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Antimicrobial Agents and Chemotherapy, September 2009, p. 3952-3962, Vol. 53, No. 9
0066-4804/09/$08.00+0     doi:10.1128/AAC.00014-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

RNA Interference-Mediated Silencing of the Respiratory Syncytial Virus Nucleocapsid Defines a Potent Antiviral Strategy{triangledown}

Rene Alvarez,1 Sayda Elbashir,1 Todd Borland,1 Ivanka Toudjarska,1 Philipp Hadwiger,2 Mathias John,2 Ingo Roehl,2 Svetlana Shulga Morskaya,1 Rick Martinello,3 Jeffrey Kahn,3 Mark Van Ranst,4 Ralph A. Tripp,5 John P. DeVincenzo,6 Rajendra Pandey,1 Martin Maier,1 Lubomir Nechev,1 Muthiah Manoharan,1 Victor Kotelianski,1 and Rachel Meyers1*

Alnylam Pharmaceuticals, 300 Third Street, Cambridge, Massachusetts 02142,1 Roche Kulmbach, Fritz-Hornschuch-Str. 9, Kulmbach 95326, Germany,2 Yale University, Department of Pediatrics, New Haven, Connecticut,3 University of Leuven, Leuven, Belgium,4 University of Georgia, Department of Infectious Diseases, Center for Disease Intervention, Athens, Georgia,5 University of Tennessee Center for Health Sciences, Departments of Pediatrics and Molecular Sciences, Memphis, Tennessee6

Received 5 January 2009/ Returned for modification 24 January 2009/ Accepted 31 May 2009

We describe the design and characterization of a potent human respiratory syncytial virus (RSV) nucleocapsid gene-specific small interfering RNA (siRNA), ALN-RSV01. In in vitro RSV plaque assays, ALN-RSV01 showed a 50% inhibitory concentration of 0.7 nM. Sequence analysis of primary isolates of RSV showed that the siRNA target site was absolutely conserved in 89/95 isolates, and ALN-RSV01 demonstrated activity against all isolates, including those with single-mismatch mutations. In vivo, intranasal dosing of ALN-RSV01 in a BALB/c mouse model resulted in potent antiviral efficacy, with 2.5- to 3.0-log-unit reductions in RSV lung concentrations being achieved when ALN-RSV01 was administered prophylactically or therapeutically in both single-dose and multidose regimens. The specificity of ALN-RSV01 was demonstrated in vivo by using mismatch controls; and the absence of an immune stimulatory mechanism was demonstrated by showing that nonspecific siRNAs that induce alpha interferon and tumor necrosis factor alpha lack antiviral efficacy, while a chemically modified form of ALN-RSV01 lacking measurable immunostimulatory capacity retained full activity in vivo. Furthermore, an RNA interference mechanism of action was demonstrated by the capture of the site-specific cleavage product of the RSV mRNA via rapid amplification of cDNA ends both in vitro and in vivo. These studies lay a solid foundation for the further investigation of ALN-RSV01 as a novel therapeutic antiviral agent for clinical use by humans.

* Corresponding author. Mailing address: Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142. Phone: (617) 551-8354. Fax: (617) 551-8102. E-mail: rmeyers{at}alnylam.com

{triangledown} Published ahead of print on 8 June 2009.

Antimicrobial Agents and Chemotherapy, September 2009, p. 3952-3962, Vol. 53, No. 9
0066-4804/09/$08.00+0     doi:10.1128/AAC.00014-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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