RNAi: A New Class of Biological Therapeutics

RNA interference (RNAi) is a technology that can selectively knockdown gene expression. In contrast to previously used knockdown technologies, RNAi is a catalytic mechanism and hence only a few molecules are needed to yield efficient silencing of gene expression. Investigation of this biological phenomenon has shown it to be present in a wide range of species including Drosophila, zebrafish and mammals.

Two approaches are currently being explored by various commercial and academic institutions worldwide. One is the use of synthetic oligonucleotides together with functional conjugates or transfection carriers, the other is inverse complementary molecules expressed from plasmid or viral vectors. Both approaches are used extensively for target discovery and validation but could also eventually be developed into therapeutics, although systemic delivery of RNAi therapies to target tissues is still a major challenge. The opportunity to harness the RNAi pathway to silence disease-causing genes holds great promise, particularly for the development of therapeutics directed against targets that are otherwise not addressable with current medicines.

The BPDG at the New York Academy of Sciences represents a diverse group of scientists and others with an interest in biochemistry, molecular biology, biomedical research, and related areas. Members are from pharmaceutical and biotechnology companies, and university and medical center research facilities across the Eastern United States. The group also serves as the Biochemical Topical Group for the American Chemical Society's New York Section. The purpose of the BPDG is to bring together diverse institutions and communities, industrial and academic, to share new and relevant information at the frontiers of research and development.

Organizers: Angelika Bonin-Debs, Boehringer Ingelheim and Huiping Jiang, Boehringer Ingelheim

Speakers: Tod Woolf, RXi Pharmaceuticals; Art Levin, Levin BioSciences; Cy Stein, Albert Einstein College of Medicine; Andrew Siwkowski, Isis Pharmaceuticals; Steven Dowdy, UCSD School of Medicine; Kent Kirshenbaum, New York University; Aimee L. Jackson, Regulus Therapeutics

Agenda

8:30 - 9:00 AM
Registration and Continental Breakfast

9:00 - 9:10 AM
Introduction
Angelika Bonin-Debs, Boehringer Ingelheim

9:10 - 9:55 AM
30 Years of Targeting RNA: A Retrospective
Tod Woolf, RXi Pharmaceuticals

9:55 - 10:40 AM
Lessons Learned from Antisense
Art Levin, Levin BioSciences

10:40 - 11:00 AM
Refreshments

11:00 - 11:45 AM
Efficient Gene Silencing by the Gymnotic Delivery of Oligonucleotides
Cy Stein, Albert Einstein College of Medicine

11:45 AM - 12:30 PM
Evolution of Antisense Technology: From Laboratory Concept to Clinical Validation
Andrew Siwkowski, Isis Pharmaceuticals

12:30 - 1:30 PM
Luncheon

1:30 - 2:15 PM
The Road to Therapeutic RNA Interference: Tackling the 800-Pound siRNA Delivery Gorilla
Steven Dowdy, University of California, San Diego, School of Medicine

2:15 - 3:00 PM
Peptidomimetic Oligomers: A Modular Scaffold for siRNA Delivery
Kent Kirshenbaum, New York University

3:00 - 3:30 PM
Refreshments

3:30 - 4:15 PM
Opportunities for MicroRNAs as Therapeutics
Aimee L. Jackson, Regulus Therapeutics

4:15 - 4:50 PM
General Discussion

4:50 - 5:00 PM
Closing Remarks
Huiping Jiang, Boehringer Ingelheim

Abstracts

30 Years of Targeting RNA: A Retrospective
Tod Woolf, RXi Pharmaceuticals

Thirty years ago Paul Zamecnik treated mammalian cells with antisense oligonucleotides. Since this seminal work, various mechanisms for blocking RNA function in cells have been employed (blocking, RNase H cleavage, ribozymes cleavage and RISC-mediated cleavage) each with practical advantages and disadvantages. A vast array of modified oligonucleotides chemistries have been explored, but a surprisingly few modified chemistries have gained general use. This core group of modified chemistries (2' modified, phosphorothioate, neutral backbones) can be used in a great variety of configurations to impart favorable characteristics on therapeutic oligonucleotides. The affect of mechanism and chemistry on specificity, toxicity and potency on RNA targeting oligonucleotides will be discussed.

Lessons Learned from Antisense
Art Levin, Levin BioSciences

Single stranded oligonucleotides have been investigated as therapeutic agents in man for nearly 15 years. There have been successes and failures. The lessons learned from the development of antisense agents can be directly applied to the development of other RNA-targeting therapies, like siRNA. The current successes of antisense therapies are an outgrowth of investments in a thorough understanding of the chemistry, pharmacokinetics, toxicity and clinical safety that have accrued over a decade. This presentation will show specific examples of how concentrated efforts to understand basic issues in drug development have contributed to the advancement of the technology, and it will provide a background to discuss the next generations of RNA-targeting therapies.

Efficient Gene Silencing by the Gymnotic Delivery of Oligonucleotides
Cy Stein, Albert Einstein College of Medicine

It has long been thought that gene silencing by antisense oligonucleotides could be accomplished only by their transfection as particulates because of the impermeability of the cell membrane to polyanions. However, if the correct cell plating conditions are employed in vitro, and if the cells are exposed for a minimum of 6 days to physiologically relevant concentrations of 3',5' LNA phosphorothioate oligodeoxyribonucleotide gapmers, >90% gene silencing at the protein and mRNA level, with minimal to no toxicity or off target effects, can be routinely (n >> 50) observed in a large number of (but not all) cell lines. The presence of the LNAs is not an absolute requirement, as certain other oligos (e.g., G3139 [Genasense]) are also efficient silencing molecules. Several genes, including Bcl-2, survivin, the androgen receptor (in LNCaP cells, which are notoriously difficult to transfect with particulates without substantial toxicity), and Her3, have been successfully, and sequence specifically, silenced. We have named the entire delivery and silencing process gymnosis (> Gk gynmos = naked), because of the manner in which the oligos are delivered. Under the correct experimental conditions (including incorporation of continuous oligo treatment, a process we call iterative readdition), silencing can be continuously maintained in tissue culture for several hundred days. Nevertheless, removal of the antisense oligo results in complete recovery of the target to basal levels of expression by 3 days. High nuclear levels of oligo are not required for gene silencing. Instead, the LNA gapmer oligos, in a length and time dependent manner, localize to GW/P bodies. By immunoprecipitation techniques, we have demonstrated that LNA gapmer phosphorothioate oligos bind to Ago2; silencing of Ago2 in 518A2 melanoma cells by a shRNA strategy almost completely abolished gymnotic silencing even though RNaseH1 levels were unchanged. Finally, in vitro target silencing by gymnosis is highly predictive of in vivo silencing. These data suggest that the RNase H mechanism of oligonucleotide-mediated silencing is active subsequent to transfection when high nuclear concentrations of oligo are attained, but that an RNAi-like Ago2-dependent silencing mechanism may be more relevant physiologically. Acknowledgement: CAS gratefully acknowledges research support from Santaris Pharma (Copenhagen, DK) and membership on their SAB.

Evolution of Antisense Technology: From Laboratory Concept to Clinical Validation
Andrew Siwkowski, Isis Pharmaceuticals

Isis Pharmaceuticals is engaged in the discovery and development of antisense oligonucleotides for human therapeutic applications. A brief overview of recent progress will be presented. The remainder of the presentation will focus on the preclinical research and development of an antisense oligonucleotide (ASO) targeting sodium dependent glucose transporter type 2 (SGLT2). SGLT2 is a high capacity low affinity glucose transporter located on the apical surface of the proximal tubule epithelial cell. Humans who are homozygous for loss of function mutations in SGLT2 have glucosuria. In these individuals, euglycemia is maintained due to the glucose absorption of SGLT1. We have identified and characterized ASOs targeting SGLT2 in multiple species. ASO treatment to deplete SGLT2 was well tolerated and resulted in benign glucosuria. The level of glucosuria was sufficient to cause a significant reduction in HbA1C in ZDF rats. Studies characterizing PK/PD in multiple species as well as pharmacological effect will be discussed.

The Road to Therapeutic RNA Interference: Tackling the 800-Pound siRNA Delivery Gorilla
Steven Dowdy, University of California, San Diego, School of Medicine

Short interfering RNA (siRNA) induced RNA interference (RNAi) has great potential for treating human disease, especially cancer and viral diseases. However, due to the ~14,000 Dalton siRNA size and extensive anionic charge, siRNAs have limited to no bioavailability to enter unperturbed cells. Thus siRNA delivery remains the rate-limiting step for development of RNAi therapeutics. To address the siRNA delivery problem, we developed a Peptide Transduction Domain-dsRNA Binding Domain (PTD-DRBD) fusion protein siRNA delivery approach. DRBDs bind siRNAs with high avidity independent of sequence, mask the negative charge and allow for PTD-mediated siRNA delivery. PTD-DRBD delivered siRNAs induced RNAi responses in the entire cell population of 20+ cell types assayed in a non-cytotoxic fashion, including primary human umbilical vein endothelial cells (HUVEC), primary human fibroblasts, primary T cells, hematopoietic lineages and human embryonic stem cells. Moreover, PTD-DRBD delivered EGFRvIII siRNAs resulted in EGF Receptor knockdown in intracerebral glioblastoma tumors in vivo. PTD-DRBD combinatorial delivery of EGFR and Akt2 siRNAs synergized to induce a synthetic lethal response that significantly increased survival in intracerebral pre-clinical models of glioblastoma, whereas delivery of irrelevant control siRNAs did not alter longevity. Taken together, these observations demonstrate the ability of PTD-DRBD mediated siRNA delivery to induce synthetic lethal responses in pre-clinical mouse models of cancer.

Peptidomimetic Oligomers: A Modular Scaffold for siRNA Delivery
Kent Kirshenbaum, New York University

The potential for RNA interference as a new therapeutic modality is becoming increasingly evident. However, delivery of RNA oligonucleotides into cells remains a critical challenge. Advanced protocols for RNA delivery may allow for improved pharmacokinetics, cell-type specific targeting, and diminished off-target effects. There is a need for non-viral reagents that will permit efficient RNA transfection with low cytotoxicity and enable administered RNA species to manifest their capability for specific gene knock-down in a wide variety of cell types. Lipitoids are cationic oligopeptoid–phospholipid conjugates that show promise for transfection of synthetic siRNA oligos in cell culture. This peptidomimetic delivery vehicle allows for efficient siRNA transfection in a variety of human cell lines with negligible toxicity and promotes extensive downregulation of the targeted genes at both the protein and the mRNA level. A critical focus is placed on investigating the mechanism of complexation and transfection. In particular, recent studies are presented to evaluate the nature of nanoparticles formed upon association of lipitoid and RNA oligos. Prospects for development of a robust platform enabling cell type-specific targeting are discussed.

Opportunities for MicroRNAs as Therapeutics
Aimee L. Jackson, Regulus Therapeutics

MicroRNAs are endogenous non-coding RNAs that post-transcriptionally regulate gene expression. Largely unknown before 2001, it is now clear that microRNAs can act as master regulators in biological pathways central to many areas of biology including development, cancer, metabolism, and immunity. The ability to modulate multiple genes in disease pathways makes microRNA an exciting new platform for drug discovery and development in a variety of disease areas. microRNA function can be inhibited through use of antisense oligonucleotides, or anti-miRs, or can be agonized through use of mimics, enabling both positive and negative regulation of microRNA function. We will discuss the potential for microRNAs as drugs, and our progress in optimization of microRNA modulators for potency and in vivo delivery. Furthermore, we will provide an update on targeting microRNAs for immunology and other therapeutic applications.