Publications

alpha-Synuclein Suppression by Targeted Small Interfering RNA in the Primate Substantia Nigra – August 11th, 2010

A.L. McCormack et al 2010 Aug 11;5(8). pii: e12122

Abstract:

The protein alpha-synuclein is involved in the pathogenesis of Parkinson's disease and other neurodegenerative disorders. Its toxic potential appears to be enhanced by increased protein expression, providing a compelling rationale for therapeutic strategies aimed at reducing neuronal alpha-synuclein burden. Here, feasibility and safety of alpha-synuclein suppression were evaluated by treating monkeys with small interfering RNA (siRNA) directed against alpha-synuclein. The siRNA molecule was chemically modified to prevent degradation by exo- and endonucleases and directly infused into the left substantia nigra. Results compared levels of alpha-synuclein mRNA and protein in the infused (left) vs. untreated (right) hemisphere and revealed a significant 40-50% suppression of alpha-synuclein expression. These findings could not be attributable to non-specific effects of siRNA infusion since treatment of a separate set of animals with luciferase-targeting siRNA produced no changes in alpha-synuclein. Infusion with alpha-synuclein siRNA, while lowering alpha-synuclein expression, had no overt adverse consequences. In particular, it did not cause tissue inflammation and did not change (i) the number and phenotype of nigral dopaminergic neurons, and (ii) the concentrations of striatal dopamine and its metabolites. The data represent the first evidence of successful anti-alpha-synuclein intervention in the primate substantia nigra and support further development of RNA interference-based therapeutics.

Summary:

The published data demonstrated that direct delivery of chemically modified siRNAs specific for alpha-synuclein resulted in significant silencing of the target gene in the substantia nigra in the non-human primate brain. A significant 40-50% suppression of both alpha-synuclein mRNA and protein levels was observed in treated animals, as compared to controls. In these preliminary studies, the siRNA was found to be well tolerated after direct CNS administration; no complications or adverse events were observed, including the absence of detectable microglial activation or change in the number of nigral dopaminergic neurons. These results suggest that RNAi therapeutics may be useful in reducing the pathogenic burden of alpha-synuclein in patients with Parkinson’s disease. The study was conducted in collaboration with scientists at Mayo Clinic and The Parkinson’s Institute, and funded by a “LEAPS” (Linked Efforts to Accelerate Parkinson’s Solutions) award from The Michael J. Fox Foundation for Parkinson’s Research.

From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus – August 5th, 2010

K. Musunuru et. al., Nature, 466, 714-719, 2010

Abstract:

Recent genome-wide association studies (GWASs) have identified a locus on chromosome 1p13 strongly associated with both plasma low-density lipoprotein cholesterol (LDL-C) and myocardial infarction (MI) in humans. Here we show through a series of studies in human cohorts and human-derived hepatocytes that a common noncoding polymorphism at the 1p13 locus, rs12740374, creates a C/EBP (CCAAT/enhancer binding protein) transcription factor binding site and alters the hepatic expression of the SORT1 gene. With small interfering RNA (siRNA) knockdown and viral overexpression in mouse liver, we demonstrate that Sort1 alters plasma LDL-C and very low-density lipoprotein (VLDL) particle levels by modulating hepatic VLDL secretion. Thus, we provide functional evidence for a novel regulatory pathway for lipoprotein metabolism and suggest that modulation of this pathway may alter risk for MI in humans. We also demonstrate that common noncoding DNA variants identified by GWASs can directly contribute to clinical phenotypes.

Summary:

The work is part of a collaborative effort focused on validating novel genes as targets for new therapies for lipid disorders and heart disease, and provides early proof of concept on how GWAS combined with RNAi technology can yield important new insights into disease biology. Recent GWAS - including a report published in the same issue of Nature - have identified a locus on chromosome 1p13 as strongly associated with both high LDL-C levels and incidence of MI and CHD. The findings published in Nature today show through a series of studies in patient cohorts and human-derived hepatocytes that a common non-coding polymorphism at the 1p13 locus creates a novel transcription factor binding site which in turn increases the expression of several liver genes. Using a combination of over-expression and RNAi silencing techniques, the gene Sort1 was identified as a candidate gene responsible for the associated CHD effects. Knockdown in mouse liver using RNAi demonstrated that Sort1 alters plasma LDL-C and very low-density lipoprotein (VLDL) particle levels by modulating hepatic VLDL secretion.

Transcriptional regulation by small RNAs at sequences downstream from 3' gene termini – June 28th, 2010

X Yue, et al., Nature Chemical Biology, 10.1038/nchembio.400

Abstract:

Transcriptome studies reveal many noncoding transcripts overlapping 3′ gene termini. The function of these transcripts is unknown. Here we have characterized transcription at the progesterone receptor (PR) locus and identified noncoding transcripts that overlap the 3′ end of the gene. Small RNAs complementary to sequences beyond the 3′ terminus of PR mRNA modulated expression of PR, recruited argonaute 2 to a 3′ noncoding transcript, altered occupancy of RNA polymerase II, induced chromatin changes at the PR promoter and affected responses to physiological stimuli. We found that the promoter and 3′ terminal regions of the PR locus are in close proximity, providing a potential mechanism for RNA-mediated control of transcription over long genomic distances. These results extend the potential for small RNAs to regulate transcription to target sequences beyond the 3′ termini of mRNA

Summary:

These data demonstrate a novel mechanism whereby gene expression can be selectively regulated by anti-gene RNAs (agRNAs), double-stranded RNAs that target non-coding RNA sequences.

Influenza A virus-generated small RNAs regulate the switch from transcription to replication – June 1st, 2010

J.T. Perez et al., Proc. Natl Acad. Sci. USA, doi: 10.1073/pnas.1001984107, 2010

Abstract:

The discovery of regulatory small RNAs continues to reshape paradigms in both molecular biology and virology. Here we describe examples of influenza A virus-derived small viral RNAs (svRNAs). svRNAs are 22-27 nt in length and correspond to the 5′ end of each of the viral genomic RNA (vRNA) segments. Expression of svRNA correlates with the accumulation of vRNA and a bias in RNA-dependent RNA polymerase (RdRp) activity from transcription toward genome replication. Synthesis of svRNA requires the RdRp, nucleoprotein and the nuclear export protein NS2. In addition, svRNA is detectable during replication of various influenza A virus subtypes across multiple host species and associates physically with the RdRp. We demonstrate that depletion of svRNA has a minimal impact on mRNA and complementary vRNA (cRNA) but results in a dramatic loss of vRNA in a segment-specific manner. We propose that svRNA triggers the viral switch from transcription to replication through interactions with the viral polymerase machinery. Taken together, the discovery of svRNA redefines the mechanistic switch of influenza virus transcription/replication and provides a potential target for broad-range, anti-influenza virus-based therapeutics.

Summary:

In the study, Alnylam and Mount Sinai scientists discovered a novel class of virus-produced small non-coding RNAs, called small viral RNAs (svRNAs), which play a critical role in the replication of influenza A virus. Antagonism of these svRNAs resulted in decreased viral mRNA and blocked viral infectivity. These new findings provide further understanding of how influenza virus is able to infect cells, and identify new targets and strategies for anti-influenza virus-based therapies.

Targeted Delivery of RNAi Therapeutics With Endogenous and Exogenous Ligand-Based Mechanisms – May 11th, 2010

A. Akinc, et. al. Molecular Therapy (2010); doi:10.1038/mt.2010.85

Abstract:

Lipid nanoparticles (LNPs) have proven to be highly efficient carriers of short-interfering RNAs (siRNAs) to hepatocytes in vivo; however, the precise mechanism by which this efficient delivery occurs has yet to be elucidated. We found that apolipoprotein E (apoE), which plays a major role in the clearance and hepatocellular uptake of physiological lipoproteins, also acts as an endogenous targeting ligand for ionizable LNPs (iLNPs), but not cationic LNPs (cLNPs). The role of apoE was investigated using both in vitro studies employing recombinant apoE and in vivo studies in wild-type and apoE-/- mice. Receptor dependence was explored in vitro and in vivo using low-density lipoprotein receptor (LDLR-/-)-deficient mice. As an alternative to endogenous apoE-based targeting, we developed a targeting approach using an exogenous ligand containing a multivalent N-acetylgalactosamine (GalNAc)-cluster, which binds with high affinity to the asialoglycoprotein receptor (ASGPR) expressed on hepatocytes. Both apoE-based endogenous and GalNAc-based exogenous targeting appear to be highly effective strategies for the delivery of iLNPs to liver.

Summary:

The study, performed in collaboration with scientists at the Max Planck Institute of Molecular Cell Biology and Genetics and AlCana Technologies, Inc., describes a mechanism for endogenous apolipoprotein E (apoE)-mediated targeting of LNPs to the liver, demonstrates alternative ligand-directed targeting strategies for liver delivery of RNAi therapeutics, and highlights potential targeting approaches for delivery to tissues and cell types beyond the liver.

A randomized, double-blind, placebo-controlled study of an RNAi-based therapy directed against respiratory syncytial virus – April 27th, 2010

J. DeVincenzo et al. DOI: 10.1073/pnas.0912186107

Abstract:

RNA interference (RNAi) is a natural mechanism regulating protein expression that is mediated by small interfering RNAs (siRNA). Harnessing RNAi has potential to treat human disease; however, clinical evidence for the effectiveness of this therapeutic approach is lacking. ALN-RSV01 is an siRNA directed against the mRNA of the respiratory syncytial virus (RSV) nucleocapsid (N) protein and has substantial antiviral activity in a murine model of RSV infection. We tested the antiviral activity of ALN-RSV01 in adults experimentally infected with wild-type RSV. Eighty-eight healthy subjects were enrolled into a randomized, double-blind, placebo-controlled trial. A nasal spray of ALN-RSV01 or saline placebo was administered daily for 2 days before and for 3 days after RSV inoculation. RSV was measured serially in nasal washes using several different viral assays. Intranasal ALN-RSV01 was well tolerated, exhibiting a safety profile similar to saline placebo. The proportion of culture-defined RSV infections was 71.4 and 44.2% in placebo and ALN-RSV01 recipients, respectively (P = 0.009), representing a 38% decrease in the number of infected and a 95% increase in the number of uninfected subjects. The acquisition of infection over time was significantly lower in ALN-RSV01 recipients (P = 0.007 and P = 0.03, viral culture and PCR, respectively). Multiple logistic regression analysis showed that the ALN-RSV01 antiviral effect was independent of other factors, including preexisting RSV antibody and intranasal proinflammatory cytokine concentrations. ALN-RSV01 has significant antiviral activity against human RSV infection, thus establishing a unique proof-of-concept for an RNAi therapeutic in humans and providing the basis for further evaluation in naturally infected children and adults.

Summary:

These data, previously presented at the International Symposium on Respiratory Viral Infections in Singapore in 2008, showed human proof of concept with an RNAi therapeutic in a randomized, double blind, placebo-controlled study. In this study, treatment with ALN-RSV01 showed statistically significant anti-viral activity, including a decreased RSV infection rate and an increase in the number of subjects who remained free of infection.

Rational design of cationic lipids for siRNA delivery – January 17th, 2010

Semple et al., Nat Biotechnol. 2010 Feb;28(2):172-6. Epub 2010 Jan 17.

Abstract:

We adopted a rational approach to design cationic lipids for use in formulations to deliver small interfering RNA (siRNA). Starting with the ionizable cationic lipid 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), a key lipid component of stable nucleic acid lipid particles (SNALP) as a benchmark, we used the proposed in vivo mechanism of action of ionizable cationic lipids to guide the design of DLinDMA-based lipids with superior delivery capacity. The best-performing lipid recovered after screening (DLin-KC2-DMA) was formulated and characterized in SNALP and demonstrated to have in vivo activity at siRNA doses as low as 0.01 mg/kg in rodents and 0.1 mg/kg in nonhuman primates. To our knowledge, this represents a substantial improvement over previous reports of in vivo endogenous hepatic gene silencing.

Summary:

The study describes the discovery of a novel lipid, known as "KC2," based on a medicinal chemistry effort to explore the structure-activity relationships in the lipid "DLinDMA," which is used in certain first generation LNPs such as Tekmira's stable nucleic acid-lipid particles (SNALP) formulations. A large number of novel lipids were synthesized to probe the relationship of lipid structure, such as the alkyl chain, linker, and head group moieties, with function as determined by screening for in vivo gene silencing activity. Additional measurements were performed to characterize the ability of the novel lipids to mediate certain physicochemical changes in lipid bilayers consistent with needed disruption of endosomal membranes. In order to explore its suitability for systemic delivery, the novel KC2 lipid was formulated with siRNA in an LNP formulation. Specifically, the in vivo data showed that: Gene silencing in rodents was achieved following a single injection at doses as low as 0.01mg/kg; potent and selective silencing of the clinically relevant gene transthyretin (TTR) was achieved at doses as low as 0.1 mg/kg in non-human primates; and, the formulation was found to be well tolerated in both rodents and non-human primates.

Lipid-like Materials for Low Dose, in vivo Gene Silencing – December 29th, 2009

K.T. Love Proc Natl Acad Sci U S A. 2010 Feb 2;107(5):1864-9. Epub 2010 Jan 11.

Abstract:

Significant effort has been applied to discover and develop vehicles which can guide small interfering RNAs (siRNAs) through the many barriers guarding the interior of target cells. While studies have demonstrated the potential of gene silencing in vivo, improvements in delivery efficacy are required to fulfill the broadest potential of RNA interference (RNAi) therapeutics. Through the combinatorial synthesis and screening of a novel class of materials, a formulation has been identified that enables siRNA-directed liver gene silencing in vivo at doses below 0.01 mg/kg, in mice. This formulation was also shown to specifically inhibit expression of five genes simultaneously, after a single injection, in mice. The potential of this formulation was further validated in non-human primates, where high levels of specific knockdown of the clinically relevant gene transthyretin (TTR) was observed at doses as low as 0.03mg/kg. To our knowledge, this novel formulation facilitates gene silencing at orders-of-magnitude lower doses than required by any previously-described siRNA liver delivery system.

Summary:

The current study is the first to report on the simultaneous and highly specific RNAi-mediated silencing of as many as five liver targets in vivo, serving as proof of principle that multiple genes involved in similar or divergent biological pathways can be silenced with a single administration of a single drug product. From a therapeutic standpoint, this could enable novel pharmaceutical strategies, where silencing of multiple targets could achieve an enhanced level of efficacy. The new pre-clinical data describe a formulation based on a lipidoid known as 'C12-200' that was shown to:
- enable gene silencing in vivo in rodents at doses below 0.01 mg/kg;
- demonstrate complete, rapid, and durable gene silencing in rodents as soon as 24 hours with protein levels returning to baseline within 20 to 35 days;
- specifically inhibit expression of as many as five target genes simultaneously after a single injection of an LNP formulation in rodents; and,
- demonstrate potent and selective silencing of the clinically relevant gene transthyretin (TTR) at doses as low as 0.03 mg/kg in non-human primates.

Development of Lipidoid-siRNA Formulations for Systemic Delivery to the Liver – March 3rd, 2009

Akinc, A., et al Mol Ther. 2009 Mar 3. [Epub ahead of print]

Abstract:

RNA interference therapeutics afford the potential to silence target gene expression specifically, thereby blocking production of disease-causing proteins. The development of safe and effective systemic small interfering RNA (siRNA) delivery systems is of central importance to the therapeutic application of siRNA. Lipid and lipid-like materials are currently the most well-studied siRNA delivery systems for liver delivery, having been utilized in several animal models, including nonhuman primates. Here, we describe the development of a multicomponent, systemic siRNA delivery system, based on the novel lipid-like material 98N12-5(1). We show that In vivo delivery efficacy is affected by many parameters, including the formulation composition, nature of particle PEGylation, degree of drug loading, and biophysical parameters such as particle size. In particular, small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids can result in significant effects on in vivo efficacy. The lead formulation developed is liver targeted (>90% injected dose distributes to liver) and can induce fully reversible, long-duration gene silencing without loss of activity following repeat administration.

Summary:

Lipidoids are a new class of lipid-based molecules which are used to form novel nanoparticle formulations for systemic delivery of RNAi therapeutics. A previous study by Alnylam scientists in collaboration with scientists from MIT (Akinc et al., Nat Biotech 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. Data published from this new study extends the work from the previous study by: identifying key parameters affecting the pharmacodynamics of this type of formulation, including increasing the anchor length of synthesized PEG lipids, maximizing siRNA loading, and reducing particle size to more efficiently access hepatocytes; demonstrating 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 over a period of several months, and indicating no evidence of neutralizing antigenicity or tachyphylaxis; and, characterizing the long-term stability of the formulation.

Durable protection from Herpes Simplex Virus-2 transmission following intravaginal application of siRNAs targeting both a viral and host gene – January 22nd, 2009

Wu, Y., et al Cell Host Microbe. 2009 Jan 22;5(1):84-94

Abstract:

A vaginal microbicide should prevent pathogen transmission without disrupting tissue barriers to infection. Ideally, it would not need to be applied immediately before sexual intercourse, when compliance is a problem. Intravaginal administration of small interfering RNA (siRNA) lipoplexes targeting Herpes Simplex Virus Type 2 (HSV-2) genes protects mice from HSV-2. However, protection is short-lived, and the transfection lipid on its own unacceptably enhances transmission. Here, we show that cholesterol-conjugated (chol)-siRNAs without lipid silence gene expression in the vagina without causing inflammation or inducing interferons. A viral siRNA prevents transmission within a day of challenge, whereas an siRNA targeting the HSV-2 receptor nectin-1 protects for a week, but protection is delayed for a few days until the receptor is downmodulated. Combining siRNAs targeting a viral and host gene protects mice from HSV-2 for a week, irrespective of the time of challenge. Therefore, intravaginal siRNAs could provide sustained protection against viral transmission.

Summary:

The work described in this study used cholesterol-conjugated small interfering RNAs (siRNA, the molecules that mediate RNAi) targeting the HSV gene UL29 and the cell surface receptor nectin-1 which has been identified as an endogenous host receptor for HSV-2. Intravaginal delivery of siRNAs demonstrated durable protection from transmission of the virus. Specifically, in vivo data from this study showed that animals treated with siRNAs targeting UL29 and nectin-1 were protected from lethal HSV infection for about one week and showed a survival rate of up to 80%. Importantly, these results showed that cholesterol-conjugated siRNAs did not activate inflammation or interferon response genes.

Direct CNS Delivery of siRNA Mediates Robust Silencing in Oligodendrocytes – December 18th, 2008

Querbes, W., et al. Oligonucleotides. 2008 Dec 18 [Epub ahead of print]

Abstract:

The most significant challenge remaining in the development of small interfering RNAs (siRNAs) as a new class of therapeutic drugs is successful delivery In vivo. The majority of reported studies describing delivery of siRNA or short hairpin RNA (shRNA) to the central nervous system (CNS) have focused on RNA interference (RNAi) in neurons. Here we show direct CNS delivery of siRNA to a different cell type-oligodendrocytes-using convection-enhanced delivery, and demonstrate robust silencing of an endogenous oligodendrocyte-specific gene, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) with siRNA formulated in saline. The silencing is not sequence-dependent as several different siRNAs are effective in inhibiting target gene expression. Furthermore, we show that CNPase mRNA reduction is dose-dependent, durable for up to 1 week, and mediated by an RNAi mechanism. Increasing the flow rate of siRNA infusion increased the distribution of mRNA suppression to encompass white matter regions distant from the infusion site. Finally, we demonstrate suppression of CNPase mRNA in the nonhuman primate CNS. Taken together, these results show for the first time robust RNAi within oligodendrocytes in vivo and demonstrate the important potential of siRNAs in the treatment of CNS disorders involving oligodendrocyte pathology.

Summary:

The research conducted by Alnylam scientists, demonstrated that In vivo silencing of an endogenous oligodendrocyte-specific gene by an RNAi therapeutic was dose dependent, durable, and mediated by an RNAi mechanism. Specifically, the data showed that: siRNAs administered in a rodent model achieved successful delivery and silencing of an endogenous oligodendrocyte gene target (2′,3′-cyclic nucleotide 3′-phosphodiesterase or "CNPase") in a specific manner as compared with a control siRNA; direct CNS delivery of siRNA in the rodent model silenced the CNPase mRNA by approximately 75 percent, as compared with a control siRNA, and was durable for up to one week; increasing the infusion rate increased the distribution of CNPase mRNA suppression in white matter regions distant from the infusion site; CNPase silencing was mediated by an RNAi mechanism as measured by 5'RACE; and, direct infusion of an siRNA into the CNS in a non-human primate model resulted in silencing of the CNPase mRNA by 55 percent.

MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts – November 30th, 2008

Thum, T. et al. Nature. 2008 Dec 18;456(7224):980-4. Epub 2008 Nov 30

Abstract:

MicroRNAs comprise a broad class of small non-coding RNAs that control expression of complementary target messenger RNAs. Dysregulation of microRNAs by several mechanisms has been described in various disease states including cardiac disease. Whereas previous studies of cardiac disease have focused on microRNAs that are primarily expressed in cardiomyocytes, the role of microRNAs expressed in other cell types of the heart is unclear. Here we show that microRNA-21 (miR-21, also known as Mirn21) regulates the ERK-MAP kinase signalling pathway in cardiac fibroblasts, which has impacts on global cardiac structure and function. miR-21 levels are increased selectively in fibroblasts of the failing heart, augmenting ERK-MAP kinase activity through inhibition of sprouty homologue 1 (Spry1). This mechanism regulates fibroblast survival and growth factor secretion, apparently controlling the extent of interstitial fibrosis and cardiac hypertrophy. In vivo silencing of miR-21 by a specific antagomir in a mouse pressure-overload-induced disease model reduces cardiac ERK-MAP kinase activity, inhibits interstitial fibrosis and attenuates cardiac dysfunction. These findings reveal that microRNAs can contribute to myocardial disease by an effect in cardiac fibroblasts. Our results validate miR-21 as a disease target in heart failure and establish the therapeutic efficacy of microRNA therapeutic intervention in a cardiovascular disease setting.

Summary:

The new study demonstrates that miR-21 is over-expressed in the failing human heart and contributes to heart failure through its regulation of a stress-response signaling pathway associated with changes in heart muscle structure and function. The study went on to demonstrate that targeting miR-21 with an anti-miR-21, antisense oligonucleotide (also called an “antagomir”) prevented heart failure in mouse models. Furthermore, administration of anti-miR-21 after established heart failure resulted in a significant treatment benefit in the animal model.

miR-296 regulates growth factor receptor overexpression in angiogenic endothelial cells. – November 4th, 2008

Würdinger, T., et al. Cancer Cell. 2008 Nov; 14(5): 382-393

Abstract:

A key step in angiogenesis is the upregulation of growth factor receptors on endothelial cells. Here, we demonstrate that a small regulatory microRNA, miR-296, has a major role in this process. Glioma cells and angiogenic growth factors elevate the level of miR-296 in primary human brain microvascular endothelial cells in culture. The miR-296 level is also elevated in primary tumor endothelial cells isolated from human brain tumors compared to normal brain endothelial cells. Growth factor-induced miR-296 contributes significantly to angiogenesis by directly targeting the hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) mRNA, leading to decreased levels of HGS and thereby reducing HGS-mediated degradation of the growth factor receptors VEGFR2 and PDGFRbeta. Furthermore, inhibition of miR-296 with antagomirs reduces angiogenesis in tumor xenografts in vivo.

Summary:

This publication demonstrates a key role of microRNAs in regulating angiogenesis pathways in cancer, specifically in malignant brain tumors. Angiogenesis is a physiological process required for the growth and spread of many cancers and the new findings show that miR-296 is a significant regulator of this pathway. Further, targeting miR-296 with anti-miR-296 antisense oligonucleotides (also called "antagomirs") blocked angiogenesis in tumor xenograft models in mice. This new research, led by Massachusetts General Hospital and VU Medical Center in Amsterdam, working in collaboration with researchers from Regulus, Brigham and Women's Hospital, and Ludwig-Maximilians-University in Munich, describes the role of endothelial cell-expressed miR-296 in regulating the formation of new blood vessels. By inhibiting miR-296 with an anti-miR, levels of the growth factor receptors were decreased resulting in decreased growth of blood vessels. The data showed that the regulation of miR-296 in human endothelial cells directly resulted in the modulation of angiogenesis in vitro. Additionally, the specific inhibition of miR-296 in an in vivo tumor xenograft model resulted in decreased neovascularization of tumors.

5'-Triphosphate-siRNA: turning gene silencing and Rig-I activation against melanoma. – November 2nd, 2008

Poeck, H., et al. Nat Med. 2008 Nov; 14(11): 1256-63. Epub 2008 Nov 2

Abstract:

Genetic and epigenetic plasticity allows tumors to evade single-targeted treatments. Here we direct Bcl2-specific siRNA with 5'-triphosphate ends (3p-siRNA) against melanoma. Recognition of 5'-triphosphate by the cytosolic antiviral helicase retinoic acid-induced protein I (RIG-I, encoded by Ddx58) activated innate immune cells such as dendritic cells and directly induced expression of interferons (IFNs) and apoptosis in tumor cells. These RIG-I-mediated activities synergized with siRNA-mediated Bcl2 silencing to provoke massive apoptosis of tumor cells in lung metastases in vivo. The therapeutic activity required natural killer cells and IFN, as well as silencing of Bcl2, as evidenced by rescue with a mutated Bcl2 target, by site-specific cleavage of Bcl2 messenger RNA in lung metastases and downregulation of Bcl-2 protein in tumor cells in vivo. Together, 3p-siRNA represents a single molecule-based approach in which RIG-I activation on both the immune- and tumor cell level corrects immune ignorance and in which gene silencing corrects key molecular events that govern tumor cell survival.

Summary:

This pioneering study, in collaboration with the University of Bonn, documents the design and evaluation of "3p-siRNAs," a new class of RNAi therapeutics comprised of siRNA which are designed and chemically modified to activate RIG-I (retinoic acid-induced gene I, a cytoplasmic immunoreceptor which strongly induces type I interferon and NK cell responses). The publication demonstrates that a 3p-siRNA specific for the anti-apoptotic factor bcl-2 may be a highly effective anti-cancer agent in several animal models. 3p-siRNAs that are designed toward other gene targets may also have applications in infectious disease and as vaccine adjuvants. With a bcl-2-specific sequence, 3p-siRNAs led to a robust in vivo apoptotic effect, provoking massive programmed cell death of tumor cells and resulting in a greater than 80 percent reduction in total lung metastases in melanoma and adenocarcinoma mouse models.

MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators. – September 28th, 2008

Garbriely, G. et al. Mol Cell Biol. 2008 Sep;28(17):5369-80. Epub 2008 Jun 30.

Abstract:

Substantial data indicate that microRNA 21 (miR-21) is significantly elevated in glioblastoma (GBM) and in many other tumors of various origins. This microRNA has been implicated in various aspects of carcinogenesis, including cellular proliferation, apoptosis, and migration. We demonstrate that miR-21 regulates multiple genes associated with glioma cell apoptosis, migration, and invasiveness, including the RECK and TIMP3 genes, which are suppressors of malignancy and inhibitors of matrix metalloproteinases (MMPs). Specific inhibition of miR-21 with antisense oligonucleotides leads to elevated levels of RECK and TIMP3 and therefore reduces MMP activities in vitro and in a human model of gliomas in nude mice. Moreover, downregulation of miR-21 in glioma cells leads to decreases of their migratory and invasion abilities. Our data suggest that miR-21 contributes to glioma malignancy by downregulation of MMP inhibitors, which leads to activation of MMPs, thus promoting invasiveness of cancer cells. Our results also indicate that inhibition of a single oncomir, like miR-21, with specific antisense molecules can provide a novel therapeutic approach for "physiological" modulation of multiple proteins whose expression is deregulated in cancer.

Summary:

Researchers at Regulus Therapeutics, Brigham and Women's Hospital, and other collaborators discovered a new role for miR-21 in glioblastoma multiforme (GBM), the most malignant and aggressive form of glioma, a primary brain tumor. In the peer-reviewed research, our collaborators and scientists showed that increased miR-21 levels in tumor cells correlated with the grade or aggressiveness of the tumor; highest levels of miR-21 were associated with the most aggressive forms of GBM. To elucidate the role that miR-21 plays in GBM, anti-miR-21 compounds were used to inhibit miR-21 function in human tumor cells. Inhibiting miR-21 function affected the expression of genes associated with tumor invasiveness, proliferation, migration and other processes exploited by tumor cells. As a whole, these data correlated miR-21 regulation with the aggressiveness of the tumor, and suggest that miR-21 is involved in the regulation of many different cellular processes necessary for tumorogenesis.

Host Scavenger Receptor SR-BI Plays a Dual Role in the Establishment of Malaria Parasite Liver Infection. – September 11th, 2008

Rodrigues, C., et al. Cell Host Microbe. 2008 Sep; 4(3): 271-282

Abstract:

An obligatory step of malaria parasite infection is Plasmodium sporozoite invasion of host hepatocytes, and host lipoprotein clearance pathways have been linked to Plasmodium liver infection. By using RNA interference to screen lipoprotein-related host factors, we show here that the class B, type I scavenger receptor (SR-BI) is the strongest regulator of Plasmodium infection among these factors. Inhibition of SR-BI function reduced P. berghei infection in Huh7 cells, and overexpression of SR-BI led to increased infection. In vivo silencing of liver SR-BI expression in mice and inhibition of SR-BI activity in human primary hepatocytes reduced infection by P. berghei and by P. falciparum, respectively. Heterozygous SR-BI+/- mice displayed reduced P. berghei infection rates correlating with liver SR-BI expression levels. Additional analyses revealed that SR-BI plays a dual role in Plasmodium infection, affecting both sporozoite invasion and intracellular parasite development, and may therefore constitute a good target for malaria prophylaxis.

Summary:

This collaborative study with Lisbon-based biomedical research centre Instituto de Medicina Molecular (IMM) describes the discovery and in vivo validation of scavenger receptor BI (SR-BI), a major regulator of cholesterol uptake by the liver, as a critical host factor for malaria infection. The new research findings could lead to new approaches for the treatment of malaria including use of RNAi therapeutics. In the study, the liver-expressed gene, SR-BI, was identified as a critical host factor for the liver infection stage of malaria using a systematic RNAi screen of known lipoprotein pathway components in a cultured human cell-based infection assay. These finding were then confirmed in animal models of malaria infection using small interfering RNAs (siRNAs), the molecules that mediate RNAi, specific for SR-BI silencing.

Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates. – August 19th, 2008

Frank-Kamenetsky, M., et al. Proc. Natl Acad. Sci. USA. 2008 Aug; 10.1073/pnas.0805434105

Abstract:

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low density lipoprotein receptor (LDLR) protein levels and function. Loss of PCSK9 increases LDLR levels in liver and reduces plasma LDL cholesterol (LDLc), whereas excess PCSK9 activity decreases liver LDLR levels and increases plasma LDLc. Here, we have developed active, cross-species, small interfering RNAs (siRNAs) capable of targeting murine, rat, nonhuman primate (NHP), and human PCSK9. For in vivo studies, PCSK9 and control siRNAs were formulated in a lipidoid nanoparticle (LNP). Liver-specific siRNA silencing of PCSK9 in mice and rats reduced PCSK9 mRNA levels by 50-70%. The reduction in PCSK9 transcript was associated with up to a 60% reduction in plasma cholesterol concentrations. These effects were shown to be mediated by an RNAi mechanism, using 5'-RACE. In transgenic mice expressing human PCSK9, siRNAs silenced the human PCSK9 transcript by >70% and significantly reduced PCSK9 plasma protein levels. In NHP, a single dose of siRNA targeting PCSK9 resulted in a rapid, durable, and reversible lowering of plasma PCSK9, apolipoprotein B, and LDLc, without measurable effects on either HDL cholesterol (HDLc) or triglycerides (TGs). The effects of PCSK9 silencing lasted for 3 weeks after a single bolus i.v. administration. These results validate PCSK9 targeting with RNAi therapeutics as an approach to specifically lower LDLc, paving the way for the development of PCSK9-lowering agents as a future strategy for treatment of hypercholesterolemia.

Summary:

This paper describes work, conducted by Alnylam scientists in collaboration with scientists at UT Southwestern Medical Center in Dallas, Alnylam Europe (now Roche Kulmbach), and the David H. Koch at the Institute for Integrative Cancer Research at the Massachusetts Institute of Technology, Here it is demonstrated for the first time that a chemically synthesized small interfering RNA (or siRNA, the molecules that mediate RNAi) targeting the gene proprotein convertase subtilisin/kexin type 9, (or PCSK9), achieves acute and durable lowering of total LDL/cholesterol levels in both mice and rats, and LDL cholesterol levels in non-human primates. PCSK9 represent a highly validated disease target with compelling human genetic data supporting its therapeutic value. This work indicates that an RNAi approach can be used to access this important target representing a potential innovative new approach to the future therapy of hypercholesterolemia.

Heme oxygenase-1 is an anti-inflammatory host factor that promotes murine plasmodium liver infection. – May 15th, 2008

Epiphanio et al. Cell Host Microbe. 2008 May 15;3(5):277-9

Abstract:

The clinically silent Plasmodium liver stage is an obligatory step in the establishment of malaria infection and disease. We report here that expression of heme oxygenase-1 (HO-1, encoded by Hmox1) is upregulated in the liver following infection by Plasmodium berghei and Plasmodium yoelii sporozoites. HO-1 overexpression in the liver leads to a proportional increase in parasite liver load, and treatment of mice with carbon monoxide and with biliverdin, each an enzymatic product of HO-1, also increases parasite liver load. Conversely, mice lacking Hmox1 completely resolve the infection. In the absence of HO-1, the levels of inflammatory cytokines involved in the control of liver infection are increased. These findings suggest that, while stimulating inflammation, the liver stage of Plasmodium also induces HO-1 expression, which modulates the host inflammatory response, protecting the infected hepatocytes and promoting the liver stage of infection.

Summary:

This publication highlights the role of a host (human) gene, namely heme oxygenase-1 (HO-1), in the cause and pathway ofas a critical factor required for the casual agent of malaria infectionparasitic infection. In vivo studies demonstrated that HO-1 is a critical host factor involved in the liver stage of malaria infection in a mouse model. In this paper studies in mice showed that RNAi therapeutics targeting the HO-1 gene mRNA can significantly inhibit the initial liver stage of malaria infection and completely block the resulting transmission of disease to red blood cells. This represents a novel approach to therapeutic intervention for a major human pathogen and highlights the importance of host or self genes in this parasitic infection.

A combinatorial library of lipid-like materials for delivery of RNAi therapeutics – April 27th, 2008

Akinc et al. Nat Biotechnol. 2008 May;26(5):561-9. Epub 2008 Apr 27

Abstract:

The safe and effective delivery of RNA interference (RNAi) therapeutics remains an important challenge for clinical development. The diversity of current delivery materials remains limited, in part because of their slow, multi-step syntheses. Here we describe a new class of lipid-like delivery molecules, termed lipidoids, as delivery agents for RNAi therapeutics. Chemical methods were developed to allow the rapid synthesis of a large library of over 1,200 structurally diverse lipidoids. From this library, we identified lipidoids that facilitate high levels of specific silencing of endogenous gene transcripts when formulated with either double-stranded small interfering RNA (siRNA) or single-stranded antisense 2'-O-methyl (2'-OMe) oligoribonucleotides targeting microRNA (miRNA). The safety and efficacy of lipidoids were evaluated in three animal models: mice, rats and nonhuman primates. The studies reported here suggest that these materials may have broad utility for both local and systemic delivery of RNA therapeutics.

Summary:

The new research documents the design and synthesis of a new class of lipid-based molecules called "lipidoids", which were used to form novel nanoparticle formulations for systemic delivery of RNAi therapeutics. The paper shows multiple examples of the use of novel new lipid formulations that mediate successful targeted gene silencing by small interfering RNAs (siRNAs), in multiple animal species that include mice, rats, and non-human primates. In addition the data in this paper demonstrate potent, specific, and durable effects on targeted gene expression in multiple tissues, including liver, lung, and peritoneal macrophages. The paper also presents data where the same approach can be used for the delivery of "antagomirs" or microRNA antagonists.

Determinants of cytokine induction by small interfering RNA in human peripheral blood mononuclear cells – April 1st, 2008

Zamanian-Daryoush et al. Journal of Interferon & Cytokine Research 2008 April;28(4), 221-233

Abstract:

Certain synthetic small interfering RNAs (siRNAs) can trigger a strong innate immune response in mammalian cells. This nonspecific side effect may hinder the application of siRNAs as tools in gene silencing. Chemically synthesized siRNAs, including traditional 19-mers with 2-nt 3' overhangs, longer duplexes with blunt or 3' overhangs, and asymmetric duplexes with a blunt end and a 2-nt 3' overhang, can evoke strong dose-dependent interferon-alpha (IFN-alpha) and tumor necrosis factor-alpha (TNF-alpha) release in human peripheral blood mononuclear cells (PBMCs). This response is independent of retinoic acid-inducible gene I but may involve endosomal toll-like receptors (TLRs). The immunostimulatory effect of the siRNAs is directly related to either or both of the strands of the duplex in a sequence-dependent manner. However, although some single-stranded RNAs and siRNAs potently evoked both IFN-alpha and TNF-alpha induction, these responses were not always coupled. In accordance with this, specific chemical modifications differentially altered cytokine production, suggesting recruitment of different TLRs in a sequence-dependent manner.

Summary:

This work build on a previous paper (Hornung V et al Nat Med 2005 Mar;11(3):263-70) and further characterizes the ability of some siRNAs to trigger the immune system. In the study, certain siRNAs were shown to induce tumor necrosis factor (TNF)-alpha and/or interferon alpha (IFN-alpha) release in human cells in vitro. The work also demonstrates that human cells can be used for screening siRNAs and that chemical modifications of siRNAs can be used to eliminate both the IFN-alpha and TNF-alpha immunostimulatory responses in these assays. This paper is important in that it demonstrates that cell-based assays can be used to identify problematic siRNAs and therefore eliminate these prior to clinical testing.

Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits – October 23rd, 2007

DiFiglia M et al. Proc Natl Acad Sci USA, 2007 Oct 23;104(43):17204-9.

Abstract:

Huntington's Disease (HD) is autosomal dominant and, in theory, amenable to therapeutic RNA silencing. We introduced cholesterol-conjugated small interfering RNA duplexes (cc-siRNA) targeting human Htt mRNA (siRNA-Htt) into mouse striata that also received adeno-associated virus containing either expanded (100 CAG) or wild-type (18 CAG) Htt cDNA encoding huntingtin (Htt) 1-400. Adeno-associated virus delivery to striatum and overlying cortex of the mutant Htt gene, but not the wild type, produced neuropathology and motor deficits. Treatment with cc-siRNA-Htt in mice with mutant Htt prolonged survival of striatal neurons, reduced neuropil aggregates, diminished inclusion size, and lowered the frequency of clasping and footslips on balance beam. cc-siRNA-Htt was designed to target human wild-type and mutant Htt and decreased levels of both in the striatum. Our findings indicate that a single administration into the adult striatum of an siRNA targeting Htt can silence mutant Htt, attenuate neuronal pathology, and delay the abnormal behavioral phenotype observed in a rapid-onset, viral transgenic mouse model of HD.

Summary:

This paper shows that an siRNA targeting the huntingtin (Htt) gene can exert therapeutic effects in a mouse model of Huntington's disease (HD). HD is a neurodegenerative disorder caused by mutations in the Htt gene. In a mouse model of HD siRNA therapy was used to reverse cellular pathology and neurological disease. These experiments indicate that the direct delivery of an siRNA to the brain may have therapeutic potential in HD.

Mechanisms and Optimization of In Vivo Delivery of Lipophilic siRNAs – October 23rd, 2007

Wolfrum et al. Nat Biotechnol. 2007 Oct;25(10):1149-57.

Abstract:

Cholesterol-conjugated siRNAs can silence gene expression in vivo. Here we synthesize a variety of lipophilic siRNAs and use them to elucidate the requirements for siRNA delivery in vivo. We show that conjugation to bile acids and long-chain fatty acids, in addition to cholesterol, mediates siRNA uptake into cells and gene silencing in vivo. Efficient and selective uptake of these siRNA conjugates depends on interactions with lipoprotein particles, lipoprotein receptors and transmembrane proteins. High-density lipoprotein (HDL) directs siRNA delivery into liver, gut, kidney and steroidogenic organs, whereas low-density lipoprotein (LDL) targets siRNA primarily to the liver. LDL-receptor expression is essential for siRNA delivery by LDL particles, and SR-BI receptor expression is required for uptake of HDL-bound siRNAs. Cellular uptake also requires the mammalian homolog of the Caenorhabditis elegans transmembrane protein Sid1. Our results demonstrate that conjugation to lipophilic molecules enables effective siRNA uptake through a common mechanism that can be exploited to optimize therapeutic siRNA delivery.

Summary:

This paper builds on the Soutschek paper (Soutschek et al. Nature. 2004 Nov 11;432(7014):173-8) where a modified siRNA was used to achieve targeted silencing on the apo-B gene in mice. Here siRNAs are modified via linkage to various lipophilic molecules (e.g. cholesterol) and their effectiveness in achieving targeted gene silencing is evaluated in mice. In addition the paper shows that these lipophilic ("fat liking") modified siRNAs bind to circulating carrier proteins such as albumin, as well as LDL and HDL particles. Further, it is shown that the distribution of the siRNA is dictated by these carrier proteins or particles. In addition it is demonstrated that another cell surface molecule, SID-1, is involved in cellular uptake which enables targeted gene silencing. This paper represents one of the most careful molecular explanations of how successful delivery can be achieved in vivo and this may allow for further improvements and innovations to be made to improve delivery.

Effective RNAi-mediated gene silencing without interruption of the endogenous microRNA pathway – October 11th, 2007

John et al. Nature. 2007 Oct 11;449(7163):745-7.

Abstract:

Systemic administration of synthetic small interfering RNAs (siRNAs) effectively silences hepatocyte gene expression in rodents and primates. Whether or not in vivo gene silencing by synthetic siRNA can disrupt the endogenous microRNA (miRNA) pathway remains to be addressed. Here we show that effective target-gene silencing in the mouse and hamster liver can be achieved by systemic administration of synthetic siRNA without any demonstrable effect on miRNA levels or activity. Indeed, siRNA targeting two hepatocyte-specific genes (apolipoprotein B and factor VII) that achieved efficient (approximately 80%) silencing of messenger RNA transcripts and a third irrelevant siRNA control were administered to mice without significant changes in the levels of three hepatocyte-expressed miRNAs (miR-122, miR-16 and let-7a) or an effect on miRNA activity. Moreover, multiple administrations of an siRNA targeting the hepatocyte-expressed gene Scap in hamsters achieved long-term mRNA silencing without significant changes in miR-122 levels. This study advances the use of siRNAs as safe and effective tools to silence gene transcripts in animal studies, and supports the continued advancement of RNA interference therapeutics using synthetic siRNA.

Summary:

This paper investigates the possibility that an siRNA could impact the endogenous microRNA pathway. microRNAs are genetically encoded or endogenous RNA molecules found in nearly every organism and appear very important in the regulation of entire gene pathways (see Li QJ et al. Cell 2007 Apr 6;129(1):147-61). MicroRNAs use components of the RNAi cellular mechanism shared by the siRNA mechanism. The paper shows that targeted gene silencing with an siRNA can readily be achieved without affecting the endogenous cellular microRNA pathway.

Interfering with Disease: A Progress Report on siRNA-Based Therapeutics – June 6th, 2007

de Fougerolles et al. Nat Rev Drug Discov. 2007 Jun;6(6):443-53.

Abstract:

RNA interference (RNAi) quietly crept into biological research in the 1990s when unexpected gene-silencing phenomena in plants and flatworms first perplexed scientists. Following the published demonstration of RNAi in mammalian cells in 2001, it was quickly realized that this highly specific mechanism of sequence-specific gene silencing might be harnessed to develop a new class of drugs that interfere with disease-causing or disease-promoting genes. Here we discuss the considerations that go into developing RNAi-based therapeutics starting from in vitro lead design and identification, to in vivo pre-clinical drug delivery and testing. We conclude by reviewing the latest clinical experience with RNAi therapeutics.

Summary:

This is a comprehensive review on the progress of siRNA products from the early discovery through to the latest developments in delivery approaches and the status of clinical trials of RNAi based therapeutics

miR-181a Is an Intrinsic Modulator of T Cell Sensitivity and Selection – April 6th, 2007

Li QJ et al. Cell 2007 Apr 6;129(1):147-61.

Abstract:

T cell sensitivity to antigen is intrinsically regulated during maturation to ensure proper development of immunity and tolerance, but how this is accomplished remains elusive. Here we show that increasing miR-181a expression in mature T cells augments the sensitivity to peptide antigens, while inhibiting miR-181a expression in the immature T cells reduces sensitivity and impairs both positive and negative selection. Moreover, quantitative regulation of T cell sensitivity by miR-181a enables mature T cells to recognize antagonists - the inhibitory peptide antigens - as agonists. These effects are in part achieved by the downregulation of multiple phosphatases, which leads to elevated steady-state levels of phosphorylated intermediates and a reduction of the T cell receptor signaling threshold. Importantly, higher miR-181a expression correlates with greater T cell sensitivity in immature T cells, suggesting that miR-181a acts as an intrinsic antigen sensitivity "rheostat" during T cell development.

Summary:

This paper describes the role of a microRNA, namely miR-181a, in regulating the activity of the T-cell in its response to immune stimuli. The data show that miR-181a acts as a rheostat, or volume control, for T-cell activation. When miR-181a is highly expressed, it suppresses negative regulators of T-cell activation and therefore makes the T-cell very sensitive or easily activated by immune signals. When miR-181a is expressed at low levels, the negative regulators are de-repressed and so the T-cell is less easy to activate and will only respond to strong immune stimuli. The role of miR-181a is discussed in the context of T-cell development, where the sensitivity of T-cells to stimuli is critical to maturation of the immune system. This helps prevent autoimmunity but allows for the recognition of pathogens. This paper is another example of how microRNA's can regulate key cellular pathways.

Evaluation of the safety, tolerability and pharmacokinetics of ALN-RSV01, a novel RNAi antiviral therapeutic directed against respiratory syncytial virus (RSV) – March 1st, 2007

DeVincenzo et al. Antiviral Res. 2008 Mar; 77(3); 225-31. Epub 2007 Dec 26

Abstract:

Small interfering RNAs (siRNAs) work through RNA interference (RNAi), the natural RNA inhibitory pathway, to down-regulate protein production by inhibiting targeted mRNA in a sequence-specific manner. ALN-RSV01 is an siRNA directed against the mRNA encoding the N-protein of respiratory syncytial virus (RSV) that exhibits specific in vitro and in vivo anti-RSV activity. The results of two safety and tolerability studies with ALN-RSV01 involving 101 healthy adults (65 active, 36 placebo, single- and multiple dose, observer-blind, randomized dose-escalation) are described. Intranasal administration of ALN-RSV01 was well tolerated over a dose range up through 150mg as a single dose and for five daily doses. Adverse events were similar in frequency and severity to placebo (normal saline) and were transient, mild to moderate, with no dose-dependent trend. The frequency or severity of adverse events did not increase with increasing ALN-RSV01 exposure. All subjects completed all treatments and assessments with no early withdrawals or serious adverse events. Physical examinations, vital signs, ECGs and laboratory tests were normal. Systemic bioavailability of ALN-RSV01 was minimal. ALN-RSV01 appears safe and well tolerated when delivered intranasally and is a promising therapeutic candidate for further clinical development.

Summary:

This publication discusses the results of two Phase I safety and tolerability studies with ALN-RSV01, Alnylam's lead clinical program for respiratory syncytial virus (RSV) infection. Here single and multiple doses of ALN-RSV01 delivered intranasally are evaluated in healthy human volunteers. The data demonstrate that ALN-RSV01 is a well tolerated by this route of administration. The data presented here established the safety and tolerability parameters of ALN-RSV01 and supported the evaluation of ALN-RSV01 in an experimental infection model in healthy human volunteers (The GEMINI study). In early 2008, Alnylam reported on the results of the GEMINI study which demonstrated that ALN-RSV01 was not only safe and well tolerated in RSV infected volunteers but that is was associated with a statistically significant reduction in infection rate when compared to placebo-treated volunteers. The GEMINI study is the first demonstration of the efficacy of an RNAi therapeutic in well-controlled human clinical trials

RNAi Therapeutics: A Potential New Class of Pharmaceutical Drugs – December 2nd, 2006

Bumcrot et al. Nat Chem Bio 2006 Dec;2(12):711-9.

Abstract:

The rapid identification of highly specific and potent drug candidates continues to be a substantial challenge with traditional pharmaceutical approaches. Moreover, many targets have proven to be intractable to traditional small-molecule and protein approaches. Therapeutics based on RNA interference (RNAi) offer a powerful method for rapidly identifying specific and potent inhibitors of disease targets from all molecular classes. Numerous proof-of-concept studies in animal models of human disease demonstrate the broad potential application of RNAi therapeutics. The major challenge for successful drug development is identifying delivery strategies that can be translated to the clinic. With advances in this area and the commencement of multiple clinical trials with RNAi therapeutic candidates, a transformation in modrn medicine may soon be realized.

Summary:

In this comprehensive review of RNAi, there is a detailed review of the design and structure of siRNAs and how we have learned to make potent, selective and stable siRNAs via the design of siRNA sequence and through chemical modification. In addition there is a review of progress on achieving successful delivery of siRNAs in in vivo models.

RNAi-mediated gene silencing in non-human primates – May 4th, 2006

Zimmermann et al. Nature 2006 May 4;441(7089):111-4.

Abstract:

The opportunity to harness the RNA interference (RNAi) pathway to silence disease-causing genes holds great promise for the development of therapeutics directed against targets that are otherwise not addressable with current medicines. Although there are numerous examples of in vivo silencing of target genes after local delivery of small interfering RNAs (siRNAs), there remain only a few reports of RNAi-mediated silencing in response to systemic delivery of siRNA, and there are no reports of systemic efficacy in non-rodent species. Here we show that siRNAs, when delivered systemically in a liposomal formulation, can silence the disease target apolipoprotein B (apoB) in non-human primates. apoB-specific siRNAs were encapsulated in stable nucleic acid lipid particles (SNALP) and administered by intravenous injection to cynomolgus monkeys at doses of 1 or 2.5 mg kg(-1). A single siRNA injection resulted in dose-dependent silencing of apoB messenger RNA expression in the liver 48 h after administration, with maximal silencing of >90%. This silencing effect occurred as a result of apoB mRNA cleavage at precisely the site predicted for the RNAi mechanism. Significant reductions in apoB protein, serum cholesterol and low-density lipoprotein levels were observed as early as 24 h after treatment and lasted for 11 days at the highest siRNA dose, thus demonstrating an immediate, potent and lasting biological effect of siRNA treatment. Our findings show clinically relevant RNAi-mediated gene silencing in non-human primates, supporting RNAi therapeutics as a potential new class of drugs.

Summary:

This landmark publication in the field of RNAi therapeutics showed therapeutic efficacy for the first time in non-human primates. Using an siRNA that targets the apoB gene and formulating in lipid nanoparticles, Zimmermann et al. show that targeted silencing of the liver expressed apoB mRNA can be achieved after IV administration of the formulated siRNA. The knockdown of the apo-B gene resulted in a substantial lowering of the LDL cholesterol of the treated animals. The effects of the drug were still apparent even 11 days after a single IV administration and indicated the RNAi can mediate durable therapeutic effects. This paper represents major supportive data for the therapeutic potential of RNAi.

Silencing of microRNAs In Vivo with 'Antagomirs' – December 1st, 2005

Krutzfeldt et al. Nature 2005 Dec 1;438(7068):685-9.

Abstract:

MicroRNAs (miRNAs) are an abundant class of non-coding RNAs that are believed to be important in many biological processes through regulation of gene expression. The precise molecular function of miRNAs in mammals is largely unknown and a better understanding will require loss-of-function studies in vivo. Here we show that a novel class of chemically engineered oligonucleotides, termed 'antagomirs', are efficient and specific silencers of endogenous miRNAs in mice. Intravenous administration of antagomirs against miR-16, miR-122, miR-192 and miR-194 resulted in a marked reduction of corresponding miRNA levels in liver, lung, kidney, heart, intestine, fat, skin, bone marrow, muscle, ovaries and adrenals. The silencing of endogenous miRNAs by this novel method is specific, efficient and long-lasting. The biological significance of silencing miRNAs with the use of antagomirs was studied for miR-122, an abundant liver-specific miRNA. Gene expression and bioinformatic analysis of messenger RNA from antagomir-treated animals revealed that the 3' untranslated regions of upregulated genes are strongly enriched in miR-122 recognition motifs, whereas downregulated genes are depleted in these motifs. Analysis of the functional annotation of downregulated genes specifically predicted that cholesterol biosynthesis genes would be affected by miR-122, and plasma cholesterol measurements showed reduced levels in antagomir-122-treated mice. Our findings show that antagomirs are powerful tools to silence specific miRNAs in vivo and may represent a therapeutic strategy for silencing miRNAs in disease.

Summary:

This paper is a landmark study in the field on microRNA therapeutics. Here Krutzfeldt et al. demonstrate that a specifically designed and modified, single-stranded RNA molecule or "Antagomir" can specifically block endogenous microRNAs. These data were instrumental in the formation of our joint venture with Isis Pharmaceuticals, Regulus Therapeutics, which is focused on the development of microRNA-based therapeutics. microRNAs are genetically encoded or endogenous RNA molecules found in nearly every organism and appear very important in the regulation of entire gene pathways (see Li QJ et al. Cell 2007 Apr 6;129(1):147-61). The paper shows that antagomirs can be made to target a range of microRNAs. Amongst other results, the study showed that silencing of miR-122 in the liver resulted in clear phenotypic changes in cholesterol metabolism. Since microRNAs have been linked to cancer, cell proliferation and differentiation, as well as viral infection, this work indicates that microRNA-based therapeutics could represent a whole new frontier of pharmaceutical research targeting entire pathways of disease, not just single disease targets.

Sequence-specific potent induction of IFN-α by short interfering RNA in plasmacytoid dendritic cells through TLR7 – March 1st, 2005

Hornung V et al. Nat Med 2005 Mar;11(3):263-70.

Abstract:

Short interfering RNA (siRNA) is used in RNA interference technology to avoid non-target-related induction of type I interferon (IFN) typical for long double-stranded RNA. Here we show that in plasmacytoid dendritic cells (PDC), an immune cell subset specialized in the detection of viral nucleic acids and production of type I IFN, some siRNA sequences, independent of their GU content, are potent stimuli of IFN-α production. Localization of the immunostimulatory motif on the sense strand of a potent IFN-α-inducing siRNA allowed dissection of immunostimulation and target silencing. Injection into mice of immunostimulatory siRNA, when complexed with cationic liposomes, induced systemic immune responses in the same range as the TLR9 ligand CpG, including IFN-α in serum and activation of T cells and dendritic cells in spleen. Immunostimulation by siRNA was absent in TLR7-deficient mice. Thus sequence-specific TLR7-dependent immune recognition in PDC needs to be considered as an additional biological activity of siRNA, which then should be termed immunostimulatory RNA (isRNA).

Summary:

This is an important paper that was the first to describe and characterize the molecular mechanisms by which some double stranded RNA molecules can trigger an immune response. Alnylam scientists working in collaboration with Dr Hartmann then at Ludwig Maximilian University Munich Hospital, Germany, demonstrate that an immune cell, the plasmocytoid dendritic cell, recognizes certain sequence motifs in dsRNA molecules through a receptor, TLR-7, and produces the inflammatory cytokine IFNalpha in response. The characterization of this biology at a molecular level allows for one to avoid this potential side effect of an siRNA via the design of appropriate siRNA sequences and the use of chemical modifications. In addition the paper shows that these in vitro data are reproduced in vivo in a mouse model system and so provides us an in vitro assays to test for potential immunostimulatory activities of siRNA's and to therefore avoid this property in the clinical phases of development.

Therapeutic Silencing of an Endogenous Gene by Systemic Administration of Modified siRNA – November 11th, 2004

Soutschek et al. Nature. 2004 Nov 11;432(7014):173-8.

Abstract:

RNA interference (RNAi) holds considerable promise as a therapeutic approach to silence disease-causing genes, particularly those that encode so-called 'non-druggable' targets that are not amenable to conventional therapeutics such as small molecules, proteins, or monoclonal antibodies. The main obstacle to achieving in vivo gene silencing by RNAi technologies is delivery. Here we show that chemically modified short interfering RNAs (siRNAs) can silence an endogenous gene encoding apolipoprotein B (apoB) after intravenous injection in mice. Administration of chemically modified siRNAs resulted in silencing of the apoB messenger RNA in liver and jejunum, decreased plasma levels of apoB protein, and reduced total cholesterol. We also show that these siRNAs can silence human apoB in a transgenic mouse model. In our in vivo study, the mechanism of action for the siRNAs was proven to occur through RNAi-mediated mRNA degradation, and we determined that cleavage of the apoB mRNA occurred specifically at the predicted site. These findings demonstrate the therapeutic potential of siRNAs for the treatment of disease.

Summary:

In this early landmark paper in the RNAi field, Soutschek et al. demonstrated for the first time the efficacy of an siRNA in an animal model via systemic administration. The paper showed that a modified siRNA targeting the apolipoprotein B (apo-B) gene, after intravenous administration into a mouse, mediates targeted gene silencing of the apo-B mRNA in the both the liver and the jejunum (part of the intestine). In turn, this gene silencing effect resulted in the expected therapeutic benefit of silencing of apo-B, a reduced LDL and cholesterol level in the blood of the mice. This paper was an important milestone in the progression of RNAi towards human therapeutic use; its significance was highlighted by the Karolinska Institute in the award of the 2006 Nobel Prize in Physiology or Medicine to Drs. Andrew Fire and Craig Mello for their contributions to the discovery of RNAi.