki AM, Wancewicz EV, et al. 2008. Comparing in vitro and in vivo activity of 2′-O-[2-(methylamino)-2-oxoethyl]- and 2′-O-methoxyethyl-modified antisense oligonucleotides. J. Med. Chem. 51: 2766-2776.

Session III

Beane R, Gabillet S, Montaillier C, et al. 2008. Recognition of chromosomal DNA inside cells by locked nucleic acids. Biochemistry 47: 13147-13149.

Douthwaite S, Kirpekar F. 2007. Identifying modifications in RNA by MALDI mass spectrometry. Methods Enzymol. 425: 1-20.

Frieden M, Ørum H. 2008. Locked nucleic acid holds promise in the treatment of cancer. Curr. Pharm. Des. 14: 1138-1142.

Seth PP, Siwkowski A, Allerson CR, et al. 2008. Short antisense oligonucleotides with novel 2′-4′ conformationally restricted nucleoside analogues show improved potency without increased toxicity in animals. J. Med. Chem. Dec 16. [Epub ahead of print]

Session IV

Abes S, Ivanova GD, Abes R, et al. 2009. Peptide-based delivery of steric-block PNA oligonucleotides. Methods Mol. Biol. 480: 1-15.

Akinc A, Zumbuehl A, Goldberg M, et al. 2008. A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat. Biotechnol. 26: 561-569.

Fuller JE, Zugates GT, Ferreira LS, et al. 2008. Intracellular delivery of core-shell fluorescent silica nanoparticles. Biomaterials 29: 1526-1532.

Sepp-Lorenzino L, Ruddy M. 2008. Challenges and opportunities for local and systemic delivery of siRNA and antisense oligonucleotides. Clin. Pharmacol. Ther. 84: 628-632.

Xia W, Hilgenbrink AR, Matteson EL, et al. 2008. A functional folate receptor is induced during macrophage activation and can be used to target drugs to activated macrophages. Blood Oct 24. [Epub ahead of print]

Session V

Allam R, Pawar RD, Kulkami OP, et al. 2008. Viral 5′-triphosphate RNA and non-CpG DNA aggravate autoimmunity and lupus nephritis via distinct TLR-independent immune responses. Eur. J. Immunol. 38: 3487-3498.

Cooper CL, Ahluwalia NK, Efler SM, et al. 2008. Immunostimulatory effects of three classes of CpG oligodeoxynucleotides on PBMC from HCV chronic carriers. J. Immune Based Ther. Vaccines 6:3. Full Text

Gilliet M, Cao W, Liu YJ. 2008. Plasmacytoid dendritic cells: sensing nucleic acids in viral infection and autoimmune diseases. Nat. Rev. Immunol. 8: 594-606.

Tian J, Avalos AM, Mao SY, et al. 2007. Toll-like receptor 9-dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE. Nat. Immunol. 8: 487-496.

Session VI

DeVincenzo J, Cehelsky JE, Alvarez R, et al. 2008. Evaluation of the safety, tolerability and pharmacokinetics of ALN-RSV01, a novel RNAi antiviral therapeutic directed against respiratory syncytial virus (RSV). Antiviral Res. 77: 225-231.

Gilbert JC, DeFeo-Fraulini T, Hutabarat RM, et al. 2007. First-in-human evaluation of anti von Willebrand factor therapeutic aptamer ARC1779 in healthy volunteers. Circulation 116: 2678-2686. Full Text

Schwartz JC, Younger ST, Nguyen NB, et al. 2008. Antisense transcripts are targets for activating small RNAs. Nat. Struct. Mol. Biol. 15: 842-848.

Stenvang J, Lindow M, Kauppinen S. 2008. Targeting of microRNAs for therapeutics. Biochem. Soc. Trans. 36: 1197-1200.

Wu B, Moulton HM, Iversen PL, et al. 2008. Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer. Proc. Natl. Acad. Sci. USA 105: 14814-14819. Full Text

Yu XX, Murray SF, Watts L, et al. 2008. Reduction of JNK1 expression with antisense oligonucleotide improves adiposity in obese mice. Am. J. Physiol. Endocrinol. Metab. 295: E436-445.

Session VII

Chan MY, Rusconi CP, Alexander JH, et al. 2008. A randomized, repeat-dose, pharmacodynamic and safety study of an antidote-controlled factor IXa inhibitor. J. Thromb. Haemost. 6: 789-796.

Dollins CM, Nair S, Sullenger BA. 2008. Aptamers in immunotherapy. Hum. Gene Ther. 19: 443-450.

Henke E, Perk J, Vider J, et al. 2008. Peptide-conjugated antisense oligonucleotides for targeted inhibition of a transcriptional regulator in vivo. Nat. Biotechnol. 26: 91-100.

Leachman SA, Hickerson RP, Hull PR, et al. 2008. Therapeutic siRNAs for dominant genetic skin disorders including pachyonychia congenital. J. Dermatol. Sci. 51: 151-157.

Long SB, Long MB, White RR, Sullenger BA. 2008. Crystal structure of an RNA aptamer bound to thrombin. RNA 14: 2504-2512.

Michalowski D, Chitima-Matsiga R, Held DM, Burke DH. 2008. Novel bimodular DNA aptamers with guanosine quadruplexes inhibit phylogenetically diverse HIV-1 reverse transcriptases. Nucleic Acids Res. 36: 7124-7135. Full Text

Sel S, Wegmann M, Dicke T, et al. Effective prevention and therapy of experimental allergic asthma using a GATA-3-specific DNAzyme. J. Allergy Clin. Immunol. 121: 910-916.e5.

Yin H, Moulton H, Seow Y, et al. 2008. Cell-penetrating peptide-conjugated antisense oligonucleotides restore systemic muscle and cardiac dystrophin expression and function. Hum. Mol. Genet. 17: 3909-3918.

Session VIII

Van Vliet L, de Winter CL, van Deutekom JC, et al. 2008. Assessment of the feasibility of exon 45-55 multiexon skipping for duchenne muscular dystrophy. BMC Med. Genet. 9:105. Full Text

Wilkinson-Berka JL, Lofthouse S, Jaworski K, et al. 2007. An antisense oligonucleotide targeting the growth hormone receptor inhibits neovascularization in a mouse model of retinopathy. Mol. Vis. 13: 1529-1538. Full Text

Session IX

Kline JN. 2007. Immunotherapy of asthma using CpG oligodeoxynucleotides. Immunol. Res. 39: 279-286.

Kline JN, Krieg AM. 2008. Toll-like receptor 9 activation with CpG oligodeoxynucleotides for asthma therapy. Drug News Perspect. 21: 434-439.

McCluskie MJ, Krieg AM. 2006. Enhancement of infectious disease vaccines through TLR9-dependent recognition of CpG DNA. Curr. Top. Microbiol. Immunol. 311: 155-178.

Tross D, Klinman DM. 2008. Effect of CpG oligonucleotides on vaccine-induced B cell memory. J. Immunol. 181: 5785-5790.

Conference Contributors

Organizers

• The New York Academy of Sciences
• The Oligonucleotide Therapeutics Society

Organizing Committee

• Fritz Eckstein, Max-Planck Institute for Experimental Medicine, Göttingen, Germany
• Michael Gait, MRC Laboratory of Molecular Biology
• Mark Kay, Stanford University
• Arthur Krieg, Pfizer Research Technology Center
• Brett Monia, Isis Pharmaceuticals, Inc.
• John Rossi, Beckman Research Institute of the City of Hope
• Cy Stein, Albert Einstein College of Medicine
• Bruce Sullenger, Duke University Medical Center
• Thomas Tuschl, Rockefeller University
• Kathy Granger, The New York Academy of Sciences

This conference has been made possible through the generous support of the following organizations:

Gold Level Sponsorship

• Santaris Pharma

Silver Level Sponsorship

• Alnylam Pharmaceuticals

• Pfizer

Bronze Level Sponsorship

• ChemGenes Corporation

• Dicerna Pharmaceuticals, Inc.

• Isis Pharmaceuticals Inc.

• Merck & Co., Inc.

Academy Friend

• Avecia OligoMedicines

• BIOO Scientific Corporation

• Biosearch Technologies

• Bristol-Myers Squibb

• Calando Pharmaceuticals

• Girindus – Member of Solvay Organics

• Glen Research Corporation

• Idera Pharmaceuticals

• Link Technologies

• Panagene Corporation

• PolyOrg, Inc.

• Promega Corporation

• Regulus Therapeutics

• TriLink Biotechnologies

• U.S. Army

The views, opinions, and/or findings presented at the conference are those of the conference participants and should not be construed as an official Department of the Army position, policy, or decision, unless so designated by other documentation.

Speakers:
Phillip Sharp, Koch Institute, MIT
David Bartel, Whitehead Institute, MIT

More microRNA targets in cycling than in resting cells?

The Oligonucleotide Therapeutics Society has a basic science core. New findings in the life sciences inform and inspire the applied research conducted by many OTS members. This year, meeting organizers sought inspiration from leaders in the fields of gene regulation and computational biology. Specifically, the meeting opened with reports covering new research on messenger RNA (mRNA) translation and the microRNA regulators that govern the translation of mRNA into proteins.

In eukaryotes, microRNAs negatively regulate translation through their direct interaction with messenger RNAs. Typically, microRNAs act on target sequences in the 3′ untranslated region (3′UTR) of the mRNA, positioned between the translation stop codon and the polyadenylated (polyA) tail. A given mRNA may have several microRNA binding sites, including multiple sites for the same microRNA as well as sites for different microRNAs.

Alternative polyadenylation site selection

Keynote speaker Phillip Sharp of the Koch Institute, MIT, presented a surprising observation about biological "state-dependent" differences in cellular potential for responsiveness to microRNAs. He described recent work from his lab showing that mRNAs from proliferating cells have fewer microRNA target sites, and therefore are less susceptible to microRNA regulation than their counterparts in resting cells.

Many, if not most, genes have multiple polyadenylation sites, Sharp said. In some genes, the different polyadenylation sites reside in different exons, while in other genes there are multiple polyadenylation signals within a single exon. Sharp's lab, in collaboration with bioinformaticists in Christopher Burge's lab at MIT, focused on the latter case, asking whether the selection of a polyA site that was proximal—that is, nearest to the stop codon—or distal depended on cellular state. The researchers started this investigation by studying T-cell activation in culture, where upon cytokine stimulation, the immature cells go from resting to proliferating.

Activated (proliferating) T-cells preferentially use proximal, not distal, polyadenylation sites to form mRNAs.

To look for changes in polyA site selection, the Sharp and Burge groups performed microarray hybridization studies with separate arrays of oligonucleotides complementary to the common and to the extended region of a large number of mRNAs. The comparison of RNA samples from resting and 48-hour activated cells showed a dramatic activation-dependent preference for the common (proximal) polyA site. Consequently, mRNAs in the resting T cells carried longer UTRs than did the same mRNAs in proliferating T cells, providing more opportunities for microRNA-dependent regulation of gene expression in the former than the latter state.

This state-dependent difference in potential for regulation by microRNAs (and also by RNA-binding proteins, as Sharp noted) is by no means restricted to T-cell activation in vitro. That realization comes from follow-up studies on many types of normal and tumor cells in culture and in animals, which gave similar results to those in the T-cell study (Sandberg et al., 2008).

Focusing on one mRNA to study microRNA-regulated action

To examine the phenomenon in greater depth, the researchers studied one particular gene (HIP2) for which the activation-dependent rise in protein level is accompanied by a change from distal to proximal use of polyadenylation site in the mRNA. Inspecting the distal portion of the 3′UTR, they identified two putative microRNA-binding sites. When the researchers mutated these sites in the mRNA, they saw a loss of suppression by the corresponding microRNAs, confirming the involvement of microRNAs in keeping HIP2 protein levels low in the resting T cells.

The researchers then did nucleotide sequence searches for putative microRNA binding sites in proximal and distal sequences of 1190 mRNAs that have multiple polyadenylation sites. They found about twice as many sites in distal as in proximal 3′UTRs. This finding further supports a role for microRNA suppression of protein production in a state-dependent fashion, namely in the resting compared with the proliferating state of cells.

Having reported this evidence, Sharp concluded with the reminder that the field of microRNA biology is still in its infancy. His work exemplified that newness since many of the subsequent talks presented work that had not taken this striking, and perhaps fundamental finding into account in their own experimental design or interpretation.

Characterizing targets of microRNAs

The talk by David Bartel of the Whitehead Institute at MIT on the characterization of microRNA targets demonstrated how new the microRNA field still is. While many researchers have reported that microRNAs can knock down protein output of their regulatory targets, the degree to which these proteins change has been largely unknown. Bartel discussed work from his group that provides such evidence, specifically focusing on mRNAs that are regulated by microRNA miR-223, which influences neutrophil maturation in mice.

To identify those gene products governed by miR-223, the researchers used miR-223 knockout (KO) and wild-type (WT) mice. They cultured immature blood cells from each mouse strain in medium that promotes differentiation, maintaining the KO cells in medium with heavy amino acids (¹³C₆-Lys and ¹³C₆-Arg) and the WT cells in medium with light amino acids. Then they pooled extracts from the two cultures for mass spectroscopy analysis, to look for proteins that were present at different amounts in WT cells compared to KO cells.

Along with the protein analysis, the researchers also quantified the levels of mRNAs whose abundance changed in the two cell populations as a result of knocking out the microRNA. Most of the mRNAs affected by the microRNA contained predicted miR-223 binding sites. Moreover, the extent of regulation correlated with how many miR-223 binding sites each of these mRNAs contained.

Further inspection showed the importance of the position of the putative microRNA target site within the regulated mRNA, given that the changes in mRNA and protein levels were greater for mRNAs carrying miR-223 sites in the 3′UTR than in the protein-coding open reading frame (ORF). In general, the effect was cumulative, so that mRNAs with multiple miR-223 sites were affected more than those with a single site.

Both mRNA translation and stability are affected by the microRNA

What about the relative contribution of miR-223 to nondestructive translational repression versus destruction of the target mRNAs? Researchers had previously accumulated evidence for both mechanisms of microRNA action, but mainly using artificial targets. In the current study, the Bartel group was able to inspect the effects of a naturally occurring microRNA on the fate of a naturally occurring mRNA. They found that few targets were translationally repressed by more than 33% and that those showing more than this modest repression had significant mRNA degradation.

There appears to be a frequent requirement for perfect Watson-Crick base-pairing.

Because this study identified specific proteins that were affected by the knockout of a given microRNA, the researchers used the data to compare the predictive powers of several different microRNA target-site algorithms. Results of the comparison showed that TargetScan, the program developed by former graduate student Ben Lewis as part of the Bartel/Burge lab collaboration, gave the best results, with the program PicTar coming in second. Bartel noted that the key factor that gave TargetScan and PicTar the advantage was their strict requirement for perfect Watson-Crick base-pairing between the seed region near the 5′ end of the microRNA and its complement in the mRNA. Compared to PicTar and the other programs, Targetscan has the additional advantage of more accurately predicting which of the predicted targets will be most responsive to the microRNA.

Having opened his talk by noting that microRNAs arose early in the evolution of multicellular life forms, Bartel closed his presentation by musing on his somewhat surprising observation that the magnitude of most microRNA effects may individually be rather modest. That might mean, he suggested, there is an evolutionarily conserved effort to very tightly control the expression levels of most genes. Alternatively, he concedes, as Sharp noted before him, perhaps the research topic is still so young that it may be premature to make such conclusions, since what applies to miR-223 in neutrophils might not apply to other microRNAs or to other cell types.

Speakers:
Greg Verdine, Harvard University
Nicholay Ferrari, Topigen Pharmaceuticals, Inc.
Cy Stein, Albert Einstein College of Medicine

Seeking small but potent ASOs

Developers of antisense therapeutics confront major challenges, including the need to improve drug uptake into cells and design compounds with greater potency. Reports on antisense development and delivery at this year's conference discussed a range of approaches for overcoming these hurdles, including strategies for making smaller antisense oligonucleotides (ASOs) and for identifying contexts in which two or more ASOs can act synergistically. Also discussed was new evidence that may overturn longstanding dogma, which assumed that naked ASOs fail to be taken efficiently into cells in culture.

Antisense researchers have generally assumed that their drugs needed to be at least 16 or so nucleotides long, and bind to the target through a near-perfect Watson-Crick base pairing in order to be efficacious. Unfortunately, molecules that large do not readily get into living cells, at least not during the 24–48 hours of a typical experiment. Greg Verdine of Harvard University and colleagues have developed a strategy for finding short oligonucleotides—8mers or smaller—that bind to a given RNA target with high affinity, and which could therefore be a more readily deliverable drug than larger molecules.

Verdine's approach, which involves screening the target RNA of interest against a microarray of all combinations of 4-8mer sequences, doesn't demand that the interaction be through Watson-Crick base pairing. Rather, there just needs to be a high affinity interaction of the oligomer and the target RNA in its folded, native state. The project to find, optimize, and validate such oligomers, called RIPTides, for RNA-interactive polynucleotides, is being done collaboratively with Glenn McGall's team at Affymetrix, the manufacturer of oligonucleotide microarray chips.

The model system—knocking out telomerase RNA function

To test the concept, the investigators screened for RIPTides that would inhibit the assembly of telomerase, a ribonucleoprotein complex involved in maintaining chromosomal ends. They chose this endpoint because preventing telomerase assembly leads to a rapid potentiation of cell death, which is a readily measurable endpoint in cultured cells.

In their initial efforts, Verdine and coworkers found that even the tightest binding oligos had relatively low affinity for the target, in the micromolar range. By uniformly substituting 2′-O-methyl RNA for DNA oligos, they increased the affinities by approximately 40-fold. With these oligos, the researchers identified several "hot spots" for oligo binding on the target RNA.

Next, the candidate 2′-O-methyl oligos that bound the target RNA in the microarray screen were fluorescently tagged and tested in solution for their ability to bind the target RNA in its full-length, native state. Using fluorescence polarization, the researchers saw that some candidate oligomers (mainly 8mers) bound the full-length target in solution with high affinities, in the nanomolar range. Other candidates did not bind at all, presumably because the binding surface in the original RNA target, a fragment of the telomerase RNA, was no longer available in the full-length RNA. Tests of mutated oligos and compensatory mutations in the target RNA showed that most, but not all, high-affinity binding oligomers interacted with the target via strict base-pairing.

The researchers started functional testing of some of the oligos that bound with high affinity. First, they verified that the candidates could inhibit the enzyme activity of telomerase in vitro. For those candidates, they were able to calculate inhibitory constants. While they have not yet tested their cell-killing capacity, Verdine and colleagues have verified that some of the oligos, when administered to living cells, inhibit telomerase activity when those cells are cracked open and tested for enzyme activity in vitro. These tests, as well as additional experiments on other RNA targets offer evidence that the RIPTide approach may yield small but potent oligonucleotide drugs worthy of further development.

Harnessing the powers of synergy and local delivery

ASOs, like many other drug classes, become toxic at high doses, but often those doses are needed for drug efficacy. One way to minimize drug toxicity involves harnessing the power of synergy; combining low doses of two or more agents that, on their own may have little effect but together are potent and efficacious. The biopharmaceutical company Topigen aims to use synergy as it develops oligonucleotide therapeutics to treat inflammatory lung diseases such as chronic obstructive pulmonary disease (COPD) and asthma.

Another strategic feature of Topigen's drug development program for lung disease involves making drugs that can be inhaled rather than delivered systemically, since local delivery offers its own potential risk/benefit ratio. Nicholay Ferrari discussed both these concepts in reporting on Topigen's progress in developing TPI 1100, its antisense drug for treating COPD.

The potency of TPI 1100 and its affinity for the target mRNA are enhanced by substituting some of the nucleotide bases with fluorinated arabinoside (specifically, 2′-fluoro-D-arabinonucleic acid; FANA). Topigen's compounds require no further packaging beyond being dissolved in saline for successful delivery into lung tissue. At least in mice, the inhaled drug finds its way into cells deep within that tissue, and little of it appears systemically beyond the lungs.

To quiet down the inflammatory response in COPD, Topigen is taking aim at the mRNAs for two phosphodiesterases, PDE4 and PDE7. These enzymes regulate intracellular levels of cyclic AMP (cAMP), which plays a multitude of roles in pulmonary and immune cell types involved in COPD.

Validating synergy in vitro and in vivo

After validating that each of the ASOs reduced the level of the intended target mRNA, the researchers tested for potential synergy between the two. For example, Ferrari showed data from an unpublished study of human lung epithelial cells in culture, that looked for effects of the FANA-ASOs, separately or together, on production of the cytokine IL-8. Neither the PDE4 nor the PDE7 antisense compound had much effect on IL-8 release from the cells, but together they dramatically reduced the cells' ability to secrete it.

For initial in vivo studies, the researchers used a mouse model in which animals are exposed to cigarette smoke, stimulating production of various inflammatory chemokines and cytokines. In this study, Ferrari and colleagues compared the inhaled PDE ASOs to roflumilast (Altana Pharma AG, Pfizer), an orally administered PDE4 inhibitor currently under development for treating COPD. As Ferrari explained, while roflumilast has shown efficacy in Phase III studies, the drug carries serious dose-limiting toxicity issues.

An antisense drug targeting phosphodiesterases has shown promise for treatment of COPD.

The mouse study looked at several endpoints, including the smoke-related invasion of neutrophils into the lung, a rise in lung neutrophil chemoattractants (KC and MIP-2, the mouse homologs of IL-8 in humans), and an increase in MMP-9, a metalloprotease with a well-defined role in lung remodeling in COPD. For all endpoints, TPI 1100, used at a 25-fold lower dose than roflumilast, outperformed the orally delivered drug.

Ferrari reported that toxicology and safety studies in both mice and monkeys have now been completed with good results, supporting the company's application for moving into a Phase I trial in humans.

Gymnotic delivery of antisense oligonucleotides

Drug development is not the only application for ASOs. Indeed, for a few decades, researchers have hoped that antisense oligonucleotide (ASO)-dependent gene silencing could be a simple and reliable tool for studying biochemical pathways at the cellular level. The notion was that any mRNA could be silenced using a synthetic oligonucleotide that binds to it by Watson-Crick base pairing, leading to RNAse H-mediated destruction of the mRNA.

While the concept was simple, its successful execution also faced many hurdles. Unmodified oligonucleotides proved to be unstable in cell culture medium. The ASOs showed weak potency because of poor cellular uptake, low affinity for the specific target, and extensive off-target activity.

Tackling these problems, investigators found that a phosphorothioate modification of the nucleotides increased ASO stability without sacrificing target selectivity; cellular uptake could be enhanced with cationic lipid carriers but worked well only for some cell types; and stability could be increased by adding "locked nucleic acids" at the 5′ and 3′ ends of the ASOs, permitting the use of lower ASO doses to achieve on-target effects and thereby minimizing the off-target interactions. Even with these and other modifications, not all cell types, nor all mRNA targets, seemed accessible to silencing by ASOs at practical doses.

The naked truth

Cy Stein of the Albert Einstein College of Medicine, who has devoted considerable effort to finding ways to improve delivery and effectiveness of ASOs, reported preliminary findings from his lab that seem to counter dogma that he and others have perpetuated about optimal conditions for using ASOs in cell culture. As he noted, investigators using modified ASOs have come to assume the need for one or another sort of in vitro carrier to get the oligonucleotides taken into cells. In addition, most researchers have assumed that their experiment failed if no silencing was seen following a few days of treatment.

Now Stein has found that gymnotic (i.e., naked) delivery of ASOs works well, obviating the need for lipids or other uptake vehicles in the cell culture medium. As he reported, the cells must be in the log phase of growth throughout the experiment, and the mRNA silencing effect of naked oligos takes time, requiring approximately 6 days to see the early signs of mRNA decline and 10 days for almost complete silencing.

Stein first saw mRNA silencing following gymnotic ASO delivery in the melanoma cell line 591.8, using two ASOs (G3139 and 2996) targeted against the BCL-2 mRNA, along with negative control ASOs. Along with this effect in the 591.8 cells, Stein saw similar BCL-2 silencing with gymnotic delivery of G3139 and 2994 to several, but not all, other cell lines from a variety of cancer types.

Now Stein and his colleagues are looking for the mechanism by which the naked ASOs silence mRNA. While most ASOs delivered by carriers are thought to work via RNase H, gymnotically delivered ASOs may act by a different mechanism. That conclusion comes from Stein's finding that a G3139 analog, modified so that its target mRNA resists RNase H, works just as well as unmodified G3139. Instead, Stein showed cytochemical evidence that naked ASOs force their target mRNAs to move into P-bodies, those newly characterized sites for mRNA storage and degradation. Knowing that P-bodies are the site where siRNA-mediated mRNA destruction is catalyzed by the enzyme Ago2, Stein and coworkers tested for Ago2 involvement in the action of gymnotically delivered ASOs. In an initial study in which Ago2 expression itself was silenced, the silencing effect of G3139 on BCL-2 was greatly attenuated. Thus, unpackaged ASOs may, in fact, work through an RNAi pathway. If these early findings hold up, the consequences of that action will need to be carefully inspected.

Speakers:
Leaf Huang, University of North Carolina
Steven Dowdy, UCSD School of Medicine, HHMI
Kevin Fitzgerald, Alnylam Pharmaceuticals

Nanopackaging of siRNAs

Whereas developers of ASO drugs have been able to chemically modify the ASOs to protect them from extracellular degradation, developers of siRNA therapeutics have opted for building protective nanochambers for their drugs. They were forced to take this more complex approach because the mode of siRNA action, as part of the intracellular RNAi machinery, has little tolerance for chemically modified siRNAs. Considerable brainpower has been devoted to devising effective ways of packaging the siRNA drugs and then delivering the packages to the target tissue and ultimately the intracellular locale of interest.

Talks this year included both strategies for targeting the siRNA-containing particles as well as ways of avoiding the need for such targeting. Until recently, the field of siRNA drug development seemed dominated by investigators working on therapies that required only local, highly restricted delivery routes. As one sign of optimism that the hurdles to siRNA delivery will be solved, speakers on the topic of siRNA aim to develop systemic siRNA treatments.

Sneaking siRNA nanoparticles through leaky blood vessels in tumors

Leaf Huang of the University of North Carolina presented a clever strategy for delivering siRNAs systemically to solid tumors. This approach involves packaging the siRNAs as cargo in modified liposomal nanoparticles. This method exploits a key feature that distinguishes many solid tumors from normal tissue, namely a difference in the structure of blood vessels that feed the tissues. Whereas in the vasculature of normal tissues the endothelial cells are tightly connected to each other, the cells of blood vessels associated with tumors tend to be loosely connected, making for leaky vessels.

Recognizing this feature of tumor-associated blood vessels, known as enhanced permeability and retention (EPR), Huang hypothesized that nanoparticle drugs administered intravenously might selectively reach tumor tissue merely by diffusing between the endothelial cells of the tumor's blood vessels. His results confirmed that idea, at least in mice. A caveat Huang mentioned is that not all solid tumors are well vascularized, limiting the potential applicability of this treatment to just certain types of cancer.

Nanoparticle-encapsulated siRNAs are preferentially taken up into tumor tissue.

Describing the self-assembling particles that he and his colleagues developed, Huang explained that they have the siRNA(s) at their core, mixed with carrier DNA or other negatively charged macromolecules along with a cationic macromolecule such as protamine. That complex is then coated with a cholesterol-containing lipid mix that forms a double bilayer. The outer bilayer contains polyethylene glycol (PEG), which can then be decorated with a ligand for a "generic" cell membrane receptor.

By taking advantage of EPR to direct the nanoparticles to the tumor tissue, Huang felt free to devise a cellular uptake approach that didn't need to be cancer cell-focused. Instead, he chose to decorate the siRNA-containing nanoparticles with anisamide, a small molecule with a high affinity for sigma receptors, a class of cell membrane receptors that are commonly but not exclusively found on cancer cell membranes. According to Huang's plan, once the particles bound to the surface of sigma receptor-containing cells, they would be endocytosed, releasing the siRNA into the cytoplasm.

Testing the nanoparticles on a mouse model of metastatic melanoma

Having confirmed that the siRNA nanoparticles behaved as expected in mice, Huang then showed the efficacy of this approach in attacking solid tumors in mouse models of cancer. Most impressively, he reported on a mouse model of metastatic melanoma, created by injecting human melanoma cells intravenously, waiting several days for the cells to colonize lung tissue and start proliferating, and then treating the animals with experimental and control siRNA-containing nanoparticles.

Effect of siRNA in nanoparticles on melanomas in mice.

To treat metastatic melanoma, the researchers used a set of three siRNAs, each directed at a different aspect of proliferation or cell survival. The mice received two injections of the drug or control and then were sacrificed for study one week later. As Huang noted, melanoma cells are typically black, making it easy to monitor the progression of the disease and treatment just by looking at the animals' lungs. Huang and colleagues found a striking effect of the experimental siRNA mix. Whereas all the control lungs appeared heavily blackened due to widespread distribution of the tumor on the lungs, lungs from the experimental siRNA-treated mice had only modest amounts of black melanoma cells on their surface.

Huang closed the presentation by summarizing results that may explain why the nanoparticles evade uptake by liver cells, explaining that their high PEG concentration in the lipid bilayer keeps them from uptake into the hepatic Kupfer cells. Because they avoid uptake into the liver, the nanoparticles may be able to be used at low doses. Therefore this intriguing molecular complex may have a future in the treatment of many different solid tumors.

Macropinocytosis—a previously untapped cellular uptake mechanism

Given the need for effective siRNA delivery systems, it is not surprising that OTS4 featured presentations on a range of different delivery strategies. For example, Steven Dowdy (HHMI and UCSD), who spent the last decade developing a system for in vivo delivery of protein drugs, is now adapting that approach for therapeutic siRNA delivery. SiRNA drugs act via the naturally occurring intracellular RNAi machinery. To interact with the RNAi pathway components, these drugs must have certain structural features, resulting in drugs that are large (~14KD) and carry a strong negative charge. These characteristics make it all-but-impossible for siRNAs to enter cells on their own.

Before describing the siRNA drug delivery system he developed, Dowdy introduced his system for delivering protein drugs. This approach takes advantage of macropinocytosis, a poorly understood fluid-based uptake mechanism used by most, if not all, types of living cells. Material taken up by this route enters cells via vacuoles rather than vesicles.

When Dowdy realized that macropinocytosis could be used to deliver positively charged peptides into cells, he merely linked such peptides (known as peptide transduction domains—PTDs) to the protein cargoes of interest. The approach worked quite well and several such PTD-modified drugs are now being tested in Phase I/II trials.

Adapting the peptide carrier for the siRNA cargo

Adapting the PTD carrier system for siRNA cargoes required some additional modifications because of the high negative charge on siRNAs. Dowdy and colleagues first tried adding long PTDs, to overcome the negative charge, but such complexes aggregated and were highly cytotoxic. That led the investigators to include in their drug complex a small (65 amino acid) protein fragment that binds to dsRNA and therefore neutralizes much of RNA's negative charge. (Such double stranded RNA binding domains—DRBDs—occur in several proteins of the RNAi machinery, including Drosha and Dicer.) Since the DRBD binds just to the central core of the siRNAs, the researchers still needed to add multiple PTDs to the siRNA ends. The complex that Dowdy and colleagues are currently testing in cells and animals contains the siRNA and the DRBD, as well as four PTDs, enough to create sufficient net positive charge to be efficiently taken up by cells.

The complex of siRNA and special protein domains effectively silenced target mRNA in cultured cells.

Tests of the siRNA/DRBD/PTD complex on cells in culture showed that it could rapidly silence the target mRNA (for example, an ectopically expressed green fluorescent protein (GFP) mRNA) in the entire cell population. The rapid silencing occurred in tests on both tumor cells and primary cells from both human and mouse. By contrast, when the siRNA against GFP was tested with the commonly used carrier, lipofectin, the effect was much less striking and complete.

Dowdy's team assessed the DRBD/PTD and lipofectin delivery vehicles for their inherent (and presumably unwanted) effects on gene expression. Using microarrays of mRNA levels in response to vehicle treatment, the researchers found that the DRBD/PTD complex perturbed the cells much less than did the lipofectin treatment.

The siRNA/DRBD/PTD complex retards glioblastoma in mice

To test the siRNA/DRBD/PTD complex in vivo, the Dowdy group infused the siRNA complexes directly into brains of mice bearing glioblastomas, mimicking the treatment mode commonly used against this virulent form of cancer in humans. After seeing that specific gene products could be silenced in the tumors by infusing the siRNA complex, the researchers demonstrated that a single treatment with two siRNAs (here, those that target EGFR and ATK2 mRNAs) substantially retards tumor growth.

Brain tumors respond to EGFR/ATK2 siRNAs in DRBD/TPD complexes.

Moreover, while treatment of tumor-bearing mice with the EGFR siRNA complex on its own extended the life of the animals from a median of 14 days to 19 days after the drug was given, treatment with the combination of siRNAs to EGFR and ATK2 mRNAs extended the median survival time to 31 days.

For the siRNA/DRBD/PTD complexes to be safe for human use, they should not activate the innate immune system. Dowdy and colleagues have now shown that in mice, treatment with the complex has no impact on two key innate immune pathways, those activated in the interferon and the TLR systems.

These and other findings have encouraged Dowdy and his colleagues to move forward. Most critically, they are scaling up production of the siRNA complexes in order to study their effects in vivo and to learn more about their mechanism of action. The results have been confirmed independently in an EORTC (European Organization for Research and Treatment of Cancer) study.

Gearing up for systemic siRNA treatment for chronic illness

SiRNA biotechnology firms, like some of the antisense companies, are now aiming to develop potent systemic compounds for treating chronic ailments, as Alnylam's Kevin Fitzgerald made clear in his presentation. Fitzgerald reported on Alnylam's progress in developing a siRNA formulation to treat chronic hyperlipidemia.

First he reviewed the company's current platform for siRNA drug development and then its scheme for drug delivery. Briefly, for each mRNA of interest, the researchers conduct an in silico walk across the gene to find the best target sequences, then modify selected nucleotides to increase stability and minimize unwanted immunostimulatory effects.

For drug delivery, Alnylam uses lipoidal carriers optimized for their ability to deliver siRNAs into cells via membrane fusion-mediated endocytosis, an approach developed by Dan Anderson, Robert Langer, and colleagues at MIT. With this scheme, the researchers generated a library of cationic lipoidal compounds by reacting various head groups and tail groups, and tested them for their ability to mediate transfection into cells. Those compounds that performed best in the uptake studies were chosen for further study in vivo.

The anti-hyperlipidemia target—an inactivator of the LDL receptor

Turning to Alnylam's anti-hyperlipidemia project, Fitzgerald explained that the target for siRNA silencing is PCSK9 (Proprotein convertase subtilisin/kexin type 9), an autocatalytic enzyme that inactivates the hepatic low density lipoprotein receptor (LDLR). Presumably, that inactivation feeds back to stimulate LDLR production, which enhances LDL-cholesterol (LDL-C) clearance from circulation. Genetic evidence also supports the link between PCSK9 and LDL-C levels.

These and other findings led Alnylam to test siRNAs against PCSK9 as a potential therapy against hypercholesterolemia. The researchers began with studies in mice, taking advantage of a PCSK9 knockout as a control. Following a single infusion into the tail vein of the animals, there was a dose-dependent reduction in total cholesterol, with a 20% reduction in the animals receiving the highest dose. Reduced levels of cholesterol continued to be seen up to 20 days after the drug was given.

In rats, siRNA against PCSK9 led to the cleavage of the liver PCSK9 mRNA at the predicted site in the molecule.

Somewhat more robust responses were seen in similar studies on rats, perhaps because rats have higher starting levels of PCSK9 than mice do. As part of the rat studies, the investigators confirmed that treatment with the siRNA led to the cleavage of the liver PCSK9 mRNA at the predicted site in the molecule. In addition, the investigators confirmed that treatment with the siRNA against PCSK9 led to increase LDLR levels in the liver. Moreover, the treatment did not cause a compensatory increase in levels of hepatic cholesterol or triglycerides.

Studies in monkeys have now extended the findings in rodents using siRNAs against two different regions of the PCSK9 mRNA. The two siRNAs showed qualitatively similar results, with one being more potent that the other. In these monkey studies, lowering of serum cholesterol levels was seen as early as 3 days after treatment and cholesterol level was still below the pretreatment value even at 20 days after the single dose injection. Collectively these findings offer strong encouragement for continuing to develop an siRNA-based systemic treatment for chronic disease.

Speakers:
Sudhir Agrawal, Idera Pharmaceuticals
Ian MacLachlan, Tekmira Pharmaceuticals Corp.

Chemistry of immunomodulatory oligonucleotides

The immunostimulatory component of oligonucleotide therapeutic research offers a stunning example of how applied science draws on and feeds into basic scientific research. Work in the past several years has characterized the mechanisms by which microbial and viral nucleic acids and their synthetic oligonucleotide mimics selectively activate various components of the immune systems of human and other higher organisms. Not only have these findings prompted some researchers to look for means of avoiding immunostimulatory effects in oligonucleotide therapeutics aimed at silencing specific mRNAs, but other researchers have sought ways to harness the immunoreactive oligomers as products of their own.

At this year's conference, topics of discussion included a new way to minimize the potential immunostimulatory effect of siRNA drugs and a report on antagonism of oligonucleotide immunostimulation, including plans to develop the concept for treating autoimmune diseases.

Structure/function relationship of immunostimulatory CpG oligonucleotides

Sudhir Agrawal of Idera Pharmaceuticals aims to develop CpG-containing oligonucleotides as therapeutics for quieting the autoimmune reactions involved in diseases such as lupus, colitis, and arthritis. The notion that oligonucleotides might be useful as anti-inflammatory drugs, originally suggested by Krieg (Redford et al.,1998), more recently occurred to Agrawal during studies of the structure/function relationship of CpG-containing oligodeoxynucleotides. Specifically, knowing that the CpG motif in oligodeoxynucleotides has immunostimulatory effects via an interaction with the Toll-like receptor TLR9 on certain cells of the immune system, Agrawal and colleagues wanted to determine what other structural features of the oligonucleotide influenced that stimulation.

One key feature turned out to be the structure of the nucleotide adjacent to the cytosine (C) of the CpG pair; converting it to a 2′-O-methyl nucleotide eliminated the immunostimulatory effect. Moreover, and unexpectedly, the 2′-O-methyl modification converted the oligonucleotide into a competitive antagonist of the parent compound. Thus, for example, the researchers showed that a certain unmodified 18mer containing a CpG motif, when given to mice, stimulated cytokine production. However, when the same 18mer, but now carrying the 2′-O-methyl modification adjacent to the CpG, was administered first, and then the test compound given a few hours later, induction of the cytokine failed to occur. Importantly, the researchers confirmed that the 2′-O-methyl modification worked only in the context of being adjacent to the CpG motif. Agrawal refers to the CpG sequence as the immune stimulatory motif and the 2′-O-methyl adjacent to the CpG as the immune regulatory motif of the oligonucleotides.

A variety of studies have suggested a general link between autoimmune diseases and inappropriate activation of TLR9, prompting Agrawal and his colleagues to see if treatment with oligonucleotides carrying the combined immune regulatory/immune stimulatory motifs might be useful for treating such conditions. The researchers are now exploring two different mouse models of autoimmunity—one mimicking lupus susceptibility and the other a model for psoriasis.

TLR antagonist in the mouse model of lupus.

In the lupus model (the MRL/lpr mouse strain, in which the animals are susceptible to lupus due to a mutation in the Fas gene), all test animals succumb to lupus in the absence of a beneficial treatment. Testing an immunomodulatory/immunostimulatory oligonucleotide treatment compared to no treatment, Agrawal and colleagues found that the oligonucleotide treatment significantly enhanced survival of the mice. Then, looking at several different markers of the disease, including development of the characteristic "butterfly" facial rash, changes in skin histology, and formation of pathologic antibody deposits in the kidney, the researchers saw beneficial effects of oligonucleotide therapy. Agrawal reported similar beneficial effect of oligonucleotide therapy in their model of psoriasis, which substantiates the idea that these compounds hold promise for treating human autoimmune diseases.

Overcoming innate immune responses to RNA

Ian MacLachlan of Tekmira offered a cautionary tale for developers of siRNA drugs. The bad news, he said, is that siRNAs packaged for in vivo delivery typically elicit unintended innate immune responses. The good news, he went on to explain, is that these unwanted effects can be eliminated without dampening the intended mRNA silencing action by chemically modifying the sense strand of the siRNA compound.

The problem starts with the fact that, for efficient in vivo delivery, siRNAs need shielding to protect them from degradation in the circulation and to facilitate their uptake into cells. The typical shielding formulation for siRNAs includes various cationic lipids, and the resulting lipid-siRNA complexes, upon being taken up by cells, empty the siRNAs into the cytoplasm where they are be recognized as RNA components of infectious agents by the intracellular Toll-like receptors (TLR7, 8, and 3). Such RNA-mediated activation of TLRs elicits a range of innate immune responses, most often including induction of type 1 interferons and various cytokines.

Neither the siRNAs nor the delivery vehicles on their own prompt these innate immune responses; only the siRNAs in complexes do. Surprisingly, the exact nature of the immune response depends on the composition of the vehicle. For example, in human blood cells tested in vitro, the Tekmira liposomal complex, known as SNALPs (for stabilized nucleic acid lipid particle), strongly stimulated interferon alpha production and had no effect on the cytokines interleukin 6 (IL-6) or tumor necrosis factor alpha (TNFα) whereas the same siRNA in a different lipoplexed formulation had minimal effects on interferon-α induction but was a potent inducer of the cytokines.

Modifications that eliminate immune effects without dampening siRNA potency

MacLachlan then explained how he and his colleagues looked for a way to selectively abrogate the immune stimulatory effect without altering the specific siRNA-mediated silencing. As a first step, they compared SNALP-complexed double-stranded RNA (dsRNA) with SNALP complexes that included just the antisense strand (i.e., the strand that will interact with the mRNA to silence it) or just the sense strand RNA. For the siRNA being tested, only the antisense, but not the sense strand RNA had the immunostimulatory effect.

MacLachlan and colleagues found that they could eliminate the siRNA-mediated immunostimulatory effects through chemical modification of the siRNA, by substituting ribonucleotide bases with 2′-O-methyl bases. Only one of the two—either the sense or the antisense—needed to carry the 2′-O-methyl substitution in order to eliminate the immunostimulatory effect.

Next, MacLachlan and colleagues needed to determine whether the modification impacted on siRNA efficacy. Indeed, for the siRNAs tested, 2′-O-methyl substitutions on the antisense strand of the duplex dampened the silencing power of the compound. By contrast, modifying the sense strand had no such dampening effect. Therefore, this approach may be generally useful for designing siRNA drugs that will be efficacious without having unwanted immunostimulatory side effects.

Speakers:
Sanjay Bhanot, Isis Pharmaceuticals
Loretta Itri, Genta, Inc.
Martin Gleave, OncoGenex Technologies
Jeremy Heidel, Calando Pharmaceuticals

ASOs for treating chronic ailments via systemic delivery

Among the ASO drugs now moving through the clinical trial pipeline are a growing number that aim to treat chronic ailment via systemic delivery.Clinical trials of oligonucleotide therapeutics this year also included the first siRNA compound that makes use of systemic delivery. Among the ASO drugs now moving through the clinical trial pipeline are a growing number that aim to treat chronic ailment via systemic delivery.

In the ASO arena, ISIS Pharmaceuticals is developing and testing systemic drugs for chronic diseases such as atherosclerosis and type 2 diabetes. To succeed in this bold effort, the company needs to make drugs that are potent and long lasting enough for once-a-week or less frequent dosing, by a route that is no more noxious than subcutaneous injection, and with a safety profile that permits use for years, perhaps for life.

Sanjay Bhanot, Isis Vice President of Metabolic Diseases R&D, opened his presentation on the development of such drugs by reviewing the key chemical feature of ISIS's current generation of ASOs, which make them more potent and more stable than earlier ASOs. Like the earlier ASOs, this class of compounds carries a central core of phosphorothioate DNA bases, which protect the compounds against extracellular degradation and permits RNase H cleavage of the target mRNA. In addition, the molecules now have modified ribonucleotides at both the 5′ and 3′ ends, which further increase their nuclease resistance and the thermal stability of the hybrids they form with their target mRNAs, giving them the needed potency and prolonged half-life.

Having tested compounds in this class in over 5000 humans, including >500 subjects treated for several months each, Bhanot reported that the researchers continue to see a good safety profile for these ASOs. Contributing to this safety, and critical for compounds intended for extended use, is the fact that ASOs are not metabolized by the liver enzyme system—the CYP450s—that breaks down most drugs, thus avoiding drug-drug interactions.

Moving into the field of drugs for treating hyperlipidemia, chronic ailment

ISIS's effort to develop ASOs for treating chronic, systemic conditions is exemplified by their work on Mipomersen, an anti-hyperlipidemia drug. To enter the anti-hyperlipidemia market, which is filled with small molecule drugs for various aspects of cholesterol and lipid lowering, ISIS is aiming at a novel target, Apo-lipoprotein B100 (Apo B-100).

This hepatic protein is the protein component of low-density lipoprotein (LDL) and other lipoproteins. No other drug has yet to target this component of the lipoprotein, cholesterol-carrying system. Consequently, the test of Mipomersen's efficacy at reducing the ApoB-100 mRNA serves as a good "proof-of-concept" candidate, and Bhanot reported encouraging results to support the concept.

He reviewed the results of two types of Phase II trials. The first trial used Mipomersen as a single agent drug for routine lipid lowering. The second used it in combination with a statin to treat individuals with congenital hypercholesterolemia who had become refractory to statin treatment even at a high dose. In both groups, treatment for 13 weeks led to marked reduction in LDL-cholesterol levels, and no unexpected adverse effects. Within the dose range tested, response to Mipomersen was linear, showing near-classic dose responses. By contrast, statins, the popular lipid-lowering drugs, show much more attenuated dose responses. Finally, Bhanot noted that in patients with familial hyperlipidemia, Mipomersen's effect was unique in that the drug reduced several different cardiovascular disease risk factors; not just the Apo B-100 target protein, total and LDL-cholesterol, but also Lipoprotein (a) and possibly trigylcerides. Based on these encouraging results, the company is now partnering with Genzyme to conduct Phase III trials of Mipomersen on individuals with familial hypercholesterolemia.

Mipomersen, as well as another promising compound Bhanot discussed—ISIS113715, which targets the mRNA for the intracellular protein phosphotyrosine phosphatase 1B (PTP-1B) for treating type II diabetes—are both 20mers and therefore require subcutaneous injection. Ideally the company would like to produce ASO drugs with sufficient potency and stability for oral administration. With that goal in mind, Bhanot ended his presentation by mentioning studies with a new 12mer, 388626, which targets the mRNA for sodium-dependent glucose transporter 2 and may prove useful in type II diabetes as well. The observed pharmacological properties of 388626, primarily in animal studies, suggest that it may be suitable for oral delivery, a goal for the ASO field that even a few years ago seemed totally unattainable.

ASO drugs set cancer cells up for the kill

Oblimersen (Genasense) was among the first antisense oligonucleotide (ASO) therapeutics to enter clinical trials. As Genta's CMO, Loretta Itri noted, Oblimersen ranks among the most extensively tested of any systemic ASO drug in clinical trials to date. Consequently, the drug's safety for intermittent short-term use in cancer treatment is well established. The challenge has been to show that the drug is efficacious enough to warrant approval by regulatory agencies for use in cancer treatments.

Oblimersen is a phosphorothioate 18mer that targets the mRNA for Bcl-2, an anti-apoptotic protein. Many cancers have elevated levels of Bcl-2, which may protect them from the cytotoxic effects of many chemotherapeutic agents. Oblimersen aims to remove the barrier to apoptosis, making tumor cells susceptible to being killed by chemotherapy drugs. Accordingly, patients receive Oblimersen just prior to, and continuing into, the start of each drug-treatment cycle.

Genta's 771-patient Phase III melanoma study showed a weak positive benefit from combining Oblimersen with standard chemotherapy. However, when the subgroup with less extensive disease—based on lactate dehydrogenase (LDH) levels in the normal rather than elevated range—was analyzed separately, the response to Oblimersen plus chemotherapy seemed more robust than the response to dacarbazine alone. Therefore, Genta is currently sponsoring the AGENDA trial, a new Phase III melanoma study that will focus on patients predicted to be able to benefit from Oblimersen, namely those with LDH levels in the normal range.

OGX-011, like Oblimersen, is an ASO designed to enhance the susceptibility of cancer cells to killing by traditional chemotherapeutic drugs. A newer compound than Oblimersen, it carries the 2′-MOE modification for enhance stability and potency. Martin Gleave (University of British Columbia, Vancouver General Hospital, OncoGenex) reported encouraging results of a Phase II study that evaluated the OGX-011 in patients with treatment-resistant prostate cancer.

OGX-011 targets the stress-induced protein clusterin, an anti-apoptotic protein. A second generation ASO, OGX-011 is a 21mer with the 4-13-4 "2′-MOE gapmer" configuration (13 phosphorothioate dNTPs at its core, with 2′-methoxyethoxymethyl dNTPs on each end).

Gleave and his colleagues identified clusterin as a target for treatment through expression-profiling studies of treatment-resistant prostate tumors. Then, using a prostate cancer cell line, they confirmed that OGX-011 would sensitize the cells to the standard chemotherapeutic agent, taxotere.

The clinical trial Gleave discussed focused on men who had castrate-resistant prostate cancer and who had subsequently failed first-line treatment with taxotere. Designed as a feasibility study, the trial assessed survival and drug tolerability of patients in two chemotherapy treatment arms, one getting OGX-011 along with taxotere and the other getting OGX-011 along with mitoxantrone. Both groups survived longer than historical controls and were able to tolerate more cycles of chemotherapy than historical controls. Secondarily, data from the study suggested that serum levels of clusterin may be a useful predictor of survival.

The results of this Phase II study were strong enough to allow the investigators to move forward with a larger trial of OGX-011 as part of combination therapy in taxotere-resistant prostate cancer patients. That trial has received FDA approval.

Solid tumor treatment: A tall order for little siRNAs

CALAA-01 is the first systemically-administered formulated siRNA approved by the FDA for clinical testing and the first siRNA compound approved for testing as a treatment for cancer, reported Jeremy Heidel, from Calando, the developer of this drug. The siRNA in CALAA-01 targets a subunit of the ribonucleotide reductase, an enzyme needed for generating deoxynucleotide triphosphates needed for DNA synthesis and repair. According to Heidel, the subunit in question, M2, is elevated in many solid tumors and, indeed, is a well-characterized target of cancer therapies.

Calando's formulation of the drug involves packaging the siRNA into nanoparticles that are tagged in order to target them to cancer cells. In this case, the targeting ligand is the protein transferrin, chosen because the transferrin receptor is elevated on the cell surface of many types of human tumors.

Heidel reported that the CALAA-01 trial started enrolling patients in May 2008. Rather than focusing on any one type of cancer, the trial is open in general to patients whose solid tumors are refractory to standard of care. This study design will enable the researchers to use the secondary endpoints to give insights into which tumor type(s) might be most responsive to CALAA-01 treatment.

What cellular signal(s) determines whether messenger RNAs will be spliced so that they contain or lack long 3′ untranslated sequences, and consequently have many or few microRNA target sites?

What features of messenger RNAs determine whether they will be reversibly or irreversibly silenced by siRNA machinery?

Will the development of drugs that combine two different ASOs intended to work synergistically need to show efficacy of each ASO on its own in order for the combination to obtain regulatory approval?

In intact animals, do naked ASOs ever work through the RNAi rather than the RNase H pathway, and what rules govern the silencing pathway that a single stranded ASO will take in vivo?

Which ASO therapeutic will be the next one to receive regulatory approval?

What will be the biggest safety hurdles that siRNA drug developers need to overcome for using these compounds in systemic treatment?

Which siRNA will be the first to receive regulatory approval?

Which immunoregulatory oligonucleotide will be the first to receive regulatory approval?

The field of oligonucleotide therapeutics could be seen simply as an effort in rational drug design—developing drugs that treat ailments by knocking down the expression of "rationally selected" messenger RNAs. But if rational drug development were so simple, dozens of oligonucleotide drugs, for treating both acute and chronic ailments by local or systemic means, would be filling pharmacists' shelves and patients' medicine cabinets today. Establishing entirely new types of drugs, which oligonucleotide therapeutics are, has turned out to be amazingly complex. A quick look at the past and a review of the present meeting of the Oligonucleotide Therapeutics Society (OTS) highlights this complexity.

At the time of the inaugural meeting (OTS1) of the society in October 2005, just one drug—Vitrivene, an antiviral antisense oligodeoxynucleotide from Isis Pharmaceuticals—carried FDA approval, and several other oligonucleotide therapeutics (ONTs) were in the pipeline. While most of the ONTs furthest along in development in 2005 fell into the antisense DNA class, an even newer group, the small interfering RNA (siRNA) drugs, garnered intense enthusiasm at that meeting.

Because siRNA drug development is based on a naturally occurring strategy for gene silencing—RNA interference (RNAi)—many investigators, both in academia and in industry, thought that the field would outpace antisense DNA once an effective means for siRNA delivery had been developed. Concerns about delivery for both antisense DNA and siRNA drugs meant that many researchers at the first OTS meeting reported plans for treating diseases such as macular degeneration, which requires local, rather than systemic drug delivery.

Rounding out the suite of ONTs discussed at OTS1 were the immunoregulatory compounds, first discovered as unwanted off-target actors in the DNA antisense field. In 2005 the immunoregulatory ONTs were just starting to form the basis of their own separate cottage industry based on mimicry of naturally occurring DNA- and RNA-based infectious agents.

Three years after that first meeting, it is evident that the oligonucleotide therapeutics field has flourished. OTS4, held in October 2008 at the Harvard Medical School Conference Center in Boston, hosted over 300 attendees gathering for three full days to accommodate a roster of 50-odd speakers and three days worth of poster presentations. The sizable turnout did not stem from investigators eager to report on recent approvals of their drugs by regulatory agencies (Vitrivene is still the only approved ONT, and it had very limited commercial success), nor was it because siRNA developments had strongly outpaced those in the antisense field, since more antisense drugs are nearer to potential regulatory approval than are the younger siRNA-based compounds. Instead, the vibrancy of this year's meeting may be due to a combination of factors in both the basic science and clinical aspects of the field:

• MicroRNAs, the major biological players in RNAi-mediated gene regulation, currently stand at the forefront of basic biomedical research because of their role in normal development and disease. Interest in applied RNAi research may be an important by-product of emphasis on microRNAs.
• Early versions of antisense drugs were weakly potent and short-lived, and therefore not very efficacious. Second-generation chemistry led to the development of antisense compounds with much greater potency and longer half-lives. Several such antisense drugs are currently performing well in mid- and late-stage clinical trials. These outcomes have not just spurred a renewed interest in antisense therapeutics for treating acute conditions that may benefit from localized delivery. They have also prompted the development of antisense therapeutics to treat chronic diseases via systemic modes of delivery.
• Developers of siRNA-based therapeutics came to appreciate that delivering such drugs was more complex than they initially envisioned and that lessons could be learned from interaction with antisense drug developers.
• SiRNA drug developers also caught up with their antisense counterparts in appreciating that all ONTs had the potential for eliciting immunostimulatory effects, and that OTS meetings were excellent forums for discussing these problems and ways to solve them.

Highlights of this year's conference included:

Basic science observations in microRNA biology

• Messenger RNAs (mRNA) in cells at rest may have longer 3? untranslated regions (3?UTRs) than do those in proliferating cells, and consequently have more microRNA-binding sites per mRNA than do those same mRNAs in dividing cells.
• Using a reverse genetics strategy, employing cells from microRNA knock-out and wild-type mice, researchers have identified on mRNAs microRNA target sites that function in vivo. These target sites have microRNA "seed sequences," predicted most effectively by the computational algorithm called TargetScan.
• MicroRNA-mediated regulation of gene expression, while crucial for many developmental and differentiation events, generally seems to be small in magnitude, at least in cell lines.
• When the magnitude of microRNA-dependent knockdown of protein expression is small, the process typically involves sequestration but not destruction of the mRNA that translates that protein. When the magnitude of microRNA-dependent silencing is large, destruction of the mRNA target seems to occur.

Antisense development and delivery

• The RIPTide (RNA-interactive polynucleotides) approach to designing oligonucleotide therapeutics seems useful for finding small oligonucleotides (8mers or smaller) that can readily enter cells and then bind with high affinity to their target macromolecule.
• Naked oligonucleotides may be able to penetrate cells in culture, and appear to silence expression of specific target mRNAs quite effectively, but slowly.
• Naked antisense ONTs taken up by cultured cells may act in the cytoplasm to silence the target mRNAs, perhaps via the siRNA pathway.
• Combining two antisense ONTs that act synergistically against two different mRNAs offers the potential to achieve therapeutic efficacy at a lower total ONT dose than either ONT could achieve alone. Researchers are exploiting this concept in developing an antisense inhalant for treating chronic obstructive pulmonary disease.

siRNA development and delivery

• Several research groups now favor packaging siRNAs into cationic lipid nanoparticles to protect the siRNAs in the circulation and enable efficient uptake into cells.
• One version of cationic lipid nanoparticles for targeting cells in solid tumors exploits the leaky vasculature typical of many solid tumors to selectively deliver the siRNA drugs to the tumor tissue.
• Nanoparticles delivered to tumor tissue through leaky vessels can be readily endocytosed by cancer cells via particle interaction with a cell surface receptor such as the sigma receptor.
• In another strategy for delivering the siRNA payload into cells, researchers made use of macropinocytosis, a pathway common to most cells, which involves uptake into vacuoles rather than vesicles.
• In one approach to siRNA entry into cells through the macropinocytosis pathway, the siRNAs are coupled to basic peptide carriers via a fragment of protein that normally binds to double-stranded RNA (dsRNA). This creates a complex that remains soluble and intact prior to cellular uptake and then dissociates once inside the cell.
• ONT developers aim to make drugs that can be delivered systemically for treating chronic ailments. In the siRNA category, these include developing an injectable antihyperlipidemic drug that works by inactivating an inhibitor of the low-density lipoprotein receptor (LDLR). Proof-of-concept studies of the siRNA formulation in animals show that single dose treatments can significantly increase LDLR, leading to a lowering of serum cholesterol levels.

Oligonucleotide therapeutics in the clinic

• Antisense ONTs carrying the 2′-methoxyethyl (2′MOE) modification, which gives them long half-lives and a high affinity for their target mRNA are now undergoing phase 3 and phase 2 trials for chronic conditions such as hypercholesterolemia and type 2 diabetes, respectively.
• Successful application of ONTs for combination chemotherapy in cancer treatment involves finding the patient subset most likely to respond. Investigators working on two different oncology ONTs each reported identifying specific biomarkers that may help to optimize the matching of drugs to patients.
• The FDA has given the first green light for clinical testing of a siRNA drug to be used systemically rather than just for local treatment. This is also the first clinical testing of a siRNA drug intended for treating cancer.

Oligonucleotide immunoreactives

• Chemical modification or base substitutions of CpG-containing oligodeoxynucleotides can convert them from immunostimulants into antagonists of immunostimulation. This property is being exploited to develop anti-inflammatory agents for treating autoimmune diseases.
• SiRNAs can elicit adverse immunostimulatory effects via interaction with Toll-like receptors (TLRs). Such unwanted side effects can be minimized without reducing on-target efficacy by chemically modifying either or both strands of the siRNA duplex.

The following pages offer a snapshot of a portion of what was covered at this comprehensive meeting on oligonucleotide therapeutics.