Alnylam and Collaborators Present Data from Multiple Pre-Clinical and Clinical Programs at RNAi Keystone Symposium

New Data Highlight Broad Applications of RNAi Therapeutics and Continued Progress in Advancing Pipeline of Innovative Medicines

CAMBRIDGE, MA, USA | February 12, 2009 | 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 “Therapeutic Modulation of RNA Using Oligonucleotides” Keystone Symposium held February 8-13, 2009 in Lake Louise, Alberta, Canada. Alnylam and its collaborators presented data from Alnylam’s therapeutic programs including respiratory syncytial virus (RSV), liver cancer, transthyretin (TTR) amyloidosis, and Ebola, as well as new data on delivery approaches for the delivery of RNAi therapeutics.

“Alnylam scientists and collaborators continue to make significant progress with RNAi therapeutics in important areas of unmet medical need. We are encouraged by the data presented at this meeting from our RSV, liver cancer, TTR amyloidosis, and Ebola programs which show potent and specific in vitro and in vivo efficacy,” said Jack Schmidt, M.D., Chief Scientific Officer of Alnylam. “We are also excited by the progress we and our collaborators are making in the delivery of RNAi therapeutics and in the further understanding of the molecular mechanisms underlying intracellular transport of siRNAs. We believe that these cumulative data demonstrate Alnylam’s scientific leadership and commitment to translating the science of RNAi into a new class of innovative medicines.”

Respiratory Syncytial Virus

Alnylam is developing its ALN-RSV program for the treatment of RSV infection; the program includes ALN-RSV01 which is currently in Phase II clinical trials. In a poster titled “RNAi-Mediated Silencing of the Respiratory Syncytial Virus Nucleocapsid Defines a Potent Anti-Viral Strategy,” Alnylam scientists and collaborators showed new data demonstrating that the target site of ALN-RSV01 is highly conserved across clinical isolates of RSV and showed new pre-clinical data regarding the ability to achieve substantially improved anti-viral potency with multi-dose administration. Further, the studies demonstrated that non-specific, immunostimulatory small interfering RNAs (siRNAs, the molecules that mediate RNAi) lack anti-viral activity in animal models and that ALN-RSV01 mediates its anti-viral activity through an RNAi-mediated mechanism as measured by 5’RACE. ALN-RSV is being developed in collaboration with Cubist Pharmaceuticals, Inc. and Kyowa Hakko Kirin Co., Ltd.

Liver Cancer

Alnylam is developing a systemically delivered RNAi therapeutic, ALN-VSP, for the treatment of liver cancers, including hepatocellular carcinoma and other solid tumors with liver involvement. ALN-VSP comprises two siRNAs formulated in a lipid nanoparticle in collaboration with Tekmira Pharmaceuticals Corporation. ALN-VSP is designed to target two genes critical for the growth and development of cancer cells: kinesin spindle protein, or KSP, required for tumor proliferation; and vascular endothelial growth factor, or VEGF, required for growth of tumor blood vessels. In a poster titled “Advancement to the Clinic of ALN-VSP, an RNAi Therapeutic for Solid Tumors,” Alnylam scientists presented new in vivo data from its ALN-VSP program.

In an orthotopic liver tumor model, pre-clinical data with ALN-VSP demonstrated the following:

* robust anti-tumor efficacy, including the inhibition of tumor growth as measured by serum levels of alpha-fetoprotein (AFP);
* proof of an RNAi mechanism of action toward both KSP and VEGF mRNAs, as determined by 5’RACE assays;
* statistically significant improvement in median survival for ALN-VSP-treated animals as compared to controls;
* statistically significant increase in survival of ALN-VSP treated animals compared to treatment with sorafenib (Nexavar®), an approved drug for the treatment of hepatocellular carcinoma; and,
* evidence for an even greater increase in survival benefit in animals treated with both ALN-VSP and sorafenib, suggesting that a combination of both treatments could provide an improved effect on overall survival.

In addition, Alnylam scientists and collaborators at the University of California, San Francisco (UCSF) and the David H. Koch Institute for Integrative Cancer Research at the Massachusetts Institute of Technology (MIT) presented new data in a poster titled “Depletion of the Mitotic Kinesin EG5/KSP by In Vivo Delivery of siRNA Inhibits Growth of De Novo Liver Tumors.” Using a transgenic model of liver cancer driven by the over-expression of the oncogene MYC, levels of KSP were found to be over-expressed in association with increased levels of MYC and de novo tumor formation. Potent anti-tumor effects were observed in mice treated with a KSP-specific siRNA as compared to controls. In addition, increased apoptosis of tumor cells was detected in association with KSP silencing.

Finally, in studies with tumor samples from 72 patients with hepatocellular carcinoma, an over three-fold increased KSP expression was documented as compared with matched control liver samples. Further, increased KSP expression was associated with more severe stages of human liver cancer. These data provide further validation of KSP as a target for ALN-VSP and its development for the treatment of liver cancers.

Alnylam expects to initiate a Phase I trial for ALN-VSP in the first half of 2009.

TTR Amyloidosis

Alnylam is developing ALN-TTR, a systemically delivered RNAi therapeutic for the treatment of TTR amyloidosis, comprised of an siRNA targeting the TTR gene and formulated in lipid nanoparticles in collaboration with Tekmira. In a poster titled “Development of an RNAi Therapeutic Targeting Transthyretin for the Treatment of Familial Amyloidotic Polyneuropathy,” Alnylam scientists presented in vivo data demonstrating the potential therapeutic benefit of ALN-TTR.

These data, which were previously presented in November 2008 at the Cambridge Healthtech Institute’s Drug Formulation conference, showed that potent RNAi therapeutics targeting TTR significantly reduced the levels of target mRNA in the liver and TTR protein levels in circulation. Specifically, data from these studies demonstrated:

* dose-dependent silencing of TTR in vitro and absence of any immune stimulatory effects;
* dose-dependent efficacy in vivo with reduced liver TTR mRNA and TTR plasma levels by greater than 90% in the V30M-hTTR transgenic mouse model; and,
* dose-dependent reductions in liver TTR mRNA levels by approximately 80% in non-human primates.

ALN-TTR is a potential investigational new drug (IND) candidate for 2009.

Alnylam BioDefense/Ebola

Alnylam is developing an RNAi therapeutic directed against the Ebola virus, which can cause a severe, usually fatal infection, and poses a potential biological safety risk and bioterrorism threat. In a poster titled “RNAi Therapeutics for the Treatment of Ebola Virus Infection,” pre-clinical studies in collaboration with scientists from Tekmira were presented.

Pre-clinical data showed the following:

* siRNAs targeting every viral gene were tested in vitro and in vivo for anti-viral activity leading to the identification of VP35 as the most potent anti-viral RNAi therapeutic target;
* a greater than 95% decrease in viral titer was seen when the VP35 siRNA was administered to mice infected with Ebola; and,
* the VP35 siRNA, as compared with a control non-specific siRNA, was able to protect both mice and guinea pigs from lethal Ebola infection.

Alnylam is working with the United States Army Medical Research Institute of Infectious Diseases (USAMRIID), an organization which is uniquely experienced in the handling, safety, and security requirements of specialized biological agents. The National Institute of Allergy and Infectious Diseases (NIAID), a component of the National Institutes of Health (NIH), is funding this work through a federal contract (No. HHSN26620060012C).

Specificity of RNAi Mechanism

In a poster titled “RNAi-Mediated Silencing Independent of TLR-Mediated Signaling,” Alnylam scientists and collaborators at MIT presented in vivo data highlighting the specificity of the RNAi mechanism in regards to toll-like receptors (TLRs), which have been shown to play a role in the activation of innate immunity and have been implicated as targets for siRNA binding that could lead to undesired immune stimulatory effects. In this study, the assessment of TLR involvement in RNAi-mediated silencing of lung- and hepatocyte-expressed target genes was explored. Using knock-out mice lacking certain key TLRs, either TLR-3 or TLR-7, anti-viral activity of siRNAs targeting RSV infection in the lung and RNAi-mediated silencing of a hepatocyte-expressed gene, Factor VII, were evaluated. Results showed that siRNAs targeting RSV demonstrated potent anti-viral efficacy in wild type mice as expected, but also retained full anti-viral efficacy in both TLR-3 and TLR-7 knock-out mice. Further, following systemic administration of liposomally formulated siRNAs, RNAi-mediated silencing of Factor VII in hepatocytes was found to be independent of TLR-3 and TLR-7. Also, RNAi-mediated, TLR-independent in vivo efficacy was achieved with both unmodified and chemically-modified siRNAs. These data clearly demonstrate that RNAi-mediated therapeutic effects can be achieved independent of immune stimulatory mechanisms.


In a presentation titled “Medicinal Chemistry of siRNAs: Towards Improving the Potency of siRNAs,” Muthiah Manoharan, Ph.D., Vice President, Drug Discovery of Alnylam presented new in vitro and in vivo data related to the effective delivery of RNAi therapeutics by evaluating chemical modifications of both the sense and antisense strands of double-stranded RNAs. Data presented showed that among the numerous modifications to improve the drug-like properties of siRNAs, 2’-Fluoro modifications in both strands consistently provided increased target binding affinity, endonuclease stability, and increased potency. In addition, destabilization of the sense strand through other modifications such as non-canonical bases, mismatches, and abasic sites at strategic regions also played a role in improving siRNA potency in vitro.

A previous study by Alnylam scientists in collaboration with scientists from MIT (Akinc et al., Nature Biotechnology 26, 561-569 (01 May 2008)), showed successful delivery of siRNAs encapsulated in “lipidoid” formulations when administered in multiple animal species including mice, rats, and non-human primates. In a poster titled “Development of a Lipidoid Formulation for the Systemic Delivery of siRNA to Liver,” new pre-clinical data showed that lipidoid formulations achieve delivery of greater than 90% of the administered siRNA dose to the liver and maintain robust in vivo activity following repeat administration indicating no evidence of neutralizing antigenicity or tachyphylaxis.

In addition, Alnylam scientists, in collaboration with the Max Planck Institute of Molecular Cell Biology and Genetics, presented data in a poster titled “Intracellular Trafficking of Liposomally Encapsulated and Cholesterol Conjugated siRNA” on the investigation of cellular uptake mechanisms for RNAi therapeutics using different delivery approaches. The study was performed using high content confocal microscopic images of single cells exposed to liposomally formulated and cholesterol-conjugated siRNAs. Data presented in the poster showed the following:

* using a lipidoid formulation, separation of the siRNA from the lipid carrier was observed within one hour, and significant mRNA silencing was detected within four hours following exposure to cells, suggesting rapid cellular uptake and release of the siRNA from the particle within the first two hours; and,
* using a cholesterol-conjugated siRNA, cellular localization revealed a perinuclear pattern of staining suggestive of association with endoplasmic reticulum, with a smaller fraction of siRNA residing in lysosomes.

microRNA Therapeutics

Alnylam is developing microRNA therapeutics through its ownership of and collaboration with Regulus Therapeutics. In a presentation titled “Therapeutic Targeting of microRNAs,” Regulus scientists presented new data on the development of microRNA antagonists, or “anti-miRs” as follows:

* modification of anti-miRs with 2’-Fluoro/MOE chemistry was found to be superior to use of MOE chemistry alone, as measured in both rodents and non-human primates for antagonism of miR-122;
* repeat dosing studies in non-human primates for anti-miR-122 demonstrated de-repression of aldolase A mRNA levels, decreases in total cholesterol and LDL-cholesterol, but also decreases in HDL-cholesterol; and,
* in mechanistic studies in mice, anti-miR-122 was found to de-repress certain enzymes in the Kreb’s cycle leading to increased hepatic levels of alpha-ketoglutarate.

In an additional presentation titled “Are Target and Off-Target Regulation by RNAi Therapeutics Conserved Across Species?,” Regulus scientists presented new data highlighting the importance of conducting studies in non-human primates and human cellular assays in order to understand the biological effects of microRNA antagonism. These studies showed that while microRNAs are highly conserved throughout evolution, microRNA target sites are more diversified.

All together, these studies highlight a methodical and comprehensive approach to micro-RNA based drug discovery that focuses on understanding the informatics and biology of microRNAs and their biological targets, as well as the in vivo pharmacology of anti-miRs to develop the most suitable clinical candidates for the appropriate therapeutic areas.

RNA Activation (RNAa)

RNAa can be achieved with double-stranded RNAs, called “antigene RNAs” or “agRNAs,” that target 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, UCSF, and the University of Texas Southwestern Medical Center (UTSW). In a poster titled “Development of Chemically-Modified Antigene RNAs (agRNAs),” Alnylam scientists and collaborators at UTSW have demonstrated for the first time the ability of using chemical modifications to stabilize agRNAs without loss of activity for target gene activation. Since chemical modifications may be required for introducing “drug like properties” to agRNAs, such as stability and specificity, this study highlights Alnylam’s progress in advancing this new platform toward in vivo proof of concept.

About RNA Interference (RNAi)

RNAi (RNA interference) is a revolution in biology, representing a breakthrough in understanding how genes are turned on and off in cells, and a completely new approach to drug discovery and development. Its discovery has been heralded as “a major scientific breakthrough that happens once every decade or so,” and represents one of the most promising and rapidly advancing frontiers in biology and drug discovery today which was awarded the 2006 Nobel Prize for Physiology or Medicine. RNAi is a natural process of gene silencing that occurs in organisms ranging from plants to mammals. By harnessing the natural biological process of RNAi occurring in our cells, the creation of a major new class of medicines, known as RNAi therapeutics, is on the horizon. RNAi therapeutics target the cause of diseases by potently silencing specific messenger RNAs (mRNAs), thereby preventing disease-causing proteins from being made. RNAi therapeutics have the potential to treat disease and help patients in a fundamentally new way.

About Alnylam Pharmaceuticals

Alnylam is a biopharmaceutical company developing novel therapeutics based on RNA interference, or RNAi. The company is applying its therapeutic expertise in RNAi to address significant medical needs, many of which cannot effectively be addressed with small molecules or antibodies, the current major classes of drugs. Alnylam is leading the translation of RNAi as a new class of innovative medicines with peer-reviewed research efforts published in the world’s top scientific journals including Nature, Nature Medicine, and Cell. The company is leveraging these capabilities to build a broad pipeline of RNAi therapeutics; its most advanced program is in Phase II human clinical trials for the treatment of respiratory syncytial virus (RSV) infection and is partnered with Cubist and Kyowa Hakko. In addition, the company is developing RNAi therapeutics for the treatment of a wide range of disease areas, including liver cancers, hypercholesterolemia, Huntington’s disease, and TTR amyloidosis. The company’s leadership position in fundamental patents, technology, and know-how relating to RNAi has enabled it to form major alliances with leading companies including Medtronic, Novartis, Biogen Idec, Roche, Takeda, Kyowa Hakko, and Cubist. To reflect its outlook for key scientific, clinical, and business initiatives, Alnylam established “RNAi 2010” in January 2008 which includes the company’s plan to significantly expand the scope of delivery solutions for RNAi therapeutics, have four or more programs in clinical development, and to form four or more new major business collaborations, all by the end of 2010. Alnylam is a joint owner of Regulus Therapeutics, a joint venture focused on the discovery, development, and commercialization of microRNA therapeutics. Founded in 2002, Alnylam maintains headquarters in Cambridge, Massachusetts. For more information, please visit

SOURCE: Alnylam Pharmaceuticals, Inc.

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