Nature：RNA干扰技术的“成功”与“磨难”

Lindholm¹, Maj Hedtjärn¹, Henrik Frydenlund Hansen¹, Urs Berger⁴, Steven Gullans³, Phil Kearney¹, Peter Sarnow², Ellen Marie Straarup¹ & Sakari Kauppinen^1,⁵

Santaris Pharma, Bøge Allé 3, DK-2970 Hørsholm, Denmark
Department of Microbiology and Immunology, 299 Campus Drive, Stanford University School of Medicine, Stanford, California 94305, USA
RxGen Inc, 100 Deepwood Drive, Hamden, Connecticut 06517, USA
UB-in situ, PO Box 463, Natick, Massachusetts 01760, USA
Wilhelm Johannsen Centre for Functional Genome Research, Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark
These authors contributed equally to this work.

Correspondence to: Sakari Kauppinen^1,⁵ Correspondence and requests for materials should be addressed to S.K. (Email: sk@santaris.com).

microRNAs (miRNAs) are small regulatory RNAs that are important in development and disease^1,^2,³ and therefore represent a potential new class of targets for therapeutic intervention⁴. Despite recent progress in silencing of miRNAs in rodents^5,⁶, the development of effective and safe approaches for sequence-specific antagonism of miRNAs in vivo remains a significant scientific and therapeutic challenge. Moreover, there are no reports of miRNA antagonism in primates. Here we show that the simple systemic delivery of a unconjugated, PBS-formulated locked-nucleic-acid-modified oligonucleotide (LNA-antimiR) effectively antagonizes the liver-expressed miR-122 in non-human primates. Acute administration by intravenous injections of 3 or 10 mg kg^-1 LNA-antimiR to African green monkeys resulted in uptake of the LNA-antimiR in the cytoplasm of primate hepatocytes and formation of stable heteroduplexes between the LNA-antimiR and miR-122. This was accompanied by depletion of mature miR-122 and dose-dependent lowering of plasma cholesterol. Efficient silencing of miR-122 was achieved in primates by three doses of 10 mg kg^-1 LNA-antimiR, leading to a long-lasting and reversible decrease in total plasma cholesterol without any evidence for LNA-associated toxicities or histopathological changes in the study animals. Our findings demonstrate the utility of systemically administered LNA-antimiRs in exploring miRNA function in rodents and primates, and support the potential of these compounds as a new class of therapeutics for disease-associated miRNAs.

（Nature），doi:10.1038/nature06765，Mark E. Kleinman, Jayakrishna Ambati）

Sequence- and target-independent angiogenesis suppression by siRNA via TLR3

Mark E. Kleinman^1,¹², Kiyoshi Yamada^1,¹², Atsunobu Takeda^1,¹², Vasu Chandrasekaran³, Miho Nozaki¹, Judit Z. Baffi¹, Romulo J. C. Albuquerque^1,², Satoshi Yamasaki⁴, Masahiro Itaya⁴, Yuzhen Pan⁵, Binoy Appukuttan⁵, Daniel Gibbs^6,⁷, Zhenglin Yang^6,⁷, Katalin Karikó⁸, Balamurali K. Ambati^6,⁹, Traci A. Wilgus¹⁰, Luisa A. DiPietro¹⁰, Eiji Sakurai⁴, Kang Zhang^6,⁷, Justine R. Smith⁵, Ethan W. Taylor¹¹ & Jayakrishna Ambati^1,²

Departments of Ophthalmology & Visual Sciences, and,
Department of Physiology, University of Kentucky, Lexington, Kentucky 40506, USA
Department of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, USA
Department of Ophthalmology, Nagoya City University Medical School, Nagoya 467-8601, Japan
Casey Eye Institute, Oregon Health and Science University, Portland, Oregon 97239, USA
Department of Ophthalmology and Visual Sciences, Moran Eye Center,
Program in Human Molecular Biology and Genetics, Eccles Institute of Human Genetics, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA
Department of Neurosurgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA
Department of Ophthalmology, Veterans Affairs Salt Lake City Healthcare System, Salt Lake City, Utah 84148, USA
Center for Wound Healing & Tissue Regeneration, University of Illinois at Chicago College of Dentistry, Chicago, Illinois 60612-7211, USA
Laboratory for Molecular Medicine, University of North Carolina at Greensboro, Greensboro, North Carolina 27402-6170, USA
These authors contributed equally to this work.

Correspondence to: Jayakrishna Ambati^1,² Correspondence and requests for materials should be addressed to J.A. (Email: jamba2@email.uky.edu).

Clinical trials of small interfering RNA (siRNA) targeting vascular endothelial growth factor-A (VEGFA) or its receptor VEGFR1 (also called FLT1), in patients with blinding choroidal neovascularization (CNV) from age-related macular degeneration, are premised on gene silencing by means of intracellular RNA interference (RNAi). We show instead that CNV inhibition is a siRNA-class effect: 21-nucleotide or longer siRNAs targeting non-mammalian genes, non-expressed genes, non-genomic sequences, pro- and anti-angiogenic genes, and RNAi-incompetent siRNAs all suppressed CNV in mice comparably to siRNAs targeting Vegfa or Vegfr1 without off-target RNAi or interferon- alpha / beta activation. Non-targeted (against non-mammalian genes) and targeted (against Vegfa or Vegfr1) siRNA suppressed CNV via cell-surface toll-like receptor 3 (TLR3), its adaptor TRIF, and induction of interferon- gamma and interleukin-12. Non-targeted siRNA suppressed dermal neovascularization in mice as effectively as Vegfa siRNA. siRNA-induced inhibition of neovascularization required a minimum length of 21 nucleotides, a bridging necessity in a modelled 2:1 TLR3–RNA complex. Choroidal endothelial cells from people expressing the TLR3 coding variant 412FF were refractory to extracellular siRNA-induced cytotoxicity, facilitating individualized pharmacogenetic therapy. Multiple human endothelial cell types expressed surface TLR3, indicating that generic siRNAs might treat angiogenic disorders that affect 8% of the world's population, and that siRNAs might induce unanticipated vascular or immune effects.