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:
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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;
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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,
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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.
Delivery
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:
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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;
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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;
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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,
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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.
Tertiary lymphoid structures linked to improved outcomes in liver cancer patients