Alnylam Pharmaceuticals presents data on multiple therapeutic programs at RNAi Symposium

Alnylam Pharmaceuticals, Inc. (Nasdaq: ALNY), a leading RNAi therapeutics company, today announced that it presented data from multiple pre-clinical and clinical programs at the “RNA Silencing: Mechanism, Biology, and Application” Keystone Symposium held January 14-19, 2010 in Keystone, Colorado. Alnylam and its collaborators presented data from Alnylam’s therapeutic programs including transthyretin (TTR)-mediated amyloidosis and Huntington’s disease, as well as new data on delivery approaches for the systemic delivery of RNAi therapeutics. Further, results were presented for the first-ever comprehensive analysis of “canonical” siRNAs compared to “dicer substrate” siRNAs, showing a superior performance of “canonical” siRNAs.

“Alnylam scientists and collaborators continue to make significant progress in advancing the translation of RNAi to create a robust drug discovery platform for innovative medicines”

“Alnylam scientists and collaborators continue to make significant progress in advancing the translation of RNAi to create a robust drug discovery platform for innovative medicines,” said Victor Kotelianski, M.D., Ph.D., D.Sc., Senior Vice President, Senior Alnylam Fellow. “We are encouraged by the data presented at this meeting from our TTR-mediated amyloidosis and Hungtinton’s disease therapeutic programs, amongst others. We are also excited by the remarkable progress we and our collaborators are making in the delivery of RNAi therapeutics. Finally, our comprehensive study comparing in vitro and in vivo properties of canonical and dicer substrate siRNAs confirms that the naturally occurring canonical siRNA structure is indeed superior.”

Structural Activity of siRNA Designs

In a poster titled “Comprehensive Evaluation of Canonical vs. Dicer-substrate siRNAs in vitro and in vivo,” Alnylam scientists presented data on the potency of dicer-substrate siRNA compared to canonical siRNA constructs. The study demonstrated comparable in vitro and in vivo potencies, including durability, for canonical and dicer substrate siRNAs. However, canonical siRNAs were found to show superior performance regarding tolerability.

Specifically, two well-characterized genes – PTEN and Factor VII – were investigated as RNAi targets, and over 300 siRNAs were designed, synthesized, and assayed in vitro and in vivo. The data showed that:

  • highly active siRNAs can be identified with both approaches and are comparable in potency, both in vitro and in vivo, including durability of target gene silencing;
  • dicer substrate siRNAs were found to be less tolerant to the introduction of chemical modifications as compared to canonical siRNAs, making it more difficult to use chemical modifications to abrogate potential immune stimulatory properties; and,
  • finally, dicer substrate siRNAs exhibited increased cellular toxicity in vitro compared to canonical siRNAs, likely due to interference with endogenous microRNA biogenesis.

Transthyretin (TTR)-Mediated Amyloidosis (ATTR)

Alnylam is developing ALN-TTR, a systemically delivered RNAi therapeutic targeting the TTR gene for the treatment of ATTR, including familial amyloidotic polyneuropathy (FAP) and familial amyloidotic cardiomyopathy (FAC). In a poster titled “Development of ALN-TTR, an RNAi therapeutic for the treatment of transthyretin amyloidosis,” Alnylam scientists and collaborators presented pre-clinical data further demonstrating the potential therapeutic benefit of an RNAi therapeutic targeting TTR for the treatment of ATTR.

The new studies were performed by Alnylam scientists in collaboration with Maria Joao Saraiva, Ph.D. of the Institute for Molecular and Cell Biology in Portugal in a transgenic mouse model where the human V30M mutated TTR is over-expressed. Data from these studies, which were presented at Alnylam’s R&D Day in November 2009, demonstrated that the administration of ALN-TTR is associated with markedly reduced pathogenic deposition of mutant TTR in tissues. In the transgenic mouse model, results showed that the administration of ALN-TTR, as compared with control siRNA treatment, led to a marked and nearly complete reduction of mutant TTR protein accumulation by over 95% in peripheral tissues affected by disease, including the sciatic nerve, sensory ganglion, intestine, esophagus, and stomach - tissues that are associated with the sensory and autonomic neuropathy and the severe gastrointestinal dysfunction observed in patients with ATTR. The therapeutic efficacy for ALN-TTR was measured approximately two months after dosing.

Alnylam intends to initiate a Phase I trial in the first half of 2010 for its systemic RNAi therapeutic, ALN-TTR01 for the treatment of ATTR. ALN-TTR01 utilizes a first generation stable nucleic acid-lipid particles (SNALP) formulation developed in collaboration with Tekmira Pharmaceuticals Corporation. In addition, Alnylam is advancing ALN-TTR02 using its second generation LNP platform.

Huntington’s Disease

Alnylam is developing ALN-HTT, a drug-device combination for the treatment of Huntington’s disease, in collaboration with Medtronic, Inc. In a poster titled “Developing RNAi therapeutics targeting huntingtin with direct CNS delivery,” the two companies, in collaboration with Professor Don M. Gash’s laboratory at the University of Kentucky College of Medicine, presented in vivo data demonstrating the potential therapeutic benefit of ALN-HTT. These pre-clinical data, some of which were previously presented at the 2009 World Congress on Huntington’s Disease in September 2009, demonstrated that an siRNA targeting the huntingtin gene achieves broad distribution in the CNS following continuous direct CNS administration, as well as robust silencing of the huntingtin gene mRNA. Data also showed that silencing was achieved at substantial distances from the infusion site, and was well tolerated following continuous direct CNS administration over a period of approximately one month. New pre-clinical research presented at this meeting showed that silencing of the huntingtin gene mRNA persists for 14 days.


In a poster titled “Lipophilic siRNA Delivery by Reconstituted Lipoprotein Particles In Vivo,” Alnylam scientists presented new research on the rational design of mimetic lipoprotein particles (MLPs) as an RNAi delivery platform. MLPs were designed to mimic the physiologic properties of endogenous lipoprotein particles and were engineered using recombinant human apolipoprotein A1 (rh-apoA1) or recombinant human apolipoprotein E (rh-apoE), phosphatidylcholine, and a cholesterol-conjugated siRNA (chol-siRNA). The resulting MLPs had biophysical properties comparable to normal high-density lipoprotein (HDL, or “good” cholesterol) particles including a mean diameter size of approximately 10nm. The new data demonstrated that administration of MLPs formulated with specific chol-siRNA resulted in silencing of multiple targets in vivo, including the PCSK9 mRNA by up to 70% and the Factor VII mRNA by 45% at the dose levels tested. These data expand previous studies that showed silencing of the apoB mRNA by up to 80% with an associated 50 to 80% reduction in levels of plasma apoB protein and cholesterol in mice. Furthermore, in vivo biodistribution data demonstrated that the MLPs were rapidly cleared from blood with significant uptake in liver, gut, and kidneys.

In addition, Alnylam scientists presented new pre-clinical data in a poster titled “Carbohydrate Conjugation to siRNA for Tissue and Cell Specific Delivery” on the design and synthesis of conjugated siRNAs using the carbohydrate ligand, N-acetylgalactosamine (GalNAc), as a ligand for the targeted delivery of RNAi therapeutics. These data, which extend previous pre-clinical research on the effects of cholesterol-conjugated siRNAs, demonstrated specific cellular binding, uptake, and gene silencing both in vitro and in vivo mediated by GalNAc-conjugated siRNA. Furthermore, siRNA conjugates containing both cholesterol and GalNAc showed further enhanced potency in vivo compared to siRNAs conjugated with the individual ligands alone. These effects were demonstrated across multiple liver-expressed gene targets, including PCSK9, Factor VII, and apoB.

Finally, in a presentation titled “Combinatorial Development of Synthetic siRNA Delivery Systems,” Alnylam collaborator Daniel Anderson, Ph.D. of the David H. Koch Institute for Integrative Research at the Massachusetts Institute of Technology (MIT) presented data recently published in the journal Proceedings of the National Academy of Sciences (PNAS) (Love et al. (January 11, 2010) Proc. Natl Acad. Sci. USA, 10.1073/pnas.0910603106). The presentation described advances in discovery and development of novel “lipidoid” formulations for the systemic delivery of RNAi therapeutics. In particular, the new pre-clinical research findings demonstrate the discovery of new lipidoid materials that facilitate significantly improved in vivo potency for RNAi therapeutics. These new lipidoid materials comprise part of Alnylam’s second generation lipid nanoparticle (LNP) platform.

RNA Activation (RNAa)

RNAa can be achieved with double-stranded RNAs, called “antigene RNAs” or “agRNAs,” that target non-coding RNA transcripts complementary to gene promoters, and represents a new approach for RNA therapeutics with gene activation. Alnylam has formed collaborations and in-licensed intellectual property for RNAa from The Salk Institute, the University of California San Francisco (UCSF), and the University of Texas Southwestern Medical Center (UTSW). In a poster titled “Regulation of Transcription by Small RNAs Complementary to Sequences Downstream from the 3’ Termini of Genes,” scientists at UTSW, in collaboration with Alnylam, presented pre-clinical data that demonstrated that certain agRNAs, called “3’-agRNAs”, complementary to the 3’ region of genes can inhibit or activate gene expression. 3’-agRNAs were found to act through sequence-specific recognition of a 3’ non-coding transcript of the target gene in a manner dependent on the protein argonaute 2, a component of the RNAi enzyme machinery. In addition, the identification of an endogenous inhibitory microRNA that may target non-coding RNA transcripts complementary to the 3’ terminal region of genes suggests a potential physiological significance for 3’-agRNAs.

microRNA Therapeutics

In addition, Regulus Therapeutics Inc. also presented new pre-clinical data from multiple therapeutic programs at the conference including:

  • a poster titled “microRNA mimics as cancer therapeutics,” in which Regulus scientists presented in vivo data demonstrating delivery of miR mimics and microRNA target repression in an orthotopic liver tumor mouse model;
  • a poster titled “Inhibition of microRNA function in macrophages by anti-miRs,” in which Regulus scientists in collaboration with its partner GlaxoSmithKline provided the initial demonstration of a pharmacological effect in immune cells by specific microRNA inhibition;
  • a presentation titled “Myeloproliferative disease, autoimmunity and cancer in mice with targeted deletion of miR-146a gene,” in which Regulus scientists and collaborators from the California Institute of Technology presented data that demonstrated the biological role of miR-146a by establishing loss- and gain-of-function mouse models; and,
  • a poster titled “Identification of miR-122 conserved targets in liver affecting cholesterol regulation,” in which Regulus scientists presented expression profiling data from livers of anti-miR-122-treated cynomolgus monkeys, mice, and rats.

Alnylam Pharmaceuticals


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