AU2009212920A1 - Nucleic acid compounds for inhibiting gene expression and uses thereof - Google Patents

Description

AUSTRALIA Patents Act COMPLETE SPECIFICATION (ORIGINAL) Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: MDRNA Research, Inc. Actual Inventor(s): Steven C Quay, James McSwiggen, Narendra K. Vaish, Mohammed Ahmadian Address for Service and Correspondence: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: NUCLEIC ACID COMPOUNDS FOR INHIBITING GENE EXPRESSION AND USES THEREOF Our Ref : 865053 POF Code: 495839/495839 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): 60064 Attorney Docket No. U /-K-Us-CW Customer No.: 36,814 NUCLEIC ACID COMPOUNDS FOR INHIBITING GENE EXPRESSION AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS 5 This application is a continuation-in-part of PCT International Application No. PCT/US2008/055371, International Filing Date February 28, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007, and 60/934,940, filed March 2, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055370 International Filing Date February 10 28, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; 60/934,941, filed April 13, 2007; and 60/934,932, filed April 20, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055362 International Filing Date February 28, 2008, claiming priority of United States Provisional Patent Applications, 15 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; 60/934,931, filed April 20, 2007; 60/934,934, filed April 24, 2007; 60/934,928, filed April 24, 2007; 60/934,943, filed April 25, 2007; and 60/934,942, filed April 25, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055380 International Filing Date February 28, 2008, claiming priority of United States 20 Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; 60/934,93 1, filed April 20, 2007; 60/934,934, filed April 24, 2007; 60/934,928, filed April 24, 2007; 60/934,943, filed April 25, 2007; and 60/934,949, filed May 3, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055383 International Filing Date February 28, 2008, 25 claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; 60/934,941, filed April 13, 2007; 60/934,932, filed April 20, 2007; 60/934,933, filed April 20, 2007; and 60/934,944, filed May 10, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055375 International Filing Date February 28, 2008, 30 claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; 60/934,946, filed May 3, 2007; 60/934,945, filed May 10, 2007; 60/934,935, filed May 15, 2007; and 60/934,922, filed 1A iiatorney LJOCKe INo. U6-Uvrk- I Customer No.: 36,814 May 17, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055360 International Filing Date February 28, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; 60/934,946, filed May 3, 2007; 5 60/934,945, filed May 10, 2007; 60/934,935, filed May 15, 2007; 60/934,922, filed May 17, 2007; and 60/932,970, filed May 22, 2007. This application is also a continuation-in part of PCT International Application No. PCT/US2008/055374 International Filing Date February 28, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; 60/934,921, filed 10 May 24, 2007; 60/932,968, filed May 30, 2007; 60/932,969, filed May 30, 2007; and 60/932,967, filed June 1, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055381 International Filing Date February 28, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; 60/934,954, filed June 6, 2007; 15 and 60/934,929, filed June 14, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055357 International Filing Date February 28, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; 60/934,954, filed June 6, 2007; 60/934,929, filed June 14, 2007; and 60/934,965, filed June 22, 2007. This 20 application is also a continuation-in-part of PCT International Application No. PCT/US2008/055378 International Filing Date February 28, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 60/949,443, filed July 12, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055372 25 International Filing Date February 28, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 60/949,444, filed July 12, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055345 International Filing Date February 28, 2008, claiming priority of United States 30 Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 60/949,448, filed July 12, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055377 International Filing Date February 28, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed 2 Attorney Docket No. 08-U9PCT Customer No.: 36,814 March 2, 2007; 60/949,450, filed July 12, 2007; 60/951,167, filed July 20, 2007; 60/951,168, filed July 20, 2007; and 60/951,170, filed July 20, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055376 International Filing Date February 28, 2008, claiming priority of United States 5 Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; 60/952,191, filed July 26, 2007; 60/952,188, filed July 26, 2007; 60/952,192, filed July 26, 2007; and 60/952,499, filed July 27, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055373 International Filing Date February 28, 2008, claiming priority of United States 10 Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; 60/952,191, filed July 26, 2007; 60/952,188, filed July 26, 2007; 60/952,192, filed July 26, 2007; 60/952,499, filed July 27, 2007; and 60/953,873, filed August 3, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055385 International Filing Date February 28, 2008, 15 claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 60/956,093, filed August 15, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055386 International Filing Date February 28, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 20 60/934,940, filed March 2, 2007; and 60/970,414, filed September 6, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055382 International Filing Date February 28, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 60/956,679, filed August 17, 2007. This 25 application is also a continuation-in-part of PCT International Application No. PCT/US2008/055333 International Filing Date February 28, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 60/970,796, filed September 7, 2007. This application is also a continuation-in-part of PCT International Application No. 30 PCT/US2008/055341 International Filing Date February 28, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 60/970,858, filed September 7, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055350 International Filing Date February 28, 2008, claiming priority of 3 Attorney Docket No. 08-U9PC[ Customer No.: 36,814 United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 60/971,476, filed September 11, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055356 International Filing Date February 28, 2008, claiming priority of 5 United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 60/971,874, filed September 12, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055366 International Filing Date February 28, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 10 60/934,940, filed March 2, 2007; and 60/973,397, filed September 18, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055339 International Filing Date February 28, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; 60/973,398, filed September 18, 2007; 60/013,212, filed 15 December 12, 2007; 60/013,239, filed December 12, 2007; and 60/973,397, filed September 18, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055365 International Filing Date February 28, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 60/983,179, filed October 27, 20 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055340 International Filing Date February 28, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; 60/983,183, filed October 27, 2007; 60/983,182, filed October 27, 2007; 60/983,180, filed October 27, 2007; 60/983,181, filed October 27, 25 2007; 60/986,910, filed November 9, 2007; 60/986,822, filed November 9, 2007; and 60/986,893, filed November 9, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055505 International Filing Date February 29, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 60/986,913, filed 30 November 9, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055556 International Filing Date February 29, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/021,491, filed November 16, 2007. This application is also a continuation-in-part of PCT International Application 4 Attorney Docket No. 08-09PCT Customer No.: 36,814 No. PCT/US2008/055515 International Filing Date February 29, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 60/989,419, filed November 20, 2007. This application is also a continuation-in-part of PCT International Application No. 5 PCT/US2008/055599 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 60/986,916, filed November 9, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055601 International Filing Date March 3, 2008, claiming priority of 10 United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 60/986,919, filed November 9, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055603 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 15 60/934,940, filed March 2, 2007; and 60/987,733, filed November 13, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055606 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 60/987,737, filed November 13, 2007. This 20 application is also a continuation-in-part of PCT International Application No. PCT/US2008/055548 International Filing Date February 29, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; 60/987,740, filed November 13, 2007; 60/988,076, filed November 14, 2007; 60/988,079, filed November 14, 2007; 60/988,082, filed November 25 14, 2007; and 60/988,083, filed November 14, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055611 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/013,979, filed December 14, 2007. This application is also a continuation-in-part 30 of PCT International Application No. PCT/US2008/055615 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 60/988,395, filed November 15, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055709 International Filing Date March 3, 2008, claiming 5 Attorney Docket No. 08-09PCT Customer No.: 36,814 priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; 60/988,397, filed November 15, 2007; and 60/988,398, filed November 15, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055618 International Filing Date March 5 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 60/988,399, filed November 15, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055644 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 10 60/934,940, filed March 2, 2007; 60/988,400, filed November 15, 2007; 60/988,401, filed November 15, 2007; and 60/988,402, filed November 15, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055651 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; 15 60/986,944, filed November 9, 2007; 60/988,403, filed November 15, 2007; and 61/013,981, filed December 14, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055649 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/015,611, filed December 20 20, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055711 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; 60/988,405, filed November 15, 2007; 60/989,427, filed November 20, 2007; 60/989,424, filed November 20, 2007; and 60/989,428, filed 25 November 20, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055635 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/014,305, filed December 17, 2007. This application is also a continuation-in-part of PCT International Application No. 30 PCT/US2008/055524 International Filing Date February 29, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/014,730, filed December 18, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055672 International Filing Date March 3, 2008, claiming priority of 6 Attorney Vocket No. 08-09PCT Customer No.: 36,814 United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/015,164, filed December 19, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055627 International Filing Date March 3, 2008, claiming priority of 5 United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/015,166, filed December 19, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055697 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 10 60/934,940, filed March 2, 2007; and 61/012,401, filed December 7, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055662 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/016,340, filed December 21, 2007. This 15 application is also a continuation-in-part of PCT International Application No. PCT/US2008/055678 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 60/992,808, filed December 6, 2007. This application is also a continuation-in-part of PCT International Application No. 20 PCT/US2008/055368 International Filing Date February 2, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/013,982, filed December 14, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055676 International Filing Date March 3, 2008, claiming priority of 25 United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/014,139, filed December 17, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055550 International Filing Date February 29, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 30 60/934,940, filed March 2, 2007; 60/934,921, filed May 24, 2007; 60/932,968, filed May 30, 2007; 60/932,969, filed May 30, 2007; and 60/932,967, filed June 1, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055560 International Filing Date February 29, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 7 Attorney Docket No. U-Uw i Customer No.: 36,814 60/934,940, filed March 2, 2007; 60/934,921, filed May 24, 2007; 60/932,968, filed May 30, 2007; 60/932,969, filed May 30, 2007; 60/932,967, filed June 1, 2007; and 61/014,733 filed December 18, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055698 International Filing Date March 5 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/012,402, filed December 7, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055695 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 10 60/934,940, filed March 2, 2007; and 61/016,343, filed December 21, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055701 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/015,616, filed December 20, 2007. This 15 application is also a continuation-in-part of PCT International Application No. PCT/US2008/055693 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/015,618, filed December 20, 2007. This application is also a continuation-in-part of PCT International Application No. 20 PCT/US2008/055704 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/014,734, filed December 18, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055708 International Filing Date March 3, 2008, claiming priority of 25 United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/015,167, filed December 19, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055597 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 30 60/934,940, filed March 2, 2007; and 61/015,170, filed December 19, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055604 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/014,735, filed December 18, 2007. This 8 Attorney Docket No. 08-09PCT Customer No.: 36,814 application is also a continuation-in-part of PCT International Application No. PCT/US2008/055608 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/014,737, filed December 18, 2007. This 5 application is also a continuation-in-part of PCT International Application No. PCT/US2008/055353 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 60/992,975, filed December 6, 2007. This application is also a continuation-in-part of PCT International Application No. 10 PCT/US2008/055631 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/015,171, filed December 19, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055563 International Filing Date February 29, 2008, claiming priority of 15 United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/015,173, filed December 19, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055612 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 20 60/934,940, filed March 2, 2007; and 61/012,404, filed December 7, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055622 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/014,738, filed December 18, 2007. This 25 application is also a continuation-in-part of PCT International Application No. PCT/US2008/055625 International Filing Date March 3, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/015,619, filed December 20, 2007. This application is also a continuation-in-part of PCT International Application No. 30 PCT/US2008/055527 International Filing Date February 29, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007 and 60/934,940, filed March 2, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055533 International Filing Date February 29, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, 9 Attorney Vocket No. UZS-U9kCTI Customer No.: 36,814 filed March 16, 2007; 60/934,940, filed March 2, 2007; and 61/016,319, filed December 21, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055554 International Filing Date February 29, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007 and 5 60/934,940, filed March 2, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055511 International Filing Date February 29, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007 and 60/934,940, filed March 2, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055532 10 International Filing Date February 29, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007 and 60/934,940, filed March 2, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055516 International Filing Date February 29, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed 15 March 16, 2007 and 60/934,940, filed March 2, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055551 International Filing Date February 29, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007 and 60/934,940, filed March 2, 2007. This application is also a continuation-in-part of PCT International 20 Application No. PCT/US2008/055519 International Filing Date February 29, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007 and 60/934,940, filed March 2, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055542 International Filing Date February 29, 2008, claiming priority of United States 25 Provisional Patent Applications, 60/934,930, filed March 16, 2007 and 60/934,940, filed March 2, 2007. This application is also a continuation-in-part of PCT International Application No. PCT/US2008/055526 International Filing Date February 29, 2008, claiming priority of United States Provisional Patent Applications, 60/934,930, filed March 16, 2007 and 60/934,940, filed March 2, 2007. All of the above identified 30 applications are incorporated herein by reference in their entirety. 10 Attorney Uocket No. UW-U9I'CT Customer No.: 36,814 TECHNICAL FIELD The present disclosure provides RNA molecules, for example, meroduplex ribonucleic acid molecules (mdRNA), and blunt ended double-stranded ribonucleic acid molecules capable of decreasing or silencing expression of a target gene or family of 5 genes. An mdRNA of this disclosure comprises at least three strands that combine to form at least two non-overlapping double-stranded regions separated by a nick or gap wherein one strand is complementary to a particular mRNA(s). Also provided are methods of decreasing expression of a target gene or family of genes in a cell or in a subject to treat a disease or condition associated with the target gene or family of genes. 10 BACKGROUND RNA interference (RNAi) refers to the cellular process of sequence specific, post-transcriptional gene silencing in animals mediated by small inhibitory nucleic acid molecules, such as a double-stranded RNA (dsRNA) that is homologous to a portion of a targeted messenger RNA (Fire et al., Nature 391:806, 1998; Hamilton et al., Science 15 286:950-951, 1999). RNAi has been observed in a variety of organisms, including mammalians (Fire et al., Nature 391:806, 1998; Bahramian and Zarbl, Mol. Cell. Biol. 19:274-283, 1999; Wianny and Goetz, Nature Cell Biol. 2:70, 1999). RNAi can be induced by introducing an exogenous synthetic 21 -nucleotide RNA duplex into cultured mammalian cells (Elbashir et al., Nature 411:494, 2001a). 20 The mechanism by which dsRNA mediates targeted gene-silencing can be described as involving two steps. The first step involves degradation of long dsRNAs by a ribonuclease III-like enzyme, referred to as Dicer, into short interfering RNAs (siRNAs) having from 21 to 23 nucleotides with double-stranded regions of about 19 base pairs and a two nucleotide, generally, overhang at each 3'-end (Berstein et al., Nature 409:363, 25 2001; Elbashir et al., Genes Dev. 15:188, 2001b; and Kim et al., Nature Biotech. 23:222, 2005). The second step of RNAi gene-silencing involves activation of a multi-component nuclease having one strand (guide or antisense strand) from the siRNA and an Argonaute protein to form an RNA-induced silencing complex ("RISC") (Elbashir et al., Genes Dev. 15:188, 2001). Argonaute initially associates with a double-stranded siRNA and then 30 endonucleolytically cleaves the non-incorporated strand (passenger or sense strand) to facilitate its release due to resulting thermodynamic instability of the cleaved duplex (Leuschner et al., EMBO 7:314, 2006). The guide strand in the activated RISC binds to a complementary target mRNA, which is then cleaved by the RISC to promote gene 11 Attorney Docket No. 08-09PCT Customer No.: 36,814 silencing. Cleavage of the target RNA occurs in the middle of the target region that is complementary to the guide strand (Elbashir et al., 2001b). There continues to be a need for alternative effective therapeutic modalities useful for treating or preventing diseases or disorders in which reduced expression of a gene or 5 family of genes (gene silencing) would be beneficial. The present disclosure meets such needs, and further provides other related advantages. BRIEF SUMMARY Briefly, the present disclosure provides nicked or gapped double-stranded RNA (dsRNA) comprising at least three strands, and a blunt ended double-stranded RNA 10 having continuous strands (i.e., not nicked or gapped) that are suitable as a substrate for Dicer or as a RISC activator to modify expression of a target messenger RNA (mRNA). In one aspect, the instant disclosure provides a meroduplex mdRNA molecule, comprising a first strand that is complementary to a human mRNA nucleotide sequence, and a second strand and a third strand that are each complementary to non-overlapping 15 regions of the first strand, wherein the second strand and third strands can anneal with the first strand to form at least two double-stranded regions spaced apart by up to 10 nucleotides and thereby forming a gap between the second and third strands, and wherein (a) the mdRNA molecule optionally includes at least one double-stranded region of 5 base pairs to 13 base pairs, or (b) the double-stranded regions combined total about 20 15 base pairs to about 40 base pairs and the mdRNA molecule optionally has blunt ends. In certain embodiments, the first strand is about 15 to about 40 nucleotides in length, and the second and third strands are each, individually, about 5 to about 20 nucleotides, wherein the combined length of the second and third strands is about 15 nucleotides to about 40 nucleotides. In other embodiments, the first strand is about 15 to about 40 25 nucleotides in length and is complementary to at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides of a human mRNA nucleotide sequence. In still further embodiments, the first strand is about 15 to about 40 nucleotides in length and is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence that is 30 complementary to at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides of a human mRNA nucleotide sequence. 12 ALLU[Cy LJOCKt INO. Uv-Uru I Customer No.: 36,814 In other embodiments, the mdRNA is a RISC activator (e.g., the first strand has about 15 nucleotides to about 25 nucleotides) or a Dicer substrate (e.g., the first strand has about 26 nucleotides to about 40 nucleotides). In some embodiments, the gap comprises at least one to ten unpaired nucleotides in the first strand positioned between the double 5 stranded regions formed by the second and third strands when annealed to the first strand, or the gap is a nick. In certain embodiments, the nick or gap is located 10 nucleotides from the 5'-end of the first (antisense) strand or at the Argonaute cleavage site. In another embodiment, the meroduplex nick or gap is positioned such that the thermal stability is maximized for the first and second strand duplex and for the first and third strand duplex 10 as compared to the thermal stability of such meroduplexes having a nick or gap in a different position. In another aspect, the instant disclosure provides an mdRNA molecule having a first strand that is complementary to human mRNA nucleotide sequence, and a second strand and a third strand that is each complementary to non-overlapping regions of the 15 first strand, wherein the second strand and third strand can anneal with the first strand to form at least two double-stranded regions spaced apart by up to 10 nucleotides and thereby forming a gap between the second and third strands, and wherein (a) the mdRNA molecule optionally includes at least one double-stranded region of 5 base pairs to 13 base pairs, or (b) the double-stranded regions combined total about 15 base pairs to about 20 40 base pairs and the mdRNA molecule optionally has blunt ends; and wherein at least one pyrimidine of the mdRNA comprises a pyrimidine nucleoside according to Formula I or II: R 0 O R1 NH 2 5 4 3NH ' N (I) R4I' R R4 R5 N 4' ' R 8

R

8 3' 2' R3 R 2 R3 R 2 25 13 Attorney Docket No. 08-09PCT Customer No.: 36,814 wherein R1 and R 2 are each independently a -H, -OH, -OCH 3 , -OCH 2

OCH

2

CH

3 ,

-OCH

2

CH

2

OCH

3 , halogen, substituted or unsubstituted C 1

-C

10 alkyl, alkoxy, alkoxyalkyl, hydroxyalkyl, carboxyalkyl, alkylsulfonylamino, aminoalkyl, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, trifluoromethyl, cycloalkyl, 5 (cycloalkyl)alkyl, substituted or unsubstituted C 2 -Cio alkenyl, substituted or unsubstituted -0-allyl, -O-CH 2

CH=CH

2 , -O-CH=CHCH 3 , substituted or unsubstituted C 2

-C

10 alkynyl, carbamoyl, carbamyl, carboxy, carbonylamino, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, -NH 2 , -NO 2 , -C-N, or heterocyclo group; R 3 and R4 are each independently a hydroxyl, a protected hydroxyl, a phosphate, or an 10 internucleoside linking group; and R5 and R8 are independently 0 or S. In certain embodiments, at least one nucleoside is according to Formula I in which R' is methyl and R2 is -OH. In certain related embodiments, at least one uridine of the dsRNA molecule is replaced with a nucleoside according to Formula I in which R' is methyl and R 2 is -OH, or R1 is methyl, R 2 is -OH, and R8 is S. In some embodiments, the at least one R, is a 15 C 1

-C

5 alkyl, such as methyl. In some embodiments, at least one R2 is selected from 2'-0

(C

1 -Cs) alkyl, 2'-O-methyl, 2'-OCH 2 0CH 2

CH

3 , 2'-OCH 2

CH

2 0CH 3 , 2'-O-allyl, or fluoro. In some embodiments, at least one pyrimidine nucleoside of the mdRNA molecule is a locked nucleic acid (LNA) in the form of a bicyclic sugar, wherein R2 is oxygen, and the 2'-O and 4'-C form an oxymethylene bridge on the same ribose ring (e.g., a 5 20 methyluridine LNA) or is a G clamp. In other embodiments, one or more of the nucleosides are according to Formula I in which R' is methyl and R 2 is a 2'-O-(C1-C 5 ) alkyl, such as 2'-O-methyl. In some embodiments, the gap comprises at least one unpaired nucleotide in the first strand positioned between the double-stranded regions formed by the second and third strands when annealed to the first strand, or the gap is a 25 nick. In certain embodiments, the nick or gap is located 10 nucleotides from the 5'-end of the first strand or at the Argonaute cleavage site. In another embodiment, the meroduplex nick or gap is positioned such that the thermal stability is maximized for the first and second strand duplex and for the first and third strand duplex as compared to the thermal stability of such meroduplexes having a nick or gap in a different position. 30 In still another aspect, the instant disclosure provide for an mdRNA having one more hydroxymethyl modified nucleomonomer(s) (see chemical formulas below monomers D, F, G, H, I, and J.). Hereunder as one such example is an acyclic nucleomonomer, more preferably an acyclic monomer selected from the group consisting 14 Attorney Docket No. 08-09PCT Customer No.: 36,814 of monomers D, F, G, H, I, and J. Additional monomers that may be incorporated into an mdRNA of this disclosure include: 0 Base O Base HO- HO 0 OH 0 -O-P=O O-P=O 5 In still another aspect, the instant disclosure provides a method for reducing the expression of a human gene in a cell, comprising administering an mdRNA molecule to a cell expressing the gene, wherein the mdRNA molecule is capable of specifically binding to an mRNA and thereby reducing the gene's level of expression in the cell. In a related aspect, there is provided a method of treating or preventing a disease associated with the 10 expression of a gene or family of genes in a subject by administering an mdRNA molecule of this disclosure. In certain embodiments, the cell or subject is human. In certain embodiments, the disease is cancer, a metabolic disease or an inflammatory disease. In any of the aspects of this disclosure, some embodiments provide an mdRNA 15 molecule having a 5-methyluridine (ribothymidine), a 2-thioribothymidine, or 2'-0 methyl-5-methyluridine in place of at least one uridine on the first, second, or third strand, or in place of each and every uridine on the first, second, or third strand. In further embodiments, the mdRNA further comprises one or more non-standard nucleoside, such as a deoxyuridine, locked nucleic acid (LNA) molecule, or a universal 20 binding nucleotide, or a G clamp. Exemplary universal-binding nucleotides include C-phenyl, C-naphthyl, inosine, azole carboxamide, 1-p-D-ribofuranosyl-4-nitroindole, 1 p-D-ribofuranosyl-5-nitroindole, 1 -p-D-ribofuranosyl-6-nitroindole, or 1 -p-D ribofuranosyl-3-nitropyrrole. In some embodiments, the mdRNA molecule further comprises a 2'-sugar substitution, such as a 2'-O-methyl, 2'-O-methoxyethyl, 25 2'-O-2-methoxyethyl, 2'-O-allyl, or halogen (e.g., 2'-fluoro). In certain embodiments, the mdRNA molecule further comprises a terminal cap substituent on one or both ends of one or more of the first strand, second strand, or third strand, such as independently an alkyl, abasic, deoxy abasic, glyceryl, dinucleotide, acyclic nucleotide, or inverted deoxynucleotide moiety. In other embodiments, the mdRNA molecule further comprises 15 Attorney Uocket No. U6-U9FU I Customer No.: 36,814 at least one modified internucleoside linkage, such as independently a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl phosphonate, alkyl phosphonate, 3'-alkylene phosphonate, 5'-alkylene phosphonate, chiral phosphonate, phosphonoacetate, thiophosphonoacetate, phosphinate, 5 phosphoramidate, 3'-amino phosphoramidate, aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate, or boranophosphate linkage. In any of the aspects of this disclosure, some embodiments provide an mdRNA comprising an overhang of one to four nucleotides on at least one 3'-end that is not part of 10 the gap, such as at least one deoxyribonucleotide or two deoxyribonucleotides (e.g., thymidine). In some embodiments, at least one or two 5'-terminal ribonucleotide of the second strand within the double-stranded region comprises a 2'-sugar substitution. In related embodiments, at least one or two 5'-terminal ribonucleotide of the first strand within the double-stranded region comprises a 2'-sugar substitution. In other related 15 embodiments, at least one or two 5'-terminal ribonucleotide of the second strand and at least one or two 5'-terminal ribonucleotide of the first strand within the double-stranded regions comprise independent 2'-sugar substitutions. In other embodiments, the mdRNA molecule comprises at least three 5-methyluridines within at least one double-stranded region. In some embodiments, the mdRNA molecule has a blunt end at one or both ends. 20 In other embodiments, the 5'-terminal of the third strand is a hydroxyl or a phosphate. In one aspect, the instant disclosure provides a double-stranded (dsRNA) molecule, comprising a first strand that is complementary to a human mRNA nucleotide sequence, and a second strand that is complementary to the first strand, wherein the double-stranded region is from about 15 base pairs to about 40 base pairs. In certain 25 aspects, the dsRNA molecule has one or more blunt ends. In certain embodiments, the first strand and the second strand are each independently from about 15 to about 40 nucleotides in length. In other embodiments, the first strand is about 15 to about 40 nucleotides in length and is complementary to at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous 30 nucleotides of a human mRNA nucleotide sequence. In still further embodiments, the first strand is about 15 to about 40 nucleotides in length and is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence that is complementary to at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 16 Attomey UocKet No. UZ5-U91F [ Customer No.: 36,814 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides of a human mRNA nucleotide sequence. In other embodiments, the dsRNA is a RISC activator (e.g., the first strand has about 15 nucleotides to about 25 nucleotides) or a Dicer substrate (e.g., the first strand has 5 about 26 nucleotides to about 40 nucleotides). In another aspect, the instant disclosure provides a dsRNA molecule having a first strand that is complementary to human mRNA nucleotide sequence, and a second strand that is complementary to the first strand, wherein the double-stranded region is from about 15 base pairs to about 40 base pairs. In certain embodiments, the dsRNA has one 10 or more blunt ends; and wherein at least one pyrimidine of the dsRNA comprises a pyrimidine nucleoside according to Formula I or II: R 0 R1 NH 2 6 3NH N (I) R 4 5R5 R Rs N 15 4' ' RR 3' 2 R3 R2 R 3 R2 wherein R 1 and R 2 are each independently a -H, -OH, -OCH 3 , -OCH 2 0CH 2

CH

3 , 20 -OCH 2

CH

2 0CH 3 , halogen, substituted or unsubstituted Ci-Cio alkyl, alkoxy, alkoxyalkyl, hydroxyalkyl, carboxyalkyl, alkylsulfonylamino, aminoalkyl, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, substituted or unsubstituted C 2 -Cio alkenyl, substituted or unsubstituted -0-allyl, -O-CH 2

CH=CH

2 , -O-CH=CHCH 3 , substituted or unsubstituted C 2 -Cio alkynyl, 25 carbamoyl, carbamyl, carboxy, carbonylamino, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, -NH 2 , -NO 2 , -C-N, or heterocyclo group; R 3 and R 4 are each independently a hydroxyl, a protected hydroxyl, a phosphate, or an intemucleoside linking group; and R 5 and R are independently 0 or S. In certain embodiments, at least one nucleoside is according to Formula I in which R' is methyl and 30 R2 is -OH. In certain related embodiments, at least one uridine of the dsRNA molecule is replaced with a nucleoside according to Formula I in which R' is methyl and R 2 is -OH, or R1 is methyl, R 2 is -OH, and R 8 is S. In some embodiments, the at least one R 1 is a 17 Attorney Docket No. 08-09PCT Customer No.: 36,814 Ci-C 5 alkyl, such as methyl. In some embodiments, at least one R2 is selected from 2'-O (Ci-C 5 ) alkyl, 2'-O-methyl, 2'-OCH 2

OCH

2

CH

3 , 2'-OCH 2

CH

2

OCH

3 , 2'-O-allyl, or fluoro. In some embodiments, at least one pyrimidine nucleoside of the dsRNA molecule is a locked nucleic acid (LNA) in the form of a bicyclic sugar, wherein R 2 is oxygen, and the 5 2'-O and 4'-C form an oxymethylene bridge on the same ribose ring (e.g., a 5 methyluridine LNA) or is a G clamp. In other embodiments, one or more of the nucleosides are according to Formula I in which R' is methyl and R 2 is a 2'-O-(C 1

-C

5 ) alkyl, such as 2'-0-methyl. In still another aspect, the instant disclosure provide for a dsRNA having one 10 more hydroxymethyl modified nucleomonomer(s) (see chemical formulas below monomers D, F, G, H, I, and J.). Hereunder as one such example is an acyclic nucleomonomer, more preferably an acyclic monomer selected from the group consisting of monomers D, F, G, H, I, and J. Additional monomers that may be incorporated into a dsRNA of this disclosure include: 0 Base - Base HO- HO 0 OH 0 -O-P=O -O-P=O 15 % In still another aspect, the instant disclosure provides a method for reducing the expression of a human gene in a cell, comprising administering a dsRNA molecule to a cell expressing the gene, wherein the dsRNA molecule is capable of specifically binding 20 to an mRNA and thereby reducing the gene's level of expression in the cell. In a related aspect, there is provided a method of treating or preventing a disease associated with the expression of a gene or family of genes in a subject by administering a dsRNA molecule of this disclosure. In certain embodiments, the cell or subject is human. In certain embodiments, the disease is cancer, a metabolic disease or inflammatory disease. 25 In any of the aspects of this disclosure, some embodiments provide a dsRNA molecule having a 5-methyluridine (ribothymidine), a 2-thioribothymidine, or 2'-0 methyl-5-methyluridine in place of at least one uridine on the first, second, or third strand, or in place of each and every uridine on the first, second, or third strand. In further embodiments, the dsRNA further comprises one or more non-standard nucleoside, 18 Attorney Docket No. U8-U9PCT Customer No.: 36,814 such as a deoxyuridine, locked nucleic acid (LNA) molecule, or a universal-binding nucleotide, or a G clamp. Exemplary universal-binding nucleotides include C-phenyl, C naphthyl, inosine, azole carboxamide, 1 -p-D-ribofuranosyl-4-nitroindole, 1 -0-D ribofuranosyl-5-nitroindole, 1 -p-D-ribofuranosyl-6-nitroindole, or 1 -- D-ribofuranosyl 5 3-nitropyrrole. In some embodiments, the dsRNA molecule further comprises a 2'-sugar substitution, such as a 2'-O-methyl, 2'-O-methoxyethyl, 2'-O-2-methoxyethyl, 2'-O-allyl, or halogen (e.g., 2'-fluoro). In certain embodiments, the dsRNA molecule further comprises a terminal cap substituent on one or both ends of one or more of the first strand or second strands, such as independently an alkyl, abasic, deoxy abasic, glyceryl, 10 dinucleotide, acyclic nucleotide, or inverted deoxynucleotide moiety. In other embodiments, the mdRNA molecule further comprises at least one modified internucleoside linkage, such as independently a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl phosphonate, alkyl phosphonate, 3'-alkylene phosphonate, 5'-alkylene 15 phosphonate, chiral phosphonate, phosphonoacetate, thiophosphonoacetate, phosphinate, phosphoramidate, 3'-amino phosphoramidate, aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate, or boranophosphate linkage. In any of the aspects of this disclosure, some embodiments provide a dsRNA 20 comprising an overhang of one to four nucleotides on at least one 3'-end, such as at least one deoxyribonucleotide or two deoxyribonucleotides (e.g., thymidine). In some embodiments, at least one or two 5'-terminal ribonucleotide of the second strand within the double-stranded region comprises a 2'-sugar substitution. In related embodiments, at least one or two 5'-terminal ribonucleotide of the first strand within the double-stranded 25 region comprises a 2'-sugar substitution. In other related embodiments, at least one or two 5'-terminal ribonucleotide of the second strand and at least one or two 5'-terminal ribonucleotide of the first strand within the double-stranded region comprise independent 2'-sugar substitutions. In other embodiments, thedsdRNA molecule comprises at least three 5-methyluridines within the double-stranded region. In some embodiments, the 30 dsRNA molecule has a blunt end at one or both ends. In other embodiments, the 5'-terminal of the third strand is a hydroxyl or a phosphate. 19 Attorney Uocket No. U0-09Pi Customer No.: 36,814 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the average gene silencing activity of intact (first bar), nicked (middle bar), and gapped (last bar) dsRNA Dicer substrate specific for each of 22 different targets (AKT, EGFR, FLTl, FRAPI, HIFlA, IL17A, 1L18, IL6, MAP2K1, 5 MAPK1, MAPK14, PDGFA, PDGFRA, PIKC3A, PKN3, RAFI, SRD5A1, TNF, TNFSF13B, VEGFA, BCR-ABL [b2a2], and BCR-ABL [b3a2]). Each bar is a graphical representation of an average activity of ten different sequences for each target, which is calculated from the data found in Table 1. Figure 2 shows knockdown activity for RISC activator lacZ dsRNA (21 10 nucleotide sense strand/21 nucleotide antisense strand; 21/21), Dicer substrate lacZ dsRNA (25 nucleotide sense strand/27 nucleotide antisense strand; 25/27), and meroduplex lacZ mdRNA (13 nucleotide sense strand and 11 nucleotide sense strand/27 nucleotide antisense strand; 13, 11/27 - the sense strand is missing one nucleotide so that a single nucleotide gap is left between the 13 nucleotide and 11 nucleotide sense strands 15 when annealed to the 27 nucleotide antisense strand. Knockdown activities were normalized to a Qneg control dsRNA and presented as a normalized value of Qneg (i.e., Qneg represents 100% or "normal" gene expression levels). A smaller value indicates a greater knockdown effect. Figure 3 shows knockdown activity of a RISC activator influenza dsRNA G1498 20 (21/21) and nicked dsRNA (10, 11/21) at 100 nM. The "wt" designation indicates an unsubstituted RNA molecule; "rT" indicates RNA having each uridine substituted with a ribothymidine; and "p" indicates that the 5'-nucleotide of that strand was phosphorylated. The 21 nucleotide sense and antisense strands of G1498 were individually nicked between the nucleotides 10 and 11 as measured from the 5'-end, and is referred to as 25 11, 10/21 and 21/10, 11, respectively. The G1498 single stranded 21 nucleotide antisense strand alone (designated AS-only) was used as a control. Figure 4 shows knockdown activity of a lacZ dicer substrate (25/27) having a nick in one of each of positions 8 to 14 and a one nucleotide gap at position 13 of the sense strand (counted from the 5'-end). A dideoxy guanosine (ddG) was incorporated at the 5' 30 end of the 3'-most strand of the nicked or gapped sense sequence at position 13. Figure 4 discloses SEQ ID NO: 3. Figure 5 shows knockdown activity ofa dicer substrate influenza dsRNA G1498DS (25/27) and this sequence nicked at one of each of positions 8 to 14 of the 20 Attorney Docket No. U6-U9PCT Customer No.: 36,814 sense strand, and shows the activity of these nicked molecules that are also phosphorylated or have a locked nucleic acid substitution. Figure 6 shows a dose dependent knockdown activity a dicer substrate influenza dsRNA G1498DS (25/27) and this sequence nicked at position 13 of the sense strand. 5 Figure 7 shows knockdown activity of a dicer substrate influenza dsRNA G1498DS having a nick or a gap of one to six nucleotides that begins at any one of positions 8 to 12 of the sense strand. Figure 8 shows knockdown activity of a LacZ RISC dsRNA having a nick or a gap of one to six nucleotides that begins at any one of positions 8 to 14 of the sense 10 strand. Figure 9 shows knockdown activity of an influenza RISC dsRNA having a nick at any one of positions 8 to 14 of the sense strand and further having one or two locked nucleic acids (LNA) per sense strand. The inserts on the right side of the graph provides a graphic depiction of the meroduplex structures (for clarity, a single antisense strand is 15 shown at the bottom of the grouping with each of the different nicked sense strands above the antisense) having different nick positions with the relative positioning of the LNAs on the sense strands. Figure 10 shows knockdown activity of a LacZ dicer substrate dsRNA having a nick at any one of positions 8 to 14 of the sense strand as compared to the same nicked 20 dicer substrates but having a locked nucleic acid substitution. Figure 11 shows the percent knockdown in influenza viral titers using influenza specific mdRNA against influenza strain WSN. Figure 12 shows the in vivo reduction in PR8 influenza viral titers using influenza specific mdRNA as measured by TCID 50 . 25 DETAILED DESCRIPTION The instant disclosure provides for a nicked or gapped double-stranded RNA comprising at least three strands, and a blunt ended double-stranded RNA having continuous strands (i.e., not nicked or gapped) that are a suitable substrate for Dicer or RISC and, therefore, may be advantageously employed for gene silencing via, for 30 example, the RNA interference pathway. That is, partially duplexed dsRNA molecules described herein (also referred to as meroduplexes having a nick or gap in at least one strand) and blunt-ended dsRNA are capable of initiating an RNA interference cascade that modifies (e.g., reduces) expression of a target messenger RNA (mRNA), such as a 21 Attorney Docket No. 08-09PCT Customer No.: 36,814 human tumor necrosis factor (TNF) mRNA, vascular endothelial growth factor (VEGF) mRNA, vascular endothelial growth factor receptor (VEGFR) mRNA, epidermal growth factor receptor (EGFR) mRNA, erythroblastic leukemia viral oncogene homolog (ERBB) mRNA, platelet derived growth factor (PDGF) mRNA, platelet derived growth factor 5 receptor (PDGFR) mRNA, breakpoint cluster region (BCR)-abelson murine leukemia viral oncogene homolog (ABL) mRNA, steroid-5-alpha-reductase, alpha polypeptide 1 (SRD5A1) mRNA, steroid-5-alpha-reductase, alpha polypeptide 2 (SRD5A2) mRNA, phosphoinositide-3-kinase, catalytic (PIK3C) mRNA, mitogen-activated protein kinase (MAPK) mRNA, p38 MAPK family mRNA, hypoxia-inducible factor 1 alpha (HIFlA) 10 mRNA, protein kinase N3 (PKN3) mRNA, interleukin 17A (IL1 7A) mRNA, interleukin 6 (IL6) mRNA, interleukin 18 (IL1 8) mRNA, tumor necrosis factor (ligand) superfamily member 13b (TNFSF13B) mRNA, mitogen-activated protein kinase 1 (MAPK1) mRNA, v-raf-1 murine leukemia viral oncogene homolog 1 (RAFi) mRNA, v-AKT murine thymoma viral oncogene (AKT) mRNA, FK506 binding protein 12-rapamycin associated 15 protein 1 (FRAP 1) mRNA, mitogen-activated protein kinase 2 (MAPK2) mRNA, cyclin dependent kinase 2 (CDK2) mRNA, ATP-binding cassette, subfamily B, member 1 (ABCB1) mRNA, B-cell CLL/lymphoma 2 (BCL2) mRNA, angiopoietin 2 (ANGPT2) mRNA, checkpoint kinase 1 homolog (CHEKI) mRNA, insulin-like growth factor 1 receptor (IGF 1 R) mRNA, signal transducer and activator of transcription 3 (STAT3) 20 mRNA, matrix metalloproteinase (MMP) mRNA, folate hydrolase (prostate-specific membrane antigen) 1 (FOLH1) mRNA, v-myc myelocytomatosis viral oncogene homolog (avian) (MYC) mRNA, telomerase RNA component (TERC) mRNA, telomerase reverse transcriptase (TERT) mRNA, protein kinase C, alpha (PRKCA) mRNA, RAS viral (v-ras) oncogene homolog (RAS) mRNA, chemokine (C-X-C motif) 25 ligand or receptor (CXC) mRNA, Wingless-Type MMTV (Murine Mammary Tumor Virus) Integration Site (WNT) mRNA, toll-like receptor (TLR) mRNA, Fc fragment of IgE, low affinity II, receptor for (CD23) (FCER2) mRNA, FOS gene, (FOS, FOSB, FOSL1, OR FOSL2) mRNA, hydroxysteroid (11-beta) dehydrogenase (HSD1 IBI) mRNA, JUN gene (cJUN, JUNB, or JUND) mRNA, thymidine phosphorylase (TYMP) 30 mRNA, early growth response (EGR) mRNA, zeste homolog 2 (EZH2) mRNA, cyclin D1 (CCND1) mRNA, Fas (TNF receptor superfamily, member 6) (FAS) mRNA, proliferating cell nuclear antigen (PCNA) mRNA, fibroblast growth factor 2 (FGF2) mRNA, tumor growth factor-beta (TGF-p) mRNA, tumor growth factor-beta receptor 22 Attorney Docket No. 08-09PCT Customer No.: 36,814 (TGF-pR) mRNA, tumor-associated calcium signal transducer 1 (TACSTD1) mRNA, Mucin 1 (MUC1) mRNA, protein tyrosine phosphatase, non-receptor-l l (Noonan Syndrome 1) (PTPN1 1) mRNA, neuregulin 1 (NRGl) mRNA, membrane metallo endopeptidase (MME) mRNA, CD19 molecule (CD19) mRNA, CD40 molecule, TNF 5 receptor superfamily member 5 (CD40) mRNA, apolipoprotein B (including Ag(x) antigen) (ApoB) mRNA, synuclein, alpha (non A4 component of amyloid precursor) (SNCA) mRNA, silent mating type information regulation 2 homolog (SIRT2) mRNA, histone deacetylase (HDAC) mRNA, membrane-spanning 4-domains, subfamily A, member 1 (MS4A1) mRNA, CD22 molecule (CD22) mRNA, diacylglycerol o 10 acyltransferase 1 (DGAT1) mRNA, diacylglycerol o-acyltransferase 2 (DGAT2) mRNA, CD3 molecule (CD3) mRNA, proprotein convertase subtilisin-like kexin type 9 (PCSK9) mRNA, MET (Mesenchymal epithelial transition factor) (c-Met proto-oncogene) mRNA, catenin (cadherin-associated protein) (beta-catenin) (CTNNB 1) mRNA, inhition of DNA binding proteins (Inhibition of Differentiation Proteins, Dominant Negative Helix-Loop 15 Helix Protein) (ID, e.g., ID-1) mRNA, protein tyrosine phosphatase, non-receptor type l(PTPN1) mRNA, tie-I (TIE1; tyrosine kinase with immunoglobulin and EGF factor homology domains 1) mRNA, tek tyrosine kinase (TEK) mRNA, fibroblast growth factor receptor (FGFR) mRNA, mitogen-activated protein kinase 3 (MAPK3) mRNA, survivin (BIRC5) mRNA, polo-like kinase family genes (PLK Family; PLK1, PLK2, and PLK3) 20 mRNA, or any one or more combination gene targets identified above. A person of skill in the art would expect the thermodynamically less stable nicked or gapped dsRNA passenger strand (as compared to an intact dsRNA) to fall apart before any gene silencing effect would result (see, e.g., Leuschner et al., EMBO 7:314, 2006). Meroduplex ribonucleic acid (mdRNA) molecules described herein include a first 25 (antisense) strand that is complementary to a human mRNA nucleotide sequence, along with second and third strands (together forming a gapped sense strand) that are each complementary to non-overlapping regions of the first strand, wherein the second and third strands can anneal with the first strand to form at least two double-stranded regions separated by a gap, and wherein at least one double-stranded region is from about 5 base 30 pairs to about 15 base pairs, or the combined double-stranded regions total about 15 base pairs to about 40 base pairs and the mdRNA is blunt-ended. The gap can be from 0 nucleotides (i.e., a nick in which only a phosphodiester bond between two nucleotides is broken in a polynucleotide molecule) up to about 10 nucleotides (i.e., the first strand will 23 Attorney Docket No. US-U9P I Customer No.: 36,814 have at least one unpaired nucleotide). In certain embodiments, the nick or gap is located 10 nucleotides from the 5'-end of the first (antisense) strand or at the Argonaute cleavage site. In another embodiment, the meroduplex nick or gap is positioned such that the thermal stability is maximized for the first and second strand duplex and for the first and 5 third strand duplex as compared to the thermal stability of such meroduplexes having a nick or gap in a different position. Also provided herein are methods of using such dsRNA to reduce expression of a gene in a cell or to treat or prevent diseases or disorders associated with gene expression, including cancer, metabolic, and inflammatory diseases. Prior to introducing more detail to this disclosure, it may be helpful to an 10 appreciation thereof to provide definitions of certain terms to be used herein. In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to 15 any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, "about" or "consisting essentially of" mean ± 20% of the indicated range, value, or structure, unless otherwise indicated. As used herein, the terms "include" and "comprise" are open ended and are used synonymously. It should be understood that the terms "a" 20 and "an" as used herein refer to "one or more" of the enumerated components. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the term "isolated" means that the referenced material (e.g., nucleic acid molecules of the instant disclosure), is removed from its original 25 environment, such as being separated from some or all of the co-existing materials in a natural environment (e.g., a natural environment may be a cell). As used herein, "complementary" refers to a nucleic acid molecule that can form hydrogen bond(s) with another nucleic acid molecule or itself by either traditional Watson-Crick base pairing or other non-traditional types of pairing (e.g., Hoogsteen or 30 reversed Hoogsteen hydrogen bonding) between complementary nucleosides or nucleotides. In reference to the nucleic molecules of the present disclosure, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid molecule to proceed, for example, RNAi activity, and there is a sufficient degree of complementarity to avoid non-specific binding 24 Attorney Uocket No. U8-U9PU1 Customer No.: 36,814 of the nucleic acid molecule (e.g., dsRNA) to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or under conditions in which the assays are performed in the case of in vitro assays (e.g., hybridization assays). Determination of 5 binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., CSH Symp. Quant. Biol. LI:123, 1987; Frier et al., Proc. Nat'l. Acad. Sci. USA 83:9373, 1986; Turner et al., J. Am. Chem. Soc. 109:3783, 1987). Thus, "complementary" or "specifically hybridizable" or "specifically binds" are terms that indicate a sufficient degree of complementarity or precise pairing such that stable and 10 specific binding occurs between a nucleic acid molecule (e.g., dsRNA) and a DNA or RNA target. It is understood in the art that a nucleic acid molecule need not be 100% complementary to a target nucleic acid sequence to be specifically hybridizable or to specifically bind. That is, two or more nucleic acid molecules may be less than fully complementary and is indicated by a percentage of contiguous residues in a nucleic acid 15 molecule that can form hydrogen bonds with a second nucleic acid molecule. For example, a first nucleic acid molecule may have 10 nucleotides and a second nucleic acid molecule may have 10 nucleotides, then base pairing of 5, 6, 7, 8, 9, or 10 nucleotides between the first and second nucleic acid molecules, which may or may not form a contiguous double-stranded region, represents 50%, 60%, 70%, 80%, 90%, and 20 100% complementarity, respectively. In certain embodiments, complementary nucleic acid molecules may have wrongly paired bases - that is, bases that cannot form a traditional Watson-Crick base pair or other non-traditional types of pair (i.e., "mismatched" bases). For instance, complementary nucleic acid molecules may be identified as having a certain number of "mismatches," such as zero or about 1, about 2, 25 about 3, about 4 or about 5. "Perfectly" or "fully" complementary nucleic acid molecules means those in which a certain number of nucleotides of a first nucleic acid molecule hydrogen bond (anneal) with the same number of residues in a second nucleic acid molecule to form a contiguous double-stranded region. For example, two or more fully complementary 30 nucleic acid molecule strands can have the same number of nucleotides (i.e., have the same length and form one double-stranded region, with or without an overhang) or have a different number of nucleotides (e.g., one strand may be shorter than but fully contained within another strand or one strand may overhang the other strand). 25 Attorney ijocKet No. Uzs-uwr i Customer No.: 36,814 By "ribonucleic acid" or "RNA" is meant a nucleic acid molecule comprising at least one ribonucleotide molecule. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'-position of a p-D-ribofuranose moiety. The term RNA includes double-stranded (ds) RNA, single-stranded (ss) RNA, isolated RNA (such as 5 partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA), altered RNA (which differs from naturally occurring RNA by the addition, deletion, substitution or alteration of one or more nucleotides), or any combination thereof. For example, such altered RNA can include addition of non-nucleotide material, such as at one or both ends of an RNA molecule, internally at one or more nucleotides of 10 the RNA, or any combination thereof. Nucleotides in RNA molecules of the instant disclosure can also comprise non-standard nucleotides, such as naturally occurring nucleotides, non-naturally occurring nucleotides, chemically-modified nucleotides, deoxynucleotides, or any combination thereof. These altered RNAs may be referred to as analogs or analogs of RNA containing standard nucleotides (i.e., standard nucleotides, as 15 used herein, are considered to be adenine, cytidine, guanidine, thymidine, and uridine). The term "dsRNA" as used herein, which is interchangeable with "mdRNA," refers to any nucleic acid molecule comprising at least one ribonucleotide molecule and capable of inhibiting or down regulating gene expression, for example, by promoting RNA interference ("RNAi") or gene silencing in a sequence-specific manner. The 20 dsRNAs (mdRNAs) of the instant disclosure may be suitable substrates for Dicer or for association with RISC to mediate gene silencing by RNAi. Examples of dsRNA molecules of this disclosure are provided in the Sequence Listing identified herein. One or both strands of the dsRNA can further comprise a terminal phosphate group, such as a 5'-phosphate or 5', 3'-diphosphate. As used herein, dsRNA molecules, in addition to at 25 least one ribonucleotide, can further include substitutions, chemically-modified nucleotides, and non-nucleotides. In certain embodiments, dsRNA molecules comprise ribonucleotides up to about 100% of the nucleotide positions. In addition, as used herein, the term dsRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence 30 specific RNAi, for example, meroduplex RNA (mdRNA), nicked dsRNA (ndsRNA), gapped dsRNA (gdsRNA), short interfering nucleic acid (siNA), siRNA, micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering substituted oligonucleotide, short interfering modified oligonucleotide, chemically-modified dsRNA, post-transcriptional gene silencing RNA (ptgsRNA), or the 26 Attorney Docket No. U8-09PCT Customer No.: 36,814 like. The term "large double-stranded RNA" ("large dsRNA") refers to any double stranded RNA longer than about 40 base pairs (bp) to about 100 bp or more, particularly up to about 300 bp to about 500 bp. The sequence of a large dsRNA may represent a segment of an mRNA or an entire mRNA. A double-stranded structure may be formed 5 by a self-complementary nucleic acid molecule or by annealing of two or more distinct complementary nucleic acid molecule strands. In one aspect, a dsRNA comprises two separate oligonucleotides, comprising a first strand (antisense) and a second strand (sense), wherein the antisense and sense strands are self-complementary (i.e., each strand comprises a nucleotide sequence that is 10 complementary to a nucleotide sequence in the other strand and the two separate strands form a duplex or double-stranded structure, for example, wherein the double-stranded region is about 15 to about 24 base pairs or about 26 to about 40 base pairs); the antisense strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof; and the sense strand comprises a 15 nucleotide sequence corresponding (i.e., homologous) to the target nucleic acid sequence or a portion thereof (e.g., a sense strand of about 15 to about 25 nucleotides or about 26 to about 40 nucleotides corresponds to the target nucleic acid or a portion thereof). In another aspect, the dsRNA is assembled from a single oligonucleotide in which the self-complementary sense and antisense strands of the dsRNA are linked together by a 20 nucleic acid based-linker or a non-nucleic acid-based linker. In certain embodiments, the first (antisense) and second (sense) strands of the dsRNA molecule are covalently linked by a nucleotide or non-nucleotide linker as described herein and known in the art. In other embodiments, a first dsRNA molecule is covalently linked to at least one second dsRNA molecule by a nucleotide or non-nucleotide linker known in the art, wherein the 25 first dsRNA molecule can be linked to a plurality of other dsRNA molecules that can be the same or different, or any combination thereof. In another embodiment, the linked dsRNA may include a third strand that forms a meroduplex with the linked dsRNA. In still another aspect, dsRNA molecules described herein form a meroduplex RNA (mdRNA) having three or more strands such as, for example, an 'A' (first or 30 antisense) strand, 'Si' (second) strand, and 'S2' (third) strand in which the 'S1' and 'S2' strands are complementary to and form base pairs (bp) with non-overlapping regions of the 'A' strand (e.g., an mdRNA can have the form of A:S1 S2). The double-stranded region formed by the annealing of the 'Si' and 'A' strands is distinct from and non overlapping with the double-stranded region formed by the annealing of the 'S2' and 'A' 27 Attorney LJocket No. US-U9FUCT Customer No.: 36,814 strands. An mdRNA molecule is a "gapped" molecule, i.e., it contains a "gap" ranging from 0 nucleotides up to about 10 nucleotides (or a gap of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides). In one embodiment, the A:SI duplex is separated from the A:S2 duplex by a gap resulting from 5 at least one unpaired nucleotide (up to about 10 unpaired nucleotides) in the 'A' strand that is positioned between the A:Sl duplex and the A:S2 duplex and that is distinct from any one or more unpaired nucleotide at the 3'-end of one or more of the 'A', 'Si', or 'S2' strands. In another embodiment, the A:S1 duplex is separated from the A:S2 duplex by a gap of zero nucleotides (i.e., a nick in which only a phosphodiester bond between two 10 nucleotides is broken or missing in the polynucleotide molecule) between the A:S 1 duplex and the A:S2 duplex - which can also be referred to as nicked dsRNA (ndsRNA). For example, A:S 1S2 may be comprised of a dsRNA having at least two double-stranded regions that combined total about 14 base pairs to about 40 base pairs and the double stranded regions are separated by a gap of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 15 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides, optionally having blunt ends, or A:S1S2 may comprise a dsRNA having at least two double-stranded regions spaced apart by up to 10 nucleotides and thereby forming a gap between the second and third strands wherein at least one of the double-stranded regions optionally has from 5 base pairs to 13 base pairs. 20 A dsRNA or large dsRNA may include a substitution or modification in which the substitution or modification may be in a phosphate backbone bond, a sugar, a base, or a nucleoside. Such nucleoside substitutions can include natural non-standard nucleosides (e.g., 5-methyluridine or 5-methylcytidine or a 2-thioribothymidine), and such backbone, sugar, or nucleoside modifications can include an alkyl or heteroatom substitution or 25 addition, such as a methyl, alkoxyalkyl, halogen, nitrogen or sulfur, or other modifications known in the art. In addition, as used herein, the term "RNAi" is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics. For example, dsRNA molecules of 30 this disclosure can be used to epigenetically silence genes at the post-transcriptional level or the pre-transcriptional level or any combination thereof. As used herein, "target nucleic acid", "target mRNA", "target RNA", and "target gene" refers to any nucleic acid sequence whose expression or activity is to be altered (e.g. a target gene may be one or more of the following: tumor necrosis factor (TNF), 28 Auomey i'ocKet iNo. U6-UY)1r i Customer No.: 36,814 vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR), erythroblastic leukemia viral oncogene homolog (ERBB), platelet derived growth factor (PDGF), platelet derived growth factor receptor (PDGFR), breakpoint cluster region (BCR)-abelson murine 5 leukemia viral oncogene homolog (ABL), steroid-5-alpha-reductase, alpha polypeptide 1 (SRD5A1), steroid-5-alpha-reductase, alpha polypeptide 2 (SRD5A2), phosphoinositide 3-kinase, catalytic (PIK3C), mitogen-activated protein kinase (MAPK), p38 MAPK family, hypoxia-inducible factor 1 alpha (HIF 1 A), protein kinase N3 (PKN3), interleukin 17A (IL1 7A), interleukin 6 (IL6), interleukin 18 (ILl 8), tumor necrosis factor (ligand) 10 superfamily member 13b (TNFSF13B), mitogen-activated protein kinase 1 (MAPK1), v raf-1 murine leukemia viral oncogene homolog 1 (RAF1), v-AKT murine thymoma viral oncogene (AKT), FK506 binding protein 12-rapamycin associated protein 1 (FRAP 1), mitogen-activated protein kinase 2 (MAPK2), cyclin-dependent kinase 2 (CDK2), ATP binding cassette, subfamily B, member 1 (ABCB 1), B-cell CLL/lymphoma 2 (BCL2), 15 angiopoietin 2 (ANGPT2), checkpoint kinase 1 homolog (CHEKI), insulin-like growth factor 1 receptor (IGF1R), signal transducer and activator of transcription 3 (STAT3), matrix metalloproteinase (MMP), folate hydrolase (prostate-specific membrane antigen) 1 (FOLH 1), v-myc myelocytomatosis viral oncogene homolog (avian) (MYC), telomerase RNA component (TERC), telomerase reverse transcriptase (TERT), protein kinase C, 20 alpha (PRKCA), RAS viral (v-ras) oncogene homolog (RAS), chemokine (C-X-C motif) ligand or receptor (CXC), Wingless-Type MMTV (Murine Mammary Tumor Virus) Integration Site (WNT), toll-like receptor (TLR), Fc fragment of IgE, low affinity II, receptor for (CD23) (FCER2), FOS gene, (FOS, FOSB, FOSLI, OR FOSL2), hydroxysteroid (11-beta) dehydrogenase (HSD1 1 B 1), JUN gene (cJUN, JUNB, or 25 JUND), thymidine phosphorylase (TYMP), early growth response (EGR), zeste homolog 2 (EZH2), cyclin Dl (CCND1), Fas (TNF receptor superfamily, member 6) (FAS), proliferating cell nuclear antigen (PCNA), fibroblast growth factor 2 (FGF2), tumor growth factor-beta (TGF-P), tumor growth factor-beta receptor (TGF-sR), tumor associated calcium signal transducer 1 (TACSTD1), Mucin 1 (MUC 1), protein tyrosine 30 phosphatase, non-receptor-Il (Noonan Syndrome 1) (PTPN 11), neuregulin 1 (NRG 1), membrane metallo-endopeptidase (MME), CD 19 molecule (CD 19), CD40 molecule, TNF receptor superfamily member 5 (CD40), apolipoprotein B (including Ag(x) antigen) (ApoB), synuclein, alpha (non A4 component of amyloid precursor) (SNCA), silent 29 Attorney Docket No. 08-09PCT Customer No.: 36,814 mating type information regulation 2 homolog (SIRT2), histone deacetylase (HDAC), membrane-spanning 4-domains, subfamily A, member 1 (MS4A1), CD22 molecule (CD22), diacylglycerol o-acyltransferase 1 (DGAT1), diacylglycerol o-acyltransferase 2 (DGAT2), CD3 molecule (CD3), proprotein convertase subtilisin-like kexin type 9 5 (PCSK9), MET (Mesenchymal epithelial transition factor) (c-Met proto-oncogene), catenin (cadherin-associated protein) (beta-catenin) (CTNNB 1), inhition of DNA binding proteins (Inhibition of Differentiation Proteins, Dominant Negative Helix-Loop-Helix Protein) (ID, e.g., ID-1), protein tyrosine phosphatase, non-receptor type 1 (PTPN 1), tie-I (TIE 1; tyrosine kinase with immunoglobulin and EGF factor homology domains 1), tek 10 tyrosine kinase (TEK), fibroblast growth factor receptor (FGFR), mitogen-activated protein kinase 3 (MAPK3), survivin (BIRC5), polo-like kinase family genes (PLK Family; PLK1, PLK2, and PLK3). The target nucleic acid can be DNA, RNA, or analogs thereof, and includes single, double, and multi-stranded forms. By "target site" or "target sequence" is meant a sequence within a target nucleic acid (e.g., mRNA) that, when 15 present in an RNA molecule, is "targeted" for cleavage by RNAi and mediated by a dsRNA construct of this disclosure containing a sequence within the antisense strand that is complementary to the target site or sequence. As used herein, "off-target effect" or "off-target profile" refers to the observed altered expression pattern of one or more genes in a cell or other biological sample not 20 targeted, directly or indirectly, for gene silencing by an mdRNA or dsRNA. For example, an off-target effect can be quantified by using a DNA microarray to determine how many non-target genes have an expression level altered by about two-fold or more in the presence of a candidate mdRNA or dsRNA, or analog thereof specific for a target sequence. A "minimal off-target effect" means that an mdRNA or dsRNA affects 25 expression by about two-fold or more of about 25% to about 1% of the non-target genes examined or it means that the off-target effect of substituted or modified mdRNA or dsRNA (e.g., having at least one uridine substituted with a 5-methyluridine or 2 thioribothymidine and optionally having at least one nucleotide modified at the 2' position), is reduced by at least about 1% to about 80% or more as compared to the effect 30 on non-target genes of an unsubstituted or unmodified mdRNA or dsRNA. By "sense region" or "sense strand" is meant one ore more nucleotide sequences of a dsRNA molecule having complementarity to one or more antisense regions of the dsRNA molecule. In addition, the sense region of a dsRNA molecule comprises a nucleic acid sequence having homology or identity to a target sequence. By "antisense region" or 30 Attorney Docket No. 08-09PCT Customer No.: 36,814 "antisense strand" is meant a nucleotide sequence of a dsRNA molecule having complementarity to a target nucleic acid sequence. In addition, the antisense region of a dsRNA molecule can comprise nucleic acid sequence region having complementarity to one or more sense strands of the dsRNA molecule. 5 "Analog" as used herein refers to a compound that is structurally similar to a parent compound (e.g., a nucleic acid molecule), but differs slightly in composition (e.g., one atom or functional group is different, added, or removed). The analog may or may not have different chemical or physical properties than the original compound and may or may not have improved biological or chemical activity. For example, the analog may be 10 more hydrophilic or it may have altered activity as compared to a parent compound. The analog may mimic the chemical or biological activity of the parent compound (i.e., it may have similar or identical activity), or, in some cases, may have increased or decreased activity. The analog may be a naturally or non-naturally occurring (e.g., chemically-modified or recombinant) variant of the original compound. An example of 15 an RNA analog is an RNA molecule having a non-standard nucleotide, such as 5-methyuridine or 5-methylcytidine or 2-thioribothymidine, which may impart certain desirable properties (e.g., improve stability, bioavailability, minimize off-target effects or interferon response). As used herein, the term "universal base" refers to nucleotide base analogs that 20 form base pairs with each of the standard DNA/RNA bases with little discrimination between them. A universal base is thus interchangeable with all of the standard bases when substituted into a nucleotide duplex (see, e.g., Loakes et al., J. Mol. Bio. 270:426, 1997). Examplary universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, or nitroazole derivatives such as 3-nitropyrrole, 25 4-nitroindole, 5-nitroindole, and 6-nitroindole (see, e.g., Loakes, Nucleic Acids Res. 29:2437, 2001). The term "gene" as used herein, especially in the context of "target gene" or "gene target" for RNAi, means a nucleic acid molecule that encodes an RNA or a transcription product of such gene, including a messenger RNA (mRNA, also referred to as structural 30 genes that encode for a polypeptide), an mRNA splice variant of such gene, a functional RNA (fRNA), or non-coding RNA (ncRNA), such as small temporal RNA (stRNA), microRNA (miRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Such non-coding RNAs can serve as target nucleic acid 31 Attorney Docket No. 08-09PCT Customer No.: 36,814 molecules for dsRNA mediated RNAi to alter the activity of the target RNA involved in functional or regulatory cellular processes. As used herein, "gene silencing" refers to a partial or complete loss-of-function through targeted inhibition of gene expression in a cell, which may also be referred to as 5 RNAi "knockdown," "inhibition," "down-regulation," or "reduction" of expression of a target gene. Depending on the circumstances and the biological problem to be addressed, it may be preferable to partially reduce gene expression. Alternatively, it might be desirable to reduce gene expression as much as possible. The extent of silencing may be determined by methods described herein and known in the art (see, e.g., PCT Publication 10 No. WO 99/32619; Elbashir et al., EMBO J. 20:6877, 2001). Depending on the assay, quantification of gene expression permits detection of various amounts of inhibition that may be desired in certain embodiments of this disclosure, including prophylactic and therapeutic methods, which will be capable of knocking down target gene expression, in terms of mRNA level or protein level or activity, for example, by equal to or greater than 15 10%, 30%, 50%, 75% 90%, 95% or 99% of baseline (i.e., normal) or other control levels, including elevated expression levels as may be associated with particular disease states or other conditions targeted for therapy. As used herein, the term "therapeutically effective amount" means an amount of dsRNA that is sufficient to result in a decrease in severity of disease symptoms, an 20 increase in frequency or duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease, in the subject (e.g., human) to which it is administered. For example, a therapeutically effective amount of dsRNA directed against an mRNA of a target nucleic acid or target gene can reduce or alleviate the signs and/or symptoms of a disease or condition by at least about 20%, at least about 40%, at least 25 about 60%, or at least about 80% relative to untreated subjects. A therapeutically effective amount of a therapeutic compound can decrease, for example, atheromatous plaque size or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such therapeutically effective amounts based on such factors as the subject's size, the severity of symptoms, and the particular composition or 30 route of administration selected. The nucleic acid molecules of the instant disclosure, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed herein. For example, to treat a particular disease, disorder, or condition, the dsRNA molecules can be administered to a patient or can be 32 Attorney Uocket No. UZS-U91C I Customer No.: 36,814 administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs, under conditions suitable for treatment. In addition, one or more dsRNA may be used to knockdown expression of an mRNA or a related mRNA splice variant. In this regard it is noted that a target gene may 5 be transcribed into two or more mRNA splice variants; and thus, for example, in certain embodiments, knockdown of one mRNA splice variant without affecting the other mRNA splice variant may be desired, or vice versa; or knockdown of all transcription products may be targeted. In addition, it should be understood that the individual compounds, or groups of 10 compounds, derived from the various combinations of the structures and substituents described herein, are disclosed by the present application to the same extent as if each compound or group of compounds was set forth individually. Thus, selection of particular structures or particular substituents is within the scope of the present disclosure. As described herein, all value ranges are inclusive over the indicated range. Thus, a range 15 of C 1

-C

4 will be understood to include the values of 1, 2, 3, and 4, such that C1, C 2 , C 3 and C 4 are included. The term "alkyl" as used herein refers to saturated straight- or branched-chain aliphatic groups containing from 1-20 carbon atoms, preferably 1-8 carbon atoms and most preferably 1-4 carbon atoms. This definition applies as well to the alkyl portion of 20 alkoxy, alkanoyl and aralkyl groups. The alkyl group may be substituted or unsubstituted. In certain embodiments, the alkyl is a (CI-C 4 ) alkyl or methyl. The term "cycloalkyl" as used herein refers to a saturated cyclic hydrocarbon ring system containing from 3 to 12 carbon atoms that may be optionally substituted. Exemplary embodiments include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl and 25 cyclohexyl. In certain embodiments, the cycloalkyl group is cyclopropyl. In another embodiment, the (cycloalkyl)alkyl groups contain from 3 to 12 carbon atoms in the cyclic portion and 1 to 6 carbon atoms in the alkyl portion. In certain embodiments, the (cycloalkyl)alkyl group is cyclopropylmethyl. The alkyl groups are optionally substituted with from one to three substituents selected from the group consisting of halogen, hydroxy and 30 amino. The terms "alkanoyl" and "alkanoyloxy" as used herein refer, respectively, to -C(O) alkyl groups and -O-C(=O)- alkyl groups, each optionally containing 2 to 10 carbon atoms. Specific embodiments of alkanoyl and alkanoyloxy groups are acetyl and acetoxy, respectively. 33 Attorney Docket No. 08-09PCT Customer No.: 36,814 The term "alkenyl" refers to an unsaturated branched, straight-chain or cyclic alkyl group having 2 to 15 carbon atoms and having at least one carbon-carbon double bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkene. The group may be in either the cis or trans conformation about the double 5 bond(s). Certain embodiments include ethenyl, 1 -propenyl, 2-propenyl, 1 -methylethenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-2-propenyl, 1 -pentenyl, 2-pentenyl, 4-pentenyl, 3-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 1-heptenyl, 2-heptenyl, 1-octenyl, 2-octenyl, 1,3-octadienyl, 2-nonenyl, 1,3-nonadienyl, 2-decenyl, etc., or the like. The alkenyl group may be substituted or unsubstituted. 10 The term "alkynyl" as used herein refers to an unsaturated branched, straight-chain, or cyclic alkyl group having 2 to 10 carbon atoms and having at least one carbon-carbon triple bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkyne. Exemplary alkynyls include ethynyl, 1 -propynyl, 2 propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 4-pentynyl, 1-octynyl, 15 6-methyl- 1 -heptynyl, 2-decynyl, or the like. The alkynyl group may be substituted or unsubstituted. The term "hydroxyalkyl" alone or in combination, refers to an alkyl group as previously defined, wherein one or several hydrogen atoms, preferably one hydrogen atom has been replaced by a hydroxyl group. Examples include hydroxymethyl, 20 hydroxyethyl and 2-hydroxyethyl. The term "aminoalkyl" as used herein refers to the group -NRR', where R and R' may independently be hydrogen or (CI-C 4 ) alkyl. The term "alkylaminoalkyl" refers to an alkylamino group linked via an alkyl group (i.e., a group having the general structure -alkyl-NH-alkyl or -alkyl 25 N(alkyl)(alkyl)). Such groups include, but are not limited to, mono- and di-(C 1 -Cs alkyl)aminoC1 -C 8 alkyl, in which each alkyl may be the same or different. The term "dialkylaminoalkyl" refers to alkylamino groups attached to an alkyl group. Examples include, but are not limited to, N,N-dimethylaminomethyl, N,N-dimethylaminoethyl N,N-dimethylaminopropyl, and the like. The term 30 dialkylaminoalkyl also includes groups where the bridging alkyl moiety is optionally substituted. The term "haloalkyl" refers to an alkyl group substituted with one or more halo groups, for example chloromethyl, 2-bromoethyl, 3-iodopropyl, trifluoromethyl, perfluoropropyl, 8-chlorononyl, or the like. 34 Attorney Docket INo. uzs-uviu i Customer No.: 36,814 The term "carboxyalkyl" as used herein refers to the substituent -R' 0 -COOH, wherein R1 0 is alkylene; and "carbalkoxyalkyl" refers to -R' 0

-C(=O)OR

1 , wherein R1 0 and R 1 are alkylene and alkyl respectively. In certain embodiments, alkyl refers to a saturated straight- or branched-chain hydrocarbyl radical of 1 to 6 carbon atoms such as 5 methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, 2-methylpentyl, n-hexyl, and so forth. Alkylene is the same as alkyl except that the group is divalent. The term "alkoxy" includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom. In one embodiment, the alkoxy group contains 1 to about 10 carbon atoms. Embodiments of alkoxy groups include, but 10 are not limited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups. Embodiments of substituted alkoxy groups include halogenated alkoxy groups. In a further embodiment, the alkoxy groups can be substituted with groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, 15 aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, 20 trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties. Exemplary halogen substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, and trichloromethoxy. The term "alkoxyalkyl" refers to an alkylene group substituted with an alkoxy 25 group. For example, methoxyethyl (CH 3 0CH 2

CH

2 -) and ethoxymethyl (CH 3

CH

2

OCH

2 -) are both C 3 alkoxyalkyl groups. The term "aryl" as used herein refers to monocyclic or bicyclic aromatic hydrocarbon groups having from 6 to 12 carbon atoms in the ring portion, for example, phenyl, naphthyl, biphenyl and diphenyl groups, each of which may be substituted with, for example, one to four 30 substituents such as alkyl; substituted alkyl as defined above, halogen, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, cycloalkyloxy, alkanoyl, alkanoyloxy, amino, alkylamino, dialkylamino, nitro, cyano, carboxy, carboxyalkyl, carbamyl, carbamoyl and aryloxy. Specific embodiments of aryl groups in accordance with the present disclosure include phenyl, substituted phenyl, naphthyl, biphenyl, and diphenyl. 35 Anomey VocKet iNo. us-Uwru i Customer No.: 36,814 The term "aroyl," as used alone or in combination herein, refers to an aryl radical derived from an aromatic carboxylic acid, such as optionally substituted benzoic or naphthoic acids. The term "aralkyl" as used herein refers to an aryl group bonded to the 2-pyridinyl 5 ring or the 4-pyridinyl ring through an alkyl group, preferably one containing 1 to 10 carbon atoms. A preferred aralkyl group is benzyl. The term "carboxy," as used herein, represents a group of the formula -C(=O)OH or -C(=0)O-. The term "carbonyl" as used herein refers to a group in which an oxygen atom is 10 double-bonded to a carbon atom -C=O. The term "trifluoromethyl" as used herein refers to -CF 3 . The term "trifluoromethoxy" as used herein refers to -OCF 3 . The term "hydroxyl" as used herein refers to -OH or -0. The term "nitrile" or "cyano" as used herein refers to the group -CN. 15 The term "nitro," as used herein alone or in combination refers to a -NO 2 group. The term "amino" as used herein refers to the group -NR 9

R

9 , wherein R9 my independently be hydrogen, alkyl, aryl, alkoxy, or heteroaryl. The term "aminoalkyl" as used herein represents a more detailed selection as compared to "amino" and refers to the group -NR'R', wherein R' may independently be hydrogen or (CI-C 4 ) alkyl. The term 20 "dialkylamino" refers to an amino group having two attached alkyl groups that can be the same or different. The term "alkanoylamino" refers to alkyl, alkenyl or alkynyl groups containing the group -C(=O)- followed by -N(H)-, for example acetylamino, propanoylamino and butanoylamino and the like. 25 The term "carbonylamino" refers to the group -NR'-CO-CH 2 -R', wherein R' may be independently selected from hydrogen or (CI-C 4 ) alkyl. The term "carbamoyl" as used herein refers to -O-C(O)NH 2 . The term "carbamyl" as used herein refers to a functional group in which a nitrogen atom is directly bonded to a carbonyl, i.e., as in -NR"C(0)R" or 30 C(=O)NR"R", wherein R" can be independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, cycloalkyl, aryl, heterocyclo, or heteroaryl. The term "alkylsulfonylamino" refers to the group -NHS(O) 2 R1 2 , wherein R1 is alkyl. 36 Attorney Docket No. US-U9U I Customer No.: 36,814 The term "halogen" as used herein refers to bromine, chlorine, fluorine or iodine. In one embodiment, the halogen is fluorine. In another embodiment, the halogen is chlorine. The term "heterocyclo" refers to an optionally substituted, unsaturated, partially 5 saturated, or fully saturated, aromatic or nonaromatic cyclic group that is a 4 to 7 membered monocyclic, or 7 to 11 membered bicyclic ring system that has at least one heteroatom in at least one carbon atom-containing ring. The substituents on the heterocyclo rings may be selected from those given above for the aryl groups. Each ring of the heterocyclo group containing a heteroatom may have 1, 2, or 3 heteroatoms selected from 10 nitrogen, oxygen or sulfur. Plural heteroatoms in a given heterocyclo ring may be the same or different. Exemplary monocyclic heterocyclo groups include pyrrolidinyl, pyrrolyl, indolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, furyl, tetrahydrofuryl, thienyl, piperidinyl, piperazinyl, azepinyl, pyrimidinyl, pyridazinyl, tetrahydropyranyl, 15 morpholinyl, dioxanyl, triazinyl and triazolyl. Preferred bicyclic heterocyclo groups include benzothiazolyl, benzoxazolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl, benzimidazolyl, benzofuryl, indazolyl, benzisothiazolyl, isoindolinyl and tetrahydroquinolinyl. In more detailed embodiments heterocyclo groups may include indolyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl and pyrimidyl. 20 "Substituted" refers to a group in which one or more hydrogen atoms are each independently replaced with the same or different substituent(s). Representative substituents include -X, -R 6 , -O-, =O, -OR, -SR6, -S-, =S, -NR 6

R

6 , =NR 6 , -CX 3 , -CF 3 , 6 -CN, -OCN, -SCN, -NO, -NO 2 , =N 2 , -N 3 , -S(=0)20-, -S(=O) 2 0H, -S(=O)2R , -OS(=0)20-, -OS(=0) 2 0H, -OS(=0) 2

R

6 , -P(=O)(O~) 2 , -P(=O)(OH)(O-), -OP(=0) 2 (0), 25 -C(-O)R 6 , -C(=S)R 6 , -C(=0)OR 6 , -C(=O)O~, -C(=S)OR 6 , -NR 6

-C(=O)-N(R

6

)

2 ,

-NR

6

-C(=S)-N(R

6

)

2 , and -C(=NR 6

)NR

6

R

6 , wherein each X is independently a halogen; and each R 6 is independently hydrogen, halogen, alkyl, aryl, arylalkyl, arylaryl, arylheteroalkyl, heteroaryl, heteroarylalkyl, NR 7

R

7 , -C(=0)R 7 , and -S(=0) 2

R

7 ; and each

R

7 is independently hydrogen, alkyl, alkanyl, alkynyl, aryl, arylalkyl, arylheteralkyl, 30 arylaryl, heteroaryl or heteroarylalkyl. Aryl containing substituents, whether or not having one or more substitutions, may be attached in a para (p-), meta (m-) or ortho (o-) conformation, or any combination thereof. 37 AUoMey IJOCKet io. Us-uw'L i Customer No.: 36,814 Target Genes and Exemplary dsRNA Molecules More detail regarding TNF and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055371 and associated 5 sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding VEGF and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent applications PCT/US2008/055362 and associated 10 sequence listing; and PCT/US2008/055380 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding VEGFR and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055370 and associated 15 sequence listing; and PCT/US2008/055383 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding ERBB and EGFR and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055375 and 20 associated sequence listing; and PCT/US2008/055360 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding PDGF and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055374 and associated 25 sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding PDGFR and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055381 and associated 30 sequence listing; and PCT/US2008/055357 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding BCR-ABL and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055378 and associated 38 Attorney Docket No. 08-09PCT Customer No.: 36,814 sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding SRD5A1 and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 5 found in the priority document patent application PCT/US2008/055372 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding SRD5A2 and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 10 found in the priority document patent application PCT/US2008/055345 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding PIK3C and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 15 found in the priority document patent application PCT/US2008/055377 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding MAPK and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 20 found in the priority document patent application PCT/US2008/055376 and associated sequence listing; and PCT/US2008/055373 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding HIF 1 A and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 25 found in the priority document patent application PCT/US2008/055385 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding PKN3 and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 30 found in the priority document patent application PCT/US2008/055386 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding ILl 7A and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 39 Attorney Docket No. 08-09PCT Customer No.: 36,814 found in the priority document patent application PCT/US2008/055382 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding IL6 and its nucleotide sequence (target mRNA), function(s), 5 diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055333 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding IL18 and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 10 found in the priority document patent application PCT/US2008/055341 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding TNFSF13B and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 15 found in the priority document patent application PCT/US2008/055350 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding MAPK1 and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 20 found in the priority document patent application PCT/US2008/055356 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding RAFI and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 25 found in the priority document patent application PCT/US2008/055366 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding AKT and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 30 found in the priority document patent application PCT/US2008/055339 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding FRAP 1 and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 40 Attorney Docket No. 08-09PCT Customer No.: 36,814 found in the priority document patent application PCT/US2008/055365 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding MAPK2 and its nucleotide sequence (target mRNA), 5 function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055340 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding CDK2 and its nucleotide sequence (target mRNA), 10 function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055505 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding ABCB 1 and its nucleotide sequence (target mRNA), 15 function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055556 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding BCL2 and its nucleotide sequence (target mRNA), 20 function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055515 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding ANGPT2 and its nucleotide sequence (target mRNA), 25 function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055599 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding CHEKI and its nucleotide sequence (target mRNA), 30 function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055601 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. 41 Attorney Iocket No. Us-UYFu i Customer No.: 36,814 More detail regarding IGF 1 R and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055603 and associated sequence listing, the contents of which are hereby incoroporated by reference in their 5 entirety. More detail regarding STAT3 and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055606 and associated sequence listing, the contents of which are hereby incoroporated by reference in their 10 entirety. More detail regarding MMP and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/U2008/055548 and associated sequence listing, the contents of which are hereby incoroporated by reference in their 15 entirety. More detail regarding FOLH 1 and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055611 and associated sequence listing, the contents of which are hereby incoroporated by reference in their 20 entirety. More detail regarding MYC and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055615 and associated sequence listing, the contents of which are hereby incoroporated by reference in their 25 entirety. More detail regarding TERC and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055709 and associated sequence listing, the contents of which are hereby incoroporated by reference in their 30 entirety. More detail regarding TERT and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055709 and associated 42 Attorney Docket No. 08-U9PCT Customer No.: 36,814 sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding PRKCA and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 5 found in the priority document patent application PCT/US2008/055618 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding RAS and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 10 found in the priority document patent application PCT/US2008/055644 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding CXC and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 15 found in the priority document patent application PCT/US2008/055651 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding WNT and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 20 found in the priority document patent application PCT/US2008/055649 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding TLR and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 25 found in the priority document patent application PCT/US2008/055711 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding FCER2 and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 30 found in the priority document patent application PCT/US2008/055635 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding FOS (FOS, FOSB, FOSLI, or FOSL2) and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example 43 Attorney Docket No. U8-U9PCI Customer No.: 36,814 siRNA sequences are found in the priority document patent application PCT/US2008/055524 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding HSD 11 B 1 and its nucleotide sequence (target mRNA), 5 function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055672 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding cJUJN, JUNB, or JUND and its nucleotide sequence (target 10 mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055627 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding TYMP and its nucleotide sequence (target mRNA), 15 function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055697 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding EGR and its nucleotide sequence (target mRNA), 20 function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055662 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding EZH2 and its nucleotide sequence (target mRNA), 25 function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055678 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding CCND 1 and its nucleotide sequence (target mRNA), 30 function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055368 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. 44 Attorney vocxet No. US-U9FU 1 Customer No.: 36,814 More detail regarding FAS and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055676 and associated sequence listing, the contents of which are hereby incoroporated by reference in their 5 entirety. More detail regarding PCNA and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055550 and associated sequence listing, the contents of which are hereby incoroporated by reference in their 10 entirety. More detail regarding FGF2 and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055560 and associated sequence listing, the contents of which are hereby incoroporated by reference in their 15 entirety. More detail regarding TGF-P and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055698 and associated sequence listing, the contents of which are hereby incoroporated by reference in their 20 entirety. More detail regarding TGF-3R and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055695 and associated sequence listing, the contents of which are hereby incoroporated by reference in their 25 entirety. More detail regarding TACSTD1 and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055701 and associated sequence listing, the contents of which are hereby incoroporated by reference in their 30 entirety. More detail regarding MUC I and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055693 and associated 45 Auromey JJOCKet iNo. u6-Uvrt- i Customer No.: 36,814 sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding PTPN1 1 and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 5 found in the priority document patent application PCT/US2008/055704 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding NGR1 and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 10 found in the priority document patent application PCT/US2008/055708 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding MME and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 15 found in the priority document patent application PCT/US2008/055597 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding CD 19 and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 20 found in the priority document patent application PCT/US2008/055604 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding CD40 and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 25 found in the priority document patent application PCT/US2008/055608 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding ApoB and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 30 found in the priority document patent application PCT/US2008/055353 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding SNCA and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are 46 Attorney Docket No. 08-09PCT Customer No.: 36,814 found in the priority document patent application PCT/US2008/055631 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding SIRT2 and its nucleotide sequence (target mRNA), 5 function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055563 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding HDAC and its nucleotide sequence (target mRNA), 10 function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055612 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding MS4A1 and its nucleotide sequence (target mRNA), 15 function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055622 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding CD22 and its nucleotide sequence (target mRNA), 20 function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055625 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding DGATl and DGAT2 and its nucleotide sequence (target 25 mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055527 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. More detail regarding CD3 and its nucleotide sequence (target mRNA), 30 function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055533 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. 47 Attorney Docket No. US-U9kC [ Customer No.: 36,814 More detail regarding PCSK9 and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055554 and associated sequence listing, the contents of which are hereby incoroporated by reference in their 5 entirety. More detail regarding MET and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055511 and associated sequence listing, the contents of which are hereby incoroporated by reference in their 10 entirety. More detail regarding CTNNB 1 and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055532 and associated sequence listing, the contents of which are hereby incoroporated by reference in their 15 entirety. More detail regarding ID and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055516 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. 20 More detail regarding PTPN1 and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055551 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. 25 More detail regarding TIEl and TEK and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055519 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. 30 More detail regarding FGFR and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055542 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. 48 Attorney Docket No. US-U9I'CT Customer No.: 36,814 More detail regarding MAPK3 and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in the priority document patent application PCT/US2008/055526 and associated sequence listing, the contents of which are hereby incoroporated by reference in their 5 entirety. More detail regarding survivin (BIRC5) and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in patent application PCT/US2009/52878 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. 10 More detail regarding PLK1, PLK2, and PLK3) and its nucleotide sequence (target mRNA), function(s), diseases and disorders related thereto, and example siRNA sequences are found in patent application PCT/US2009/52888 and associated sequence listing, the contents of which are hereby incoroporated by reference in their entirety. As used herein, reference to target mRNA or target RNA sequences or sense 15 strands means an RNA isoform of a nucleotide sequence of a gene identified herein, as well as variants and homologs having at least 80% or more identity with the target mRNA. The "percent identity" between two or more nucleic acid sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of 20 identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap that needs to be introduced to optimize alignment of two or more sequences. The comparison of sequences and determination of percent identity between two or more sequences can be accomplished using a mathematical algorithm, such as BLAST and Gapped BLAST programs at their default parameters (e.g., 25 BLASTN, see www.ncbi.nlm.nih.gov/BLAST; see also Altschul et al., J. Mol. Biol. 215:403-410, 1990). In one aspect, the instant disclosure provides an mdRNA molecule, comprising a first strand that is complementary to a target mRNA, and a second strand and a third strand that are each complementary to non-overlapping regions of the first strand, 30 wherein the second strand and third strands can anneal with the first strand to form at least two double-stranded regions spaced apart by up to 10 nucleotides and thereby forming a gap between the second and third strands, and wherein (a) the mdRNA molecule optionally has at least one double-stranded region of 5 base pairs to 13 base pairs, or (b) the combined double-stranded regions total about 15 base pairs to about 49 Attorney Docket No. U8-09PC1 Customer No.: 36,814 40 base pairs and the mdRNA molecule optinally has blunt ends; wherein at least one pyrimidine of the mdRNA is substituted with a pyrimidine nucleoside according to Formula I or II: R 0 RI NH 2 /5 4 6 3NH N (I) R 4 5' R 5 N 4' l'R8 R 8 3' 2' R3 R2 R3 R2 5 wherein R' and R 2 are each independently a -H, -OH, -OCH 3 , -OCH 2 0CH 2

CH

3 ,

-OCH

2

CH

2 0CH 3 , halogen, substituted or unsubstituted C 1 -C1o alkyl, alkoxy, alkoxyalkyl, hydroxyalkyl, carboxyalkyl, alkylsulfonylamino, aminoalkyl, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, substituted or unsubstituted C 2

-C

1 0 alkenyl, substituted or unsubstituted 10 -0-allyl, -O-CH 2

CH=CH

2 , -O-CH=CHCH 3 , substituted or unsubstituted C 2 -Cio alkynyl, carbamoyl, carbamyl, carboxy, carbonylamino, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, -NH 2 , -NO 2 , -C=N, or heterocyclo group; R3 and R4 are each independently a hydroxyl, a protected hydroxyl, a phosphate, or an internucleoside linking group; and R and R are each independently 0 or S. In certain 15 embodiments, at least one nucleoside is according to Formula I in which R' is methyl and R2 is -OH, or R1 is methyl, R 2 is -OH, and R8 is S. In other embodiments, the internucleoside linking group covalently links from about 5 to about 40 nucleosides. In some embodiments, the gap comprises at least one unpaired nucleotide in the first strand positioned between the double-stranded regions formed by the second and third strands 20 when annealed to the first strand, or the gap is a nick. In certain embodiments, the nick or gap is located 10 nucleotides from the 5'-end of the first (antisense) strand or at the Argonaute cleavage site. In another embodiment, the meroduplex nick or gap is positioned such that the thermal stability is maximized for the first and second strand duplex and for the first and third strand duplex as compared to the thermal stability of 25 such meroduplexes having a nick or gap in a different position. In still another aspect, the instant disclosure provides an mdRNA molecule, comprising a first strand that is complementary to a target mRNA, and a second strand and a third strand that are each complementary to non-overlapping regions of the first 50 Attorney Locket No. Uw-uw' i Customer No.: 36,814 strand, wherein the second strand and third strands can anneal with the first strand to form at least two double-stranded regions spaced apart by up to 10 nucleotides and thereby forming a gap between the second and third strands, and wherein the mdRNA molecule optionally includes at least one double-stranded region of 5 base pairs to 13 base pairs. In 5 a further aspect, the instant disclosure provides an mdRNA molecule having a first strand that is complementary to a target mRNA, and a second strand and a third strand that are each complementary to non-overlapping regions of the first strand, wherein the second strand and third strands can anneal with the first strand to form at least two double stranded regions spaced apart by up to 10 nucleotides and thereby forming a gap between 10 the second and third strands, and wherein the combined double-stranded regions total about 15 base pairs to about 40 base pairs and the mdRNA molecule optinally has blunt ends. In some embodiments, the gap comprises at least one unpaired nucleotide in the first strand positioned between the double-stranded regions formed by the second and third strands when annealed to the first strand, or the gap is a nick. In certain 15 embodiments, the nick or gap is located 10 nucleotides from the 5'-end of the first (antisense) strand or at the Argonaute cleavage site. In another embodiment, the meroduplex nick or gap is positioned such that the thermal stability is maximized for the first and second strand duplex and for the first and third strand duplex as compared to the thermal stability of such meroduplexes having a nick or gap in a different position. 20 As provided herein, any of the aspects or embodiments disclosed herein would be useful in treating a target mRNA-associated diseases or disorders, such as cancer, a metabolic disease and/or inflammatory disease. In some embodiments, the dsRNA comprises at least three strands in which the first strand comprises about 5 nucleotides to about 40 nucleotides, and the second and 25 third strands include each, individually, about 5 nucleotides to about 20 nucleotides, wherein the combined length of the second and third strands is about 15 nucleotides to about 40 nucleotides. In other embodiments, the dsRNA comprises at least two strands in which the first strand comprises about 15 nucleotides to about 24 nucleotides or about 25 nucleotides to about 40 nucleotides. In yet other embodiments, the first strand 30 comprises about 15 to about 24 nucleotides or about 25 nucleotides to about 40 nucleotides and is complementary to at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides of a human target mRNA. In alternative embodiments, the first strand comprises about 15 to about 24 nucleotides or about 25 nucleotides to about 40 nucleotides and is at least about 51 Attorney UocKet No. uw-Uyru i Customer No.: 36,814 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence that is complementary to at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides of a human target mRNA. 5 In further embodiments, the first strand will be complementary to a second strand or a second and third strand or to a plurality of strands. The first strand and its complements will be able to form dsRNA and mdRNA molecules of this disclosure, but only about 19 to about 25 nucleotides of the first strand comprise a sequence complementary to a target mRNA. For example, a Dicer substrate dsRNA can have about 10 25 nucleotides to about 40 nucleotides, but with only 19 nucleotides of the antisense (first) strand being complementary to a target mRNA. In further embodiments, the first strand having complementarity to a target mRNA in about 19 nucleotides to about 25 nucleotides will have one, two, or three mismatches with a target mRNA, or the first strand of 19 nucleotides to about 25 nucleotides, that for example activates or is capable 15 of loading into RISC, will have at least 80% identity with the corresponding nucleotides found in a target mRNA. Certain illustrative dsRNA molecules, which can be used to design mdRNA or dsRNA molecules and can optionally include substitutions or modifications as described herein are provided in the Sequence Listings as attached herewith, which is herein 20 incorporated by reference (text file named "07-R-US-CIP_Sequence Listing"). In addition, the content of Table B as disclosed in U.S. Provisional Patent Application No. 60/934,930 (filed March 16, 2007), which was submitted with that application as a separate text file named "Table_B_HumanRefSeq_AccessionNumbers.txt" (created March 16, 2007 and having a size of 3,604 kilobytes), is incorporated herein by reference 25 in its entirety. An entire printable copy of Table B also accompanies the specification as one text file recorded on a single compact disk (CD). The CD (labeled as CD#1) is also labelled with (i) the applicant's name, (ii) the title of invention, (iii) a reference number, (iv) the date on which the data was recorded on the CD and (v) the relevant computer operating system. 30 Substituting and Modifying Target dsRNA Molecules The introduction of substituted and modified nucleotides into mdRNA and dsRNA molecules of this disclosure provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to native RNA molecules (i.e., having 52 Attorney Vocket No. US-U91UI Customer No.: 36,814 standard nucleotides) that are exogenously delivered. For example, the use of dsRNA molecules of this disclosure can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect (e.g., reducing or silencing target gene expression) since dsRNA molecules of this disclosure tend to have a longer half-life in serum. 5 Furthermore, certain substitutions and modifications can improve the bioavailability of dsRNA by targeting particular cells or tissues or improving cellular uptake of the dsRNA molecules. Therefore, even if the activity of a dsRNA molecule of this disclosure is reduced as compared to a native RNA molecule, the overall activity of the substituted or modified dsRNA molecule can be greater than that of the native RNA molecule due to 10 improved stability or delivery of the molecule. Unlike native unmodified dsRNA, substituted and modified dsRNA can also minimize the possibility of activating the interferon response in, for example, humans. In certain embodiments, a dsRNA molecule of this disclosure has at least one uridine, at least three uridines, or each and every uridine (i.e., all uridines) of the first 15 (antisense) strand of the dsRNA substituted or replaced with 5-methyluridine or 2 thioribothymidine. In a related embodiment, the dsRNA molecule or analog thereof of this disclosure has at least one uridine, at least three uridines, or each and every uridine of the second (sense) strand of the dsRNA substituted or replaced with 5-methyluridine or 2 thioribothymidine. In a related embodiment, the dsRNA molecule or analog thereof of 20 this disclosure has at least one uridine, at least three uridines, or each and every uridine of the third (sense) strand of the dsRNA substituted or replaced with 5-methyluridine or 2 thioribothymidine. In still another embodiment, the dsRNA molecule or analog thereof of this disclosure has at least one uridine, at least three uridines, or each and every uridine of both the first (antisense) and second (sense) strands; of both the first (antisense) and third 25 (sense) strands; of both the second (sense) and third (sense) strands; or of all of the first (antisense), second (sense) and third (sense) strands of the dsRNA substituted or replaced with 5-methyluridine or 2-thioribothymidine. In some embodiments, the double-stranded region of a dsRNA molecule has at least three 5-methyluridines or 2-thioribothymidines. In certain embodiments, dsRNA molecules comprise ribonucleotides at about 5% to about 30 95% of the nucleotide positions in one strand, both strands, or any combination thereof. In further embodiments, a dsRNA molecule that decreases expression of a target gene by RNAi according to the instant disclosure further comprises one or more natural or synthetic non-standard nucleoside. In related embodiments, the non-standard nucleoside is one or more deoxyuridine, locked nucleic acid (LNA) molecule, a modified 53 Aromey vocKet No. u6-uYri Customer No.: 36,814 base (e.g., 5-methyluridine), a universal-binding nucleotide, a 2'-O-methyl nucleotide, a modified intemucleoside linkage (e.g., phosphorothioate), a G clamp, or any combination thereof. In certain embodiments, the universal-binding nucleotide can be C-phenyl, C-naphthyl, inosine, azole carboxamide, 1 -S-D-ribofuranosyl-4-nitroindole, 1-3-D 5 ribofuranosyl-5-nitroindole, 1-S-D-ribofuranosyl-6-nitroindole, or 1-0-D-ribofuranosyl 3-nitropyrrole. Substituted or modified nucleotides present in dsRNA molecules, preferably in the sense or antisense strand, but also optionally in both the antisense and sense strands, comprise modified or substituted nucleotides according to this disclosure having 10 properties or characteristics similar to natural or standard ribonucleotides. For example, this disclosure features dsRNA molecules including nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle; see, e.g., Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemically modified nucleotides present in dsRNA molecules of this disclosure, preferably in the antisense 15 strand, but also optionally in the sense or both the antisense and sense strands, are resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi. Exemplary nucleotides having a Northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2'-O, 4'-C-methylene-(D-ribofuranosyl) nucleotides), 2'-methoxyethyl (MOE) nucleotides, 2'-methyl-thio-ethyl, 2'-deoxy-2' 20 fluoro nucleotides, 2'-deoxy-2'-chloro nucleotides, 2'-azido nucleotides, 5-methyluridines, or 2'-O-methyl nucleotides. In certain embodiments, the LNA is a 5-methyluridine LNA or 2-thio-5-methyluridine LNA. In any of these embodiments, one or more substituted or modified nucleotides can be a G clamp (e.g., a cytosine analog that forms an additional hydrogen bond to guanine, such as 9-(aminoethoxy)phenoxazine; see, e.g., Lin and 25 Mateucci, J. Am. Chem. Soc. 120:8531, 1998). As described herein, the first and one or more second strands of a dsRNA molecule or analog thereof provided by this disclosure can anneal or hybridize together (i.e., due to complementarity between the strands) to form at least one double-stranded region having a length of about 4 to about 10 base pairs, about 5 to about 13 base pairs, or 30 about 15 to about 40 base pairs. In some embodiments, the dsRNA has at least one double-stranded region ranging in length from about 15 to about 24 base pairs or about 19 to about 23 base pairs. In other embodiments, the dsRNA has at least one double stranded region ranging in length from about 26 to about 40 base pairs or about 27 to about 30 base pairs or about 30 to about 35 base pairs. In other embodiments, the two or 54 Auorney LjocKet INo. us-uyr- i Customer No.: 36,814 more strands of a dsRNA molecule of this disclosure may optionally be covalently linked together by nucleotide or non-nucleotide linker molecules. In certain embodiments, the dsRNA molecule or analog thereof comprises an overhang of one to four nucleotides on one or both 3'-ends of the dsRNA, such as an 5 overhang comprising a deoxyribonucleotide or two deoxyribonucleotides (e.g., thymidine, adenine). In certain embodiments, the 3'-end comprising one or more deoxyribonucleotide is in an mdRNA molecule and is either in the gap, not in the gap, or any combination thereof. In some embodiments, dsRNA molecules or analogs thereof have a blunt end at one or both ends of the dsRNA. In certain embodiments, the 5'-end of 10 the first or second strand is phosphorylated. In any of the embodiments of dsRNA molecules described herein, the 3'-terminal nucleotide overhangs can comprise ribonucleotides or deoxyribonucleotides that are chemically-modified at a nucleic acid sugar, base, or backbone. In any of the embodiments of dsRNA molecules described herein, the 3'-terminal nucleotide overhangs can comprise one or more universal base 15 ribonucleotides. In any of the embodiments of dsRNA molecules described herein, the 3'-terminal nucleotide overhangs can comprise one or more acyclic nucleotides. In any of the embodiments of dsRNA molecules described herein, the dsRNA can further comprise a terminal phosphate group, such as a 5'-phosphate (see Martinez et al., Cell. 110:563 574, 2002; and Schwarz et al., Molec. Cell 10:537-568, 2002) or a 5',3'-diphosphate. 20 As set forth herein, the terminal structure of dsRNAs of this disclosure that decrease expression of a target gene by, for example, RNAi may either have blunt ends or one or more overhangs. In certain embodiments, the overhang may be at the 3'-end or the 5'-end. The total length of dsRNAs having overhangs is expressed as the sum of the length of the paired double-stranded portion together with the overhanging nucleotides. 25 For example, if a 19 base pair dsRNA has a two nucleotide overhang at both ends, the total length is expressed as 21-mer. Furthermore, since the overhanging sequence may have low specificity to a target gene, it is not necessarily complementary (antisense) or identical (sense) to a target gene sequence. In further embodiments, a dsRNA of this disclosure that decreases expression of a target gene by RNAi may further comprise a low 30 molecular weight structure (e.g., a natural RNA molecule such as a tRNA, rRNA or viral RNA, or an artificial RNA molecule) at, for example, one or more overhanging portion of the dsRNA. In further embodiments, a dsRNA molecule that decreases expression of an target gene by RNAi according to the instant disclosure further comprises a 2'-sugar 55 Attorney Docket No. 08-09PCT Customer No.: 36,814 substitution, such as 2'-deoxy, 2'-0-methyl, 2'-O-methoxyethyl, 2'-0-2-methoxyethyl, halogen, 2'-fluoro, 2'-0-allyl, or the like, or any combination thereof. In still further embodiments, a dsRNA molecule that decreases expression of a target gene by RNAi according to the instant disclosure further comprises a terminal cap substituent on one or 5 both ends of the first strand or one or more second strands, such as an alkyl, abasic, deoxy abasic, glyceryl, dinucleotide, acyclic nucleotide, inverted deoxynucleotide moiety, or any combination thereof. In certain embodiments, at least one or two 5'-terminal ribonucleotides of the sense strand within the double-stranded region have a 2'-sugar substitution. In certain other embodiments, at least one or two 5'-terminal ribonucleotides 10 of the antisense strand within the double-stranded region have a 2'-sugar substitution. In certain embodiments, at least one or two 5'-terminal ribonucleotides of the sense strand and the antisense strand within the double-stranded region have a 2'-sugar substitution. In other embodiments, a dsRNA molecule that decreases expression of one or more target gene by RNAi according to the instant disclosure comprises one or more 15 substitutions in the sugar backbone, including any combination of ribosyl, 2'-deoxyribosyl, a tetrofuranosyl (e.g., L-a-threofuranosyl), a hexopyranosyl (e.g., p-allopyranosyl, p-altropyranosyl, and p-glucopyranosyl), a pentopyranosyl (e.g., s-ribopyranosyl, a-lyxopyranosyl, p-xylopyranosyl, and a-arabinopyranosyl), a carbocyclic (carbon only ring) analog, a pyranose, a furanose, a morpholino, or analogs or 20 derivatives thereof. In yet other embodiments, a dsRNA molecule that decreases expression of a target gene (including a mRNA splice variant thereof) by RNAi according to the instant disclosure further comprises at least one modified internucleoside linkage, such as independently a phosphorothioate, chiral phosphorothioate, phosphorodithioate, 25 phosphotriester, aminoalkylphosphotriester, methyl phosphonate, alkyl phosphonate, 3' alkylene phosphonate, 5'-alkylene phosphonate, chiral phosphonate, phosphonoacetate, thiophosphonoacetate, phosphinate, phosphoramidate, 3'-amino phosphoramidate, aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate, boranophosphate linkage, or any 30 combination thereof. A modified internucleotide linkage, as described herein, can be present in one or more strands of a dsRNA molecule of this disclosure, for example, in the sense strand, the antisense strand, both strands, or a plurality of strands (e.g., in an mdRNA). The dsRNA molecules of this disclosure can comprise one or more modified internucleotide linkages 56 Attorney Docket No. 08-09PCT Customer No.: 36,814 at the 3'-end, the 5'-end, or both of the 3'- and 5'-ends of the second sense strand, the third sense strand, the antisense strand or any combination of the antisense strand and one or more of the sense strands. In one embodiment, a dsRNA molecule capable of decreasing expression of a target gene (including a specific or selected mRNA splice variant thereof) 5 by RNAi has one modified internucleotide linkage at the 3'-end, such as a phosphorothioate linkage. For example, this disclosure provides a dsRNA molecule capable of decreasing expression of a target gene by RNAi having about 1 to about 8 or more phosphorothioate internucleotide linkages in one dsRNA strand. In yet another embodiment, this disclosure provides a dsRNA molecule capable of decreasing 10 expression of a target gene by RNAi having about 1 to about 8 or more phosphorothioate internucleotide linkages in the dsRNA strands. In other embodiments, an exemplary dsRNA molecule of this disclosure can comprise from about 1 to about 5 or more consecutive phosphorothioate internucleotide linkages at the 5'-end of the sense strand, the antisense strand, both strands, or a plurality of strands. In another example, an 15 exemplary dsRNA molecule of this disclosure can comprise one or more pyrimidine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, either strand, or a plurality of strands. In yet another example, an exemplary dsRNA molecule of this disclosure comprises one or more purine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, either strand, or a plurality of strands. 20 Many exemplary modified nucleotide bases or analogs thereof useful in the dsRNA of the instant disclosure include 5-methylcytosine; 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl, 2-propyl, or other alkyl derivatives of adenine and guanine; 8-substituted adenines and guanines (such as 8-aza, 8-halo, 8 amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, or the like); 7-methyl, 7-deaza, and 3-deaza 25 adenines and guanines; 2-thiouracil; 2-thiothymine; 2-thiocytosine; 5-methyl, 5-propynyl, 5-halo (such as 5-bromo or 5-fluoro), 5-trifluoromethyl, or other 5-substituted uracils and cytosines; and 6-azouracil. Further useful nucleotide bases can be found in Kurreck, Eur. J. Biochem. 270:1628, 2003; Herdewijn, Antisense Nucleic Acid Develop. 10:297, 2000; Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, 30 J. I., ed. John Wiley & Sons, 1990; U.S. Patent No. 3,687,808, and similar references. Certain nucleotide base moieties are particularly useful for increasing the binding affinity of the dsRNA molecules of this disclosure to complementary targets. These include 5-substituted pyrimidines; 6-azapyrimidines; and N-2, N-6, or 0-6 substituted purines (including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine). For 57 Attorney Docket No. U8-U9PCTI Customer No.: 36,814 example, 5-methyluridine and 5-methylcytosine substitutions are known to increase nucleic acid duplex stability, which can be combined with 2'-sugar modifications (such as 2'-methoxy or 2'-methoxyethyl) or internucleoside linkages (e.g., phosphorothioate) that provide nuclease resistance to the modified or substituted dsRNA. 5 In another aspect of the instant disclosure, there is provided a dsRNA that decreases expression of a target gene, comprising a first strand that is complementary to a target mRNA and a second strand that is complementary to the first strand, wherein the first and second strands form a double-stranded region of about 15 to about 40 base pairs; wherein at least one pyrimidine of the dsRNA is substituted with a pyrimidine nucleoside 10 according to Formula I or II: R 0 R NH 2 5 4 6 3NH N (I) R 4 s' Rs R4 Rs N 4' 1'8 (1 3' 2' R3R2 R3 R2 wherein R' and R 2 are each independently a -H, -OH, -OCH 3 , -OCH 2 0CH 2

CH

3 ,

-OCH

2

CH

2 0CH 3 , halogen, substituted or unsubstituted CI-Cio alkyl, alkoxy, alkoxyalkyl, hydroxyalkyl, carboxyalkyl, alkylsulfonylamino, aminoalkyl, dialkylamino, 15 alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, substituted or unsubstituted C 2 -Cio alkenyl, substituted or unsubstituted -0-allyl, -O-CH 2

CH=CH

2 , -O-CH=CHCH 3 , substituted or unsubstituted C 2 -Cio alkynyl, carbamoyl, carbamyl, carboxy, carbonylamino, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, -NH 2 , -NO 2 , -C=N, or heterocyclo group; R 3 and R 4 20 are each independently a hydroxyl, a protected hydroxyl, or an internucleoside linking group; and R 5 and R 8 are each independently 0 or S. In certain embodiments, at least one nucleoside is according to Formula I in which R 1 is methyl and R 2 is -OH, or R 1 is methyl, R 2 is -OH, and R 8 is S. In other embodiments, the internucleoside linking group covalently links from about 2 to about 40 nucleosides. 25 In certain embodiments, the first and one or more second strands of a dsRNA, which decreases expression of a target gene by RNAi and has at least one pyrimidine 58 Attorney IJOCKet NO. UZ-U9U I Customer No.: 36,814 substituted with a pyrimidine nucleoside according to Formula I or II, can anneal or hybridize together (i.e., due to complementarity between the strands) to form at least one double-stranded region having a length or a combined length of about 15 to about 40 base pairs. In some embodiments, the dsRNA has at least one double-stranded region ranging 5 in length from about 4 base pairs to about 10 base pairs or about 5 to about 13 base pairs or about 15 to about 25 base pairs or about 19 to about 23 base pairs. In other embodiments, the dsRNA has at least one double-stranded region ranging in length from about 26 to about 40 base pairs or about 27 to about 30 base pairs or about 30 to about 35 base pairs. In certain embodiments, the dsRNA molecule or analog thereof has an 10 overhang of one to four nucleotides on one or both 3'-ends, such as an overhang comprising a deoxyribonucleotide or two deoxyribonucleotides (e.g., thymidine). In some embodiments, dsRNA molecule or analog thereof has a blunt end at one or both ends of the dsRNA. In certain embodiments, the 5'-end of the first or second strand is phosphorylated. 15 In certain embodiments, at least one R' is a CI-C 5 alkyl, such as methyl or ethyl. Within other exemplary embodiments of this disclosure, compounds of Formula I are a 5-alkyluridine (i.e., R 1 is alkyl, R2 is -OH, and R 3 , R4, and R 5 are as defined herein) or compounds of Formula II are a 5-alkylcytidine (i.e., R1 is alkyl, R 2 is -OH, and R', R 4, and R5 are as defined herein). In related embodiments, the 5-alkyluridine is a 20 5-methyluridine (also referred to as ribothymidine or T- i.e., R1 is methyl and R2 is -OH), and the 5-alkylcytidine is a 5-methylcytidine. In other embodiments, at least one, at least three, or all uridines of the first strand of the dsRNA are replaced with 5 methyluridine, or at least one, at least three, or all uridines of the second strand of the dsRNA are replaced with 5-methyluridine, or any combination thereof (e.g., such changes 25 are made on more than one strand). In certain embodiments, at least one pyrimidine nucleoside of Formula I or Formula II has an R 5 that is S or R' that is S. In further embodiments, at least one pyrimidine nucleoside of the dsRNA is a locked nucleic acid (LNA) in the form of a bicyclic sugar, wherein R2 is oxygen, and the 2'-O and 4'-C form an oxymethylene bridge on the same ribose ring. In a related 30 embodiment, the LNA comprises a base substitution, such as a 5-methyluridine LNA or 2-thio-5-methyluridine LNA. In other embodiments, at least one, at least three, or all uridines of the first strand of the dsRNA are replaced with 5-methyluridine or 2 thioribothymidine or 5-methyluridine LNA or 2-thio-5-methyluridine LNA, or at least one, at least three, or all uridines of the second strand of the dsRNA are replaced with 5 59 Attorney Docket No. 08-09PCT Customer No.: 36,814 methyluridine, 2-thioribothymidine, 5-methyluridine LNA, 2-thio-5-methyluridine LNA, or any combination thereof (e.g., such changes are made on both strands, or some substitutions include 5-methyluridine only, 2-thioribothymidine only, 5-methyluridine LNA only, 2-thio-5-methyluridine LNA only, or one or more 5-methyluridine or 2 5 thioribothymidine with one or more 5-methyluridine LNA or 2-thio-5-methyluridine LNA). In further embodiments, a ribose of the pyrimidine nucleoside or the internucleoside linkage can be optionally modified. For example, compounds of Formula I or II are provided wherein R2 is alkoxy, such as a 2'-O-methyl substitution (e.g., which 10 may be in addition to a 5-alkyluridine or a 5-alkylcytidine, respectively). In certain embodiments, R 2 is selected from 2'-O-(Ci-C 5 ) alkyl, 2'-O-methyl, 2'-OCH 2

OCH

2

CH

3 , 2'-OCH 2

CH

2

OCH

3 , 2'-O-allyl, or 2'-fluoro. In further embodiments, one or more of the pyrimidine nucleosides are according to Formula I in which R' is methyl and R 2 is a 2'-0 (CI-Cs) alkyl (e.g., 2'-O-methyl), or in which R' is methyl, R2 is a 2'O-(CI-C 5 ) alkyl (e.g., 15 2'O-methyl), and R 2 is S, or any combination thereof In other embodiments, one or more, or at least two, pyrimidine nucleosides according to Formula I or II have an R 2 that is not -H or -OH and is incorporated at a 3'-end or 5'-end and not within the gap of one or more strands within the double-stranded region of the dsRNA molecule. In further embodiments, a dsRNA molecule or analog thereof comprising a 20 pyrimidine nucleoside according to Formula I or Formula II in which R2 is not -H or -OH and an overhang, further comprises at least two of pyrimidine nucleosides that are incorporated either at a 3'-end or a 5'-end or both of one strand or two strands within the double-stranded region of the dsRNA molecule. In a related embodiment, at least one of the at least two pyrimidine nucleosides in which R2 is not -H or -OH is located at a 3'-end 25 or a 5'-end within the double-stranded region of at least one strand of the dsRNA molecule, and wherein at least one of the at least two pyrimidine nucleosides in which R2 is not -H or -OH is located internally within a strand of the dsRNA molecule. In still further embodiments, a dsRNA molecule or analog thereof that has an overhang has a first of the two or more pyrimidine nucleosides in which R2 is not -H or -OH that is 30 incorporated at a 5'-end within the double-stranded region of the sense strand of the dsRNA molecule and a second of the two or more pyrimidine nucleosides is incorporated at a 5'-end within the double-stranded region of the antisense strand of the dsRNA molecule. In any of these embodiments, one or more substituted or modified nucleotides can be a G clamp (e.g., a cytosine analog that forms an additional hydrogen bond to 60 LMrney VOCKM INO. u6-uvru_ I Customer No.: 36,814 guanine, such as 9-(aminoethoxy)phenoxazine; see, e.g., Lin and Mateucci, 1998). In any of these embodiments, provided the one or more pyrimidine nucleosides are not within the gap. In yet other embodiments, a dsRNA molecule or analog thereof of Formula I or II 5 according to the instant disclosure that has an overhang that comprises four or more independent pyrimidine nucleosides or four or more independent pyrimidine nucleosides in which R2 is not -H or -OH, wherein (a) a first pyrimidine nucleoside is incorporated into a 3'-end within the double-stranded region of the sense (second) strand of the dsRNA, (b) a second pyrimidine nucleoside is incorporated into a 5'-end within the 10 double-stranded region of the sense (second) strand, (c) a third pyrimidine nucleoside is incorporated into a 3'-end within the double-stranded region of the antisense (first) strand of the dsRNA, and (d) a fourth pyrimidine nucleoside is incorporated into a 5'-end within the double-stranded region of the antisense (first) strand. In any of these embodiments, provided the one or more pyrimidine nucleosides are not within the gap. 15 In further embodiments, a dsRNA molecule or analog thereof comprising a pyrimidine nucleoside according to Formula I or Formula II in which R2 is not -H or -OH and is blunt-ended, further comprises at least two of pyrimidine nucleosides that are incorporated either at a 3'-end or a 5'-end or both of one strand or two strands of the dsRNA molecule. In a related embodiment, at least one of the at least two pyrimidine 20 nucleosides in which R 2 is not -H or -OH is located at a 3'-end or a 5'-end of at least one strand of the dsRNA molecule, and wherein at least one of the at least two pyrimidine nucleosides in which R 2 is not -H or -OH is located internally within a strand of the dsRNA molecule. In still further embodiments, a dsRNA molecule or analog thereof that is blunt-ended has a first of the two or more pyrimidine nucleosides in which R 2 is not -H 25 or -OH that is incorporated at a 5'-end of the sense strand of the dsRNA molecule and a second of the two or more pyrimidine nucleosides is incorporated at a 5'-end of the antisense strand of the dsRNA molecule. In any of these embodiments, provided the one or more pyrimidine nucleosides are not within the gap. In yet other embodiments, a dsRNA molecule comprising a pyrimidine nucleoside 30 according to Formula I or Formula II and that is blunt-ended comprises four or more independent pyrimidine nucleosides or four or more independent pyrimidine nucleosides in which R2 is not -H or -OH, wherein (a) a first pyrimidine nucleoside is incorporated into a 3'-end within the double-stranded region of the sense (second) strand of the dsRNA, (b) a second pyrimidine nucleoside is incorporated into a 5'-end within the 61 Attorney Docket No. U8-09PCT Customer No.: 36,814 double-stranded region of the sense (second) strand, (c) a third pyrimidine nucleoside is incorporated into a 3'-end within the double-stranded region of the antisense (first) strand of the dsRNA, and (d) a fourth pyrimidine nucleoside is incorporated into a 5'-end within the double-stranded region of the antisense (first) strand. In any of these embodiments, 5 provided the one or more pyrimidine nucleosides are not within the gap. In still further embodiments, a dsRNA molecule or analog thereof of Formula I or II according to the instant disclosure further comprises a terminal cap substituent on one or both ends of the first strand or second strand, such as an alkyl, abasic, deoxy abasic, glyceryl, dinucleotide, acyclic nucleotide, inverted deoxynucleotide moiety, or 10 any combination thereof. In further embodiments, one or more internucleoside linkage can be optionally modified. For example, a dsRNA molecule or analog thereof of Formula I or II according to the instant disclosure wherein at least one internucleoside linkage is modified to a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl phosphonate, alkyl phosphonate, 15 3'-alkylene phosphonate, 5'-alkylene phosphonate, chiral phosphonate, phosphonoacetate, thiophosphonoacetate, phosphinate, phosphoramidate, 3'-amino phosphoramidate, aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate, boranophosphate linkage, or any combination thereof. 20 In still another embodiment, a nicked or gapped dsRNA molecule (ndsRNA or gdsRNA, respectively) that decreases expression of a target gene by RNAi, comprising a first strand that is complementary to a target mRNA and two or more second strands that are complementary to the first strand, wherein the first and at least one of the second strands form a non-overlapping double-stranded region of about 5 to about 13 base pairs. 25 Any of the substitutions or modifications described herein is contemplated within this embodiment as well. In another exemplary of this disclosure, the dsRNAs comprise at least two or more substituted pyrimidine nucleosides can each be independently selected wherein R' comprises any chemical modification or substitution as contemplated herein, for example an alkyl (e.g., 30 methyl), halogen, hydroxy, alkoxy, nitro, amino, trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, alkanoyl, alkanoyloxy, aryl, aroyl, aralkyl, nitrile, dialkylamino, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, carboxyalkyl, alkoxyalkyl, carboxy, carbonyl, alkanoylamino, carbamoyl, carbonylamino, alkylsulfonylamino, or heterocyclo group. When two or more modified ribonucleotides are present, each modified 62 AttOrney UoCket No. UZ-U9P I Customer No.: 36,814 ribonucleotide can be independently modified to have the same, or different, modification or substitution at R' or R. In other detailed embodiments, one or more substituted pyrimidine nucleosides according to Formula I or II can be located at any ribonucleotide position, or any combination 5 of ribonucleotide positions, on either or both of the sense and antisense strands of a dsRNA molecule of this disclosure, including at one or more multiple terminal positions as noted above, or at any one or combination of multiple non-terminal ("internal") positions. In this regard, each of the sense and antisense strands can incorporate about 1 to about 6 or more of the substituted pyrimidine nucleosides. 10 In certain embodiments, when two or more substituted pyrimidine nucleosides are incorporated within a dsRNA of this disclosure, at least one of the substituted pyrimidine nucleosides will be at a 3'- or 5'-end of one or both strands, and in certain embodiments at least one of the substituted pyrimidine nucleosides will be at a 5'-end of one or both strands. In other embodiments, the substituted pyrimidine nucleosides are located at a position 15 corresponding to a position of a pyrimidine in an unmodified dsRNA that is constructed as a homologous sequence for targeting a cognate mRNA, as described herein. In addition, the terminal structure of the dsRNAs of this disclosure may have a stem-loop structure in which ends of one side of the dsRNA molecule are connected by a linker nucleic acid, e.g., a linker RNA. The length of the double-stranded region (stem 20 loop portion) can be, for example, about 15 to about 49 bp, about 15 to about 35 bp, or about 21 to about 30 bp long. Alternatively, the length of the double-stranded region that is a final transcription product of dsRNAs to be expressed in a target cell may be, for example, approximately about 15 to about 49 bp, about 15 to about 35 bp, or about 21 to about 30 bp long. When linker segments are employed, there is no particular limitation in 25 the length of the linker as long as it does not hinder pairing of the stem portion. For example, for stable pairing of the stem portion and suppression of recombination between DNAs coding for this portion, the linker portion may have a clover-leaf tRNA structure. Even if the linker has a length that would hinder pairing of the stem portion, it is possible, for example, to construct the linker portion to include introns so that the introns are 30 excised during processing of a precursor RNA into mature RNA, thereby allowing pairing of the stem portion. In the case of a stem-loop dsRNA, either end (head or tail) of RNA with no loop structure may have a low molecular weight RNA. As described above, these low molecular weight RNAs may include a natural RNA molecule, such as tRNA, rRNA or viral RNA, or an artificial RNA molecule. 63 Attorney Docket No. 08-09PCT Customer No.: 36,814 A dsRNA molecule may be comprised of a circular nucleic acid molecule, wherein the dsRNA is about 38 to about 70 nucleotides in length having from about 18 to about 23 base pairs (e.g., about 19 to about 21 bp) wherein the circular oligonucleotide forms a dumbbell shaped structure having about 19 base pairs and two loops. In certain 5 embodiments, a circular dsRNA molecule contains two loop motifs wherein one or both loop portions of the dsRNA molecule is biodegradable. For example, a circular dsRNA molecule of this disclosure is designed such that degradation of the loop portions of the dsRNA molecule in vivo can generate a dsRNA molecule with 3'-terminal overhangs, such as 3'-terminal nucleotide overhangs comprising from about 1 to about 4 (unpaired) 10 nucleotides. Substituting or modifying nucleosides of a dsRNA according to this disclosure can result in increased resistance to enzymatic degradation, such as exonucleolytic degradation, including 5'-exonucleolytic or 3'-exonucleolytic degradation. As such, in some embodiments, the dsRNAs described herein will exhibit significant resistance to 15 enzymatic degradation compared to a corresponding dsRNA having standard nucleotides, and will thereby possess greater stability, increased half-life, and greater bioavailability in physiological environments (e.g., when introduced into a eukaryotic target cell). In addition to increasing resistance of the substituted or modified dsRNAs to exonucleolytic degradation, the incorporation of one or more pyrimidine nucleosides according to 20 Formula I or II will render dsRNAs more resistant to other enzymatic or chemical degradation processes and thus more stable and bioavailable than otherwise identical dsRNAs that do not include the substitutions or modifications. In related aspects of this disclosure, dsRNA substitutions or modifications described herein will often improve stability of a modified dsRNA for use within research, diagnostic and treatment methods 25 wherein the modified dsRNA is contacted with a biological sample, for example, a mammalian cell, intracellular compartment, serum or other extracellular fluid, tissue, or other in vitro or in vivo physiological compartment or environment. In one embodiment, diagnosis is performed on an isolated biological sample. In another embodiment, the diagnostic method is performed in vitro. In a further embodiment, the diagnostic method 30 is not performed (directly) on a human or animal body. In addition to increasing stability of substituted or modified dsRNAs, incorporation of one or more pyrimidine nucleosides according to Formula I or II in a dsRNA designed for gene silencing can provide additional desired functional results, including increasing a melting point of a substituted or modified dsRNA compared to a corresponding 64 attorney vocKet No. uzs-unL i Customer No.: 36,814 unmodified dsRNA. In another aspect of this disclosure, certain substitutions or modifications of dsRNAs described herein can reduce "off-target effects" of the substituted or modified dsRNA molecules when they are contacted with a biological sample (e.g., when introduced into a target eukaryotic cell having specific, and non 5 specific mRNA species present as potential specific and non-specific targets). In yet another aspect of this disclosure, the dsRNA substitutions or modifications described herein can reduce interferon activation by the dsRNA molecule when the dsRNA is contacted with a biological sample, e.g., when introduced into a eukaryotic cell. In further embodiments, dsRNAs of this disclosure can comprise one or more 10 sense (second) strand that is homologous or corresponds to a sequence of a target gene and an antisense (first) strand that is complementary to the sense strand and a sequence of the target gene. In exemplary embodiments, at least one strand of the dsRNA incorporates one or more pyrimidines substituted according to Formula I or II (e.g., wherein the pyrimidine is one or more 5-methyluridines or 2-thioribothymidines, the 15 ribose is modified to incorporate one or more 2'-O-methyl substitutions, or any combination thereof). These and other multiple substitutions or modifications according to Formula I or II can be introduced into one or more pyrimidines, or into any combination and up to all pyrimidines present in one or more strands of a dsRNA of the instant disclosure, so long as the dsRNA has or retains RNAi activity similar to or better 20 than the activity of an unmodified dsRNA. In any of the embodiments described herein, the dsRNA may include multiple modifications. For example, a dsRNA having at least one ribothymidine or 2'-O-methyl 5-methyluridine may further comprise at least one LNA, 2'-methoxy, 2'-fluoro, 2'-deoxy, phosphorothioate linkage, an inverted base terminal cap, or any combination thereof. In 25 certain embodiments, a dsRNA will have from one to all ribothymidines and have up to 75% LNA. In other embodiments, a dsRNA will have from one to all ribothymidines and have up to 75% 2'-methoxy (e.g., not at the Argonaute cleavage site). In still other embodiments, a dsRNA will have from one to all ribothymidines and have up to 100% 2' fluoro. In further embodiments, a dsRNA will have from one to all ribothymidines and 30 have up to 75% 2'-deoxy. In further embodiments, a dsRNA will have up to 75% LNA and have up to 75% 2'-methoxy. In still other embodiments, a dsRNA will have up to 75% LNA and have up to 100% 2'-fluoro. In further embodiments, a dsRNA will have up to 75% LNA and have up to 75% 2'-deoxy. In other embodiments, a dsRNA will have up to 75% 2'-methoxy and have up to 100% 2'-fluoro. In more embodiments, a dsRNA 65 Attorney Docket No. US-U9PICI Customer No.: 36,814 will have up to 75% 2'-methoxy and have up to 75% 2'-deoxy. In further embodiments, a dsRNA will have up to 100% 2'-fluoro and have up to 75% 2'-deoxy. In further multiple modification embodiments, a dsRNA will have from one to all ribothymidines, up to 75% LNA, and up to 75% 2'-methoxy. In still further 5 embodiments, a dsRNA will have from one to all ribothymidines, up to 75% LNA, and up to 100% 2'-fluoro. In further embodiments, a dsRNA will have from one to all ribothymidines, up to 75% LNA, and up to about 75% 2'-deoxy. In further embodiments, a dsRNA will have from one to all ribothymidines, up to 75% 2'-methoxy, and up to 75% 2'-fluoro. In further embodiments, a dsRNA will have from one to all ribothymidines, up 10 to 75% 2'-methoxy, and up to 75% 2'-deoxy. In further embodiments, a dsRNA will have from one to all ribothymidines, up to 100% 2'-fluoro, and up to 75% 2'-deoxy. In yet further embodiments, a dsRNA will have from one to all ribothymidines, up to 75% LNA substitutions, up to 75% 2'-methoxy, up to 100% 2'-fluoro, and up to 75% 2'-deoxy. In other embodiments, a dsRNA will have up to 75% LNA, up to 75% 2'-methoxy, and up to 15 100% 2'-fluoro. In further embodiments, a dsRNA will have up to 75% LNA, up to 75% 2'-methoxy, and up to about 75% 2'-deoxy. In further embodiments, a dsRNA will have up to 75% LNA, up to 100% 2'-fluoro, and up to 75% 2'-deoxy. In still further embodiments, a dsRNA will have up to 75% 2'-methoxy, up to 100% 2'-fluoro, and up to 75% 2'-deoxy. 20 In any of these exemplary methods for using multiply modified dsRNA, the dsRNA may further comprise up to 100% phosphorothioate internucleoside linkages, from one to ten or more inverted base terminal caps, or any combination thereof. Additionally, any of these dsRNA may have these multiple modifications on one strand, two strands, three strands, a plurality of strands, or all strands, or on the same or different 25 nucleoside within a dsRNA molecule. Finally, in any of these multiple modification dsRNA, the dsRNA must have gene silencing activity. Within certain aspects, the present disclosure provides dsRNA that decreases expression of a target gene by RNAi and compositions comprising one or more dsRNA, wherein at least one dsRNA comprises one or more universal-binding nucleotide(s) in the 30 first, second or third position in the anti-codon of the antisense or sense strand of the dsRNA and wherein the dsRNA is capable of specifically binding to a target sequence, such as an RNA expressed by a target cell. In cases wherein the sequence of a target target RNA includes one or more single nucleotide substitutions, dsRNA comprising a universal-binding nucleotide retains its capacity to specifically bind a target RNA, 66 Icorney JJOCKe iNo. u6-uyru i Customer No.: 36,814 thereby mediating gene silencing and, as a consequence, overcoming escape of the target gene from dsRNA-mediated gene silencing. Examplary universal-binding nucleotides that may be suitably employed in the compositions and methods disclosed herein include inosine, 1-p-D-ribofuranosyl-5-nitroindole, or 1- -D-ribofuranosyl-3-nitropyrrole. 5 In certain aspects, dsRNA disclosed herein can include between about 1 universal-binding nucleotide and about 10 universal-binding nucleotides. Within other aspects, the presently disclosed dsRNA may comprise a sense strand that is homologous to a sequence of a target gene and an antisense strand that is complementary to the sense strand, with the proviso that at least one nucleotide of the antisense or sense strand of the 10 otherwise complementary dsRNA duplex has one or more universal-binding nucleotide. In certain aspects, dsRNA disclosed herein comprises one or more hydroxymethyl modified nucleomonomer(s) (see chemical formulas below). Hereunder as one such example is an acyclic nucleomonomer, more preferably an acyclic monomer selected from the group consisting of monomers D, F, G, H, I, and J. Thus, the embodiments 15 described in the first aspect with regards to hydroxymethyl modified nucleomonomers will apply for other embodiments relating to acyclic nucleomonomers. As used herein, the terms "hydroxylmethyl substituted nucleomonomers", "hydroxylmethyl substituted monomers", "acyclic nucleomonomers", "acyclic monomers", "acyclic hydroxymethyl subsitituted nucleomoners", "nucleobase analog 20 monomers" may be used interchangeably throughout. O- Base O H Base O Base O Base OH 0 OH 0 O-R 0 S-R ~O--P=O OO-P=O -O-P=O Monomer D Monomer E Monomer F Monomer G 67 Atorney UocKet iNo. u6-uvra_ Customer No.: 36,814 O0 Base O O Base O0 Base 0 NH 0 NH 0 N -P=0 0-=0 R -- 0 R N Monomer H Monomer O R Monomer J R, in the above structures, is selected from the group consisting of hydrogen, methyl group, C(1-10) alkyl, cholesterol, naturally or non-naturally occurring amino acid, sugar, vitamin, flurophore, polyamine and fatty acid. 5 In certain aspects, a dsRNA having one or more hydroxymethyl modified nucleomonomer(s) has increased potency, reduced off-target effects, reduced immune stimulation, increased stability for storage, increased stability in biological media like serum, increased duration of action and/or improved pharmacokinetic properties, all relative to the native unmodified form of the dsRNA. 10 In certan aspects, the antisense (guide strand) of a dsRNA comprises one or more hydroxymethyl modified nucleomonomer(s). In certain aspects, the antisense of a dsRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hydroxymethyl modified nucleomonomer(s). In certain aspects, the entire antisense of a dsRNA comprises hydroxymethyl modified nucleomonomer(s). In certain aspects, a hydroxymethyl 15 modified nucleomonomer in the antisense strand is present in positions 1, 2, 3, 4, 5, 6, 7, and/or 8 wherein the positions are counted from the 5'-end of the antisense strand. In certain aspects, a hydroxymethyl modified nucleomonomer in the antisense strand is present in positions 3, 4, 5, 6, 7, and/or 8 wherein the positions are counted from the 5' end of the antisense strand. In certain aspects, a hydroxymethyl modified 20 nucleomonomer in the antisense strand is present in positions 7 and/or 8 wherein the positions are counted from the 5'-end of the antisense strand. In certain aspects, a hydroxymethyl modified nucleomonomer in the antisense strand is present in positions 9, 10, 11, 12, 13, 14, 15, and/or 16 wherein the positions are counted from the 5'-end of the antisense strand. In certain aspects, a hydroxymethyl 25 modified nucleomonomer in the antisense strand is present in positions 9, 10, and/or 11, wherein the positions are counted from the 5'-end of the antisense strand. In certain aspects, a hydroxymethyl modified nucleomonomer in the antisense strand is present in 68 Attorney Docket No. 08-09PCT Customer No.: 36,814 positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and/or 17, wherein the positions are counted from the 5'-end of the antisense strand. In certain aspects, a hydroxymethyl modified nucleomonomer in the antisense strand is present in positions 1, 2, 3, 4, 5, 6, 7, 8, 9 and/or 10, wherein the positions are counted from the 3'-end of the 5 antisense strand. In certain aspects, the sense (passenger strand) of a dsRNA comprises one or more hydroxymethyl modified nucleomonomer(s). In certain aspects, the sense (passenger strand) of a dsRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hydroxymethyl modified nucleomonomer(s). In certain aspects, the entire sense (passenger strand) of a dsRNA 10 comprises hydroxymethyl modified nucleomonomer(s). In certain aspects, a hydroxymethyl modified nucleomonomer in the sense strand is present in positions 1, 2, 3, 4, 5, 6, 7, and/or 8 wherein the positions are counted from the 5'-end of the sense strand. In certain aspects, a hydroxymethyl modified nucleomonomer in the sense strand is present in positions 3, 4, 5, 6, 7, and/or 8 wherein the positions are counted from the 5' 15 end of the sense strand. In certain aspects, a hydroxymethyl modified nucleomonomer in the sense strand is present in positions 7 and/or 8 wherein the positions are counted from the 5'-end of the sense strand. In certain aspects, a hydroxymethyl modified nucleomonomer in the sense strand is present in positions 9, 10, 11, 12, 13, 14, 15, and/or 16 wherein the positions are counted from the 5'-end of the sense strand. In certain 20 aspects, a hydroxymethyl modified nucleomonomer in the sense strand is present in positions 9, 10, and/or 11, wherein the positions are counted from the 5'-end of the sense strand. ). In certain aspects, a hydroxymethyl modified nucleomonomer in the sense strand is present in positions 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and/or 32 wherein the positions are counted from the 5'-end of the sense strand. In certain aspects, a 25 hydroxymethyl modified nucleomonomer in the sense strand is present in positions 1, 2, 3, 4, 5, 6, 7, 8, 9 and/or 10, wherein the positions are counted from the 3'-end of the sense strand. In certain aspects, the first, second and/or third strands of an mdRNA having a nick or gap comprises one or more hydroxymethyl modified nucleomonomer(s). In 30 certain aspects, the first, second and/or third strands of an mdRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hydroxymethyl modified nucleomonomer(s). In certain aspects, the entire first strand of an mdRNA comprises hydroxymethyl modified nucleomonomer(s). In certain aspects, a hydroxymethyl modified nucleomonomer in the first strand is present in positions 1, 2, 3, 4, 5, 6, 7, and/or 8 wherein the positions are counted from the 5'-end of 69 Attorney Docket No. 08-09PCT Customer No.: 36,814 the first strand strand. In certain aspects, a hydroxymethyl modified nucleomonomer in the first strand is present in positions 9, 10, 11, 12, 13, 14, 15, and/or 16 wherein the positions are counted from the 5'-end of the first strand. In certain aspects, the dsRNA has at least one blunt end having one or more a 5 hydroxymethyl modified nucleomonomer(s) covalently linked to the blunt end. In certain aspects, the dsRNA has two blunt ends each having one or more a hydroxymethyl modified nucleomonomer(s) covalently linked to each blunt end. In certain aspects, a blunt end has 1, 2, 3, 4, 5, 6, 7, 8 or more hydroxymethyl modified nucleomonomers covalently linked to the blunt end. In certain aspects, a blunt end has two hydroxymethyl 10 modified nucleomonomers covalently linked to the blunt end. In certain aspects, the one or more a hydroxymethyl modified nucleomonomer(s) are covalently linked to the 5'-end of the antisense strand. In certain aspects, the one or more a hydroxymethyl modified nucleomonomer(s) are covalently linked to the 3'-end of the antisense strand. In certain aspects, the one or more a hydroxymethyl modified nucleomonomer(s) are covalently 15 linked to the 5'-end and the 3'-end of the antisense strand. In certain aspects, the one or more a hydroxymethyl modified nucleomonomer(s) are covalently linked to the 5'-end of the sense strand. In certain aspects, the one or more a hydroxymethyl modified nucleomonomer(s) are covalently linked to the the 3'-end of the sense strand. In certain aspects, the one or more a hydroxymethyl modified nucleomonomer(s) are covalently 20 linked to the 5'-end and the 3'-end of the sense strand. In certain aspects, the one or more a hydroxymethyl modified nucleomonomer(s) are covalently linked to the 3'-end of the sense strand and the 3'-end of the antisense strand. In certain aspects, the one or more a hydroxymethyl modified nucleomonomer(s) are covalently linked to the 5'-end of the sense strand and the 5'-end of the antisense strand. In certain aspects, the one or more a 25 hydroxymethyl modified nucleomonomer(s) are covalently linked to the 3'-end of the sense strand and the 5'-end of the antisense strand. In certain aspects, the one or more a hydroxymethyl modified nucleomonomer(s) are covalently linked to the 5'-end of the sense strand and the 3'-end of the antisense strand. In certain aspects, the mdRNA has at least one blunt end having one or more a 30 hydroxymethyl modified nucleomonomer(s) covalently linked to the blunt end. In certain aspects, the mdRNA has two blunt ends each having one or more a hydroxymethyl modified nucleomonomer(s) covalently linked to each blunt end. In certain aspects, a blunt end has 1, 2, 3, 4, 5, 6, 7, 8 or more hydroxymethyl modified nucleomonomers covalently linked to the blunt end. In certain aspects, a blunt end has two hydroxymethyl 70 Attorney Docket No. 08-09PCT Customer No.: 36,814 modified nucleomonomers covalently linked to the blunt end. In certain aspects, the one or more a hydroxymethyl modified nucleomonomer(s) are covalently linked to the 5'-end of the antisense strand. In certain aspects, the one or more a hydroxymethyl modified nucleomonomer(s) are covalently linked to the 3'-end of the antisense strand. In certain 5 aspects, the one or more a hydroxymethyl modified nucleomonomer(s) are covalently linked to the 5'-end and the 3'-end of the antisense strand. In certain aspects, the one or more a hydroxymethyl modified nucleomonomer(s) are covalently linked to the 5'-end of one or both of the sense strands of an mdRNA. In certain aspects, the one or more a hydroxymethyl modified nucleomonomer(s) are covalently linked to the the 3'-end of one 10 or both of the sense strands of an mdRNA. In certain aspects, the one or more a hydroxymethyl modified nucleomonomer(s) are covalently linked to the 5'-end and the 3'-end of one or both of the the sense strands of an mdRNA. In certain aspects, the one or more a hydroxymethyl modified nucleomonomer(s) are covalently linked to the 3'-end of one or both of the sense strands, and the 3'-end of the antisense strand of an mdRNA. 15 In certain aspects, the one or more a hydroxymethyl modified nucleomonomer(s) are covalently linked to the 5'-end of one or both of the sense strands, and the 5'-end of the antisense strand of an mdRNA. In certain aspects, the one or more a hydroxymethyl modified nucleomonomer(s) are covalently linked to the 3'-end of one or both of the sense strands, and the 5'-end of the antisense strand of the mdRNA. In certain aspects, 20 the one or more a hydroxymethyl modified nucleomonomer(s) are covalently linked to the 5'-end of one or both of the sense strands, and the 3'-end of the antisense strand of an mdRNA. In certain aspects, the dsRNA comprises hydroxymethyl substituted monomers at one or more position(s) that prevent and/or reduce dicer enzyme processing of the dsRNA 25 compared to an unmodified form of the dsRNA. In certain aspects, the dsRNA comprises hydroxymethyl substituted monomers at one or more position(s) that prevent and/or reduce cytokine induction by the dsRNA compared to an unmodified form of the dsRNA. In certain aspects, the dsRNA comprises hydroxymethyl substituted monomers at 30 one or more position(s) that improves and/or enhances the potency or target message knockdown activity of the dsRNA compared to an unmodified form of the dsRNA. In certain aspects, the dsRNA comprises hydroxymethyl substituted monomers at one or more position(s) that prevent and/or reduce off-target effect by the dsRNA compared to an unmodified form of the dsRNA. 71 Attorney Docket No. 08-U91C I Customer No.: 36,814 In certain aspects, the dsRNA comprises hydroxymethyl substituted monomers at one or more position(s) that improves and/or enhances the the stabilility of the dsRNA in serum compared to an unmodified form of the dsRNA. The contents of PCT patent application PCT/US2008/064417, for example figure 5 1, are hereby incoroporated by reference in its entirety. Monomers disclosed in PCT/US2008/064417, for example figure 1, may be used in combination the dsRNA molecules disclosed herein and used in combination with any modification disclosed herein. Examples monomers include the following: 0 0 Base 0, 0 Base HO- HO 0 OH 0 -O-P=O -O-P=O 10 Synthesis of Nucleic Acid Molecules Exemplary molecules of the instant disclosure are recombinantly produced, chemically synthesized, or a combination thereof. Oligonucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using protocols known in the art, for example as described in Caruthers et al., 15 Methods in Enzymol. 211:3-19, 1992; Thompson et al., PCT Publication No. WO 99/54459, Wincott et al., Nucleic Acids Res. 23:2677-2684, 1995; Wincott et al., Methods Mol. Bio. 74:59, 1997; Brennan et al., Biotechnol Bioeng. 61:33-45, 1998; and Brennan, U.S. Patent No. 6,001,311. Synthesis of RNA, including certain dsRNA molecules and analogs thereof of this disclosure, can be made using the procedure as 20 described in Usman et al., J. Am. Chem. Soc. 109:7845, 1987; Scaringe et al., Nucleic Acids Res. 18:5433, 1990; and Wincott et al., Nucleic Acids Res. 23:2677-2684, 1995; Wincott et al., Methods Mol. Bio. 74:59, 1997. In certain embodiments, the nucleic acid molecules of the present disclosure can be synthesized separately and joined together post-synthetically, for example, by ligation 25 (Moore et al., Science 256:9923, 1992; Draper et al., PCT Publication No. WO 93/23569; Shabarova et al., Nucleic Acids Res. 19:4247, 1991; Bellon et al., Nucleosides & Nucleotides 16:951, 1997; Bellon et al., Bioconjugate Chem. 8:204, 1997), or by hybridization following synthesis or deprotection. 72 AtTomey LvocKet iNo. uzs-unL i Customer No.: 36,814 In further embodiments, dsRNAs of this disclosure that decrease expression of a target gene by RNAi can be made as single or multiple transcription products expressed by a polynucleotide vector encoding one or more dsRNAs and directing their expression within host cells. In these embodiments the double-stranded portion of a final 5 transcription product of the dsRNAs to be expressed within the target cell can be, for example, about 5 to about 40 bp, about 15 to about 24 bp, or about 25 to about 40 bp long. Within exemplary embodiments, double-stranded portions of dsRNAs, in which two or more strands pair up, are not limited to completely paired nucleotide segments, and may contain non-pairing portions due to a mismatch (the corresponding nucleotides 10 are not complementary), bulge (lacking in the corresponding complementary nucleotide on one strand), overhang, or the like. Non-pairing portions can be contained to the extent that they do not interfere with dsRNA formation and function. In certain embodiments, a "bulge" may comprise 1 to 2 non-pairing nucleotides, and the double-stranded region of dsRNAs in which two strands pair up may contain from about 1 to 7, or about 1 to 5 15 bulges. In addition, "mismatch" portions contained in the double-stranded region of dsRNAs may include from about 1 to 7, or about 1 to 5 mismatches. In other embodiments, the double-stranded region of dsRNAs of this disclosure may contain both bulge and mismatched portions in the approximate numerical ranges specified herein. A dsRNA or analog thereof of this disclosure may be further comprised of a 20 nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide linker that joins the sense region of the dsRNA to the antisense region of the dsRNA. In one embodiment, a nucleotide linker can be a linker of more than about 2 nucleotides length up to about 10 nucleotides in length. In another embodiment, the nucleotide linker can be a nucleic acid aptamer. By "aptamer" or "nucleic acid aptamer" as used herein is meant a nucleic 25 acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that comprises a sequence recognized by the target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule wherein the target molecule does not naturally bind to a nucleic acid. The target molecule can be any molecule of interest. For example, the aptamer can be used to 30 bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art (see, e.g., Gold et al., Annu. Rev. Biochem. 64:763, 1995; Brody and Gold, J Biotechnol. 74:5, 2000; Sun, Curr. Opin. Mol. Ther. 2:100, 2000; 73 Attorney ijociet No. us-uwJu i Customer No.: 36,814 Kusser, J. Biotechnol. 74:27, 2000; Hermann and Patel, Science 287:820, 2000; and Jayasena, Clinical Chem. 45:1628, 1999). A non-nucleotide linker may be comprised of an abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric 5 compounds (e.g., polyethylene glycols such as those having between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 18:6353, 1990, and Nucleic Acids Res. 15:3113, 1987; Cload and Schepartz, J. Am. Chem. Soc. 113:6324, 1991; Richardson and Schepartz, J. Am. Chem. Soc. 113:5109, 1991; Ma et al., Nucleic Acids Res. 21:2585, 1993, and Biochemistry 32:1751, 1993; 10 Durand et al., Nucleic Acids Res. 18:6353, 1990; McCurdy et al., Nucleosides & Nucleotides 10:287, 1991; Jaschke et al., Tetrahedron Lett. 34:301, 1993; Ono et al., Biochemistry 30:9914, 1991; Arnold et al., PCT Publication No. WO 89/02439; Usman et al., PCT Publication No. WO 95/06731; Dudycz et al., PCT Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem. Soc. 113:4000, 1991. The 15 synthesis of a dsRNA molecule of this disclosure, which can be further modified, comprises: (a) synthesis of a first (antisense) strand and synthesis of a second (sense) strand and a third (sense) strand that are each complementary to non-overlapping regions of the first strand; and (b) annealing the first, second and third strands together under conditions suitable to obtain a dsRNA molecule. In another embodiment, synthesis of the 20 first, second and thirdstrands of a dsRNA molecule is by solid phase oligonucleotide synthesis. In yet another embodiment, synthesis of the first, second ,and third strands of a dsRNA molecule is by solid phase tandem oligonucleotide synthesis. Chemically synthesizing nucleic acid molecules with substitutions or modifications (base, sugar, phosphate, or any combination thereof) can prevent their 25 degradation by serum ribonucleases, which may lead to increased potency. See, e.g., Eckstein et al., PCT Publication No. WO 92/07065; Perrault et al., Nature 344:565, 1990; Pieken et al., Science 253:314, 1991; Usman and Cedergren, Trends in Biochem. Sci. 17:334, 1992; Usman et al., Nucleic Acids Symp. Ser. 31:163, 1994; Beigelman et al., J. Biol. Chem. 270:25702, 1995; Burgin et al., Biochemistry 35:14090, 1996; Burlina et al., 30 Bioorg. Med. Chem. 5:1999, 1997; Thompson et al., Karpeisky et al., Tetrahedron Lett. 39:1131, 1998; Earnshaw and Gait, Biopolymers (Nucleic Acid Sciences) 48:39-55, 1998; Verma and Eckstein, Annu. Rev. Biochem. 67:99-134, 1998; Herdewijn, Antisense Nucleic Acid Drug Dev. 10:297, 2000; Kurreck, Eur. J. Biochem. 270:1628, 2003; Dorsett and Tuschl, Nature Rev. Drug Discov. 3:318, 2004; PCT Publication Nos. 74 aTorney vocKet iNo. us-uvrt_ i Customer No.: 36,814 WO 91/03162; WO 93/15187; WO 97/26270; WO 98/13526; U.S. Patent Nos. 5,334,711; 5,627,053; 5,716,824; 5,767, 264; 6,300,074. Each of the above references discloses various substitutions and chemical modifications to the base, phosphate, or sugar moieties of nucleic acid molecules, which can be used in the dsRNAs described herein. For 5 example, oligonucleotides can be modified at the sugar moiety to enhance stability or prolong biological activity by increasing nuclease resistance. Representative sugar modifications include 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-O-allyl, or 2'-H. Other modifications to enhance stability or prolong biological activity can be internucleoside linkages, such as phosphorothioate, or base-modifications, such as locked 10 nucleic acids (see, e.g., U.S. Patent Nos. 6,670,461; 6,794,499; 6,268,490), or 5-methyluridine or 2'-O-methyl-5-methyluridine in place of uridine (see, e.g., U.S. Patent Application Publication No. 2006/0142230). Hence, dsRNA molecules of the instant disclosure can be modified to increase nuclease resistance or duplex stability while substantially retaining or having enhanced RNAi activity as compared to unmodified 15 dsRNA. In one embodiment, this disclosure features substituted or modified dsRNA molecules, such as phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, 20 sulfamate, formacetal, thioformacetal, or alkylsilyl substitutions. For a review of oligonucleotide backbone modifications, see Hunziker and Leumann, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, 1995; and Mesmaeker et al., A CS, 24-39, 1994. In another embodiment, a conjugate molecule can be optionally attached to a 25 dsRNA or analog thereof that decreases expression of a target gene by RNAi. For example, such conjugate molecules may be polyethylene glycol, human serum albumin, polyarginine, Gln-Asn polymer, or a ligand for a cellular receptor that can, for example, mediate cellular uptake (e.g., HIV TAT, see Vocero-Akbani et al., Nature Med. 5:23, 1999; see also U.S. Patent Application Publication No. 2004/0132161).. Examples of 30 specific conjugate molecules contemplated by the instant disclosure that can be attached to a dsRNA or analog thereof of this disclosure are described in Vargeese et al., U.S. Patent Application Publication No. 2003/0130186, and U.S. Patent Application Publication No. 2004/0110296. In another embodiment, a conjugate molecule is covalently attached to a dsRNA or analog thereof that decreases expression of a target 75 Attorney Docket No. 08-09PCT Customer No.: 36,814 gene by RNAi via a biodegradable linker. In certain embodiments, a conjugate molecule can be attached at the 3'-end of either the sense strand, the antisense strand, or both strands of a dsRNA molecule provided herein. In another embodiment, a conjugate molecule can be attached at the 5'-end of either the sense strand, the antisense strand, or 5 both strands of the dsRNA or analog thereof. In yet another embodiment, a conjugate molecule is attached at both the 3'-end and 5'-end of either the sense strand, the antisense strand, or both strands of a dsRNA molecule, or any combination thereof. In further embodiments, a conjugate molecule of this disclosure comprises a molecule that facilitates delivery of a dsRNA or analog thereof into a biological system, such as a cell. 10 A person of skill in the art can screen dsRNA of this disclosure having various conjugates to determine whether the dsRNA-conjugate possesses improved properties (e.g., pharmacokinetic profiles, bioavailability, stability) while maintaining the ability to mediate RNAi in, for example, an animal model as described herein or generally known in the art. 15 Methods for Selecting dsRNA Molecules Specific for a Target Gene As indicated herein, the present disclosure also provides methods for selecting dsRNA and analogs thereof that are capable of specifically binding to a target gene (including a mRNA splice variant thereof) while being incapable of specifically binding or minimally binding to non- target genes (i. e, any nucleic acid sequence whose 20 expression or activity is not to be altered). Thus, any of the target genes identified herein may be a non-target gene depending on what target gene(s) is identified for selecting dsRNAs capable of specifically binding to the target gene. The selection process disclosed herein is useful, for example, in eliminating dsRNAs analogs that are cytotoxic due to non-specific binding to, and subsequent degradation of, one or more non- target 25 genes. Methods of the present disclosure do not require a priori knowledge of the nucleotide sequence of every possible gene variant (including mRNA splice variants) targeted by the dsRNA or analog thereof In one embodiment, the nucleotide sequence of the dsRNA is selected from a conserved region or consensus sequence of a target gene. 30 In another embodiment, the nucleotide sequence of the dsRNA may be selectively or preferentially targeted to a certain sequence contained in an mRNA splice variant of a target gene. 76 Attorney ijocKet iNo. Um-UWU i Customer No.: 36,814 In certain embodiments, methods are provided for selecting one or more dsRNA molecule that decreases expression of a target gene by RNAi, comprising a first strand that is complementary to a target mRNA and a second strand that is complementary to the first strand, wherein the first and second strands form a double-stranded region of 5 about 15 to about 40 base pairs (see, e.g., target nucleotide sequences in the Sequence Listing identified herein), and wherein at least one uridine of the dsRNA molecule is replaced with a 5-methyluridine or 2-thioribothymidine or 2'-O-methyl-5-methyluridine, which methods employ "off-target" profiling whereby one or more dsRNA provided herein is contacted with a cell, either in vivo or in vitro, and total mRNA is collected for 10 use in probing a microarray comprising oligonucleotides having one or more nucleotide sequence from a panel of known genes, including non-target genes (e.g., interferon). Within related embodiments, one or more dsRNA molecule that decreases expressioni of a target gene by RNAi may further comprise a third strand that is complementary to the first strand, wherein the first and third strands form a double-stranded region wherein the 15 double-stranded region formed by the first and third strands is non-overlapping with a double-stranded region formed by the first and second strands. The "off-target" profile of the dsRNA provided herein is quantified by determining the number of non-target genes having reduced expression levels in the presence of the candidate dsRNAs. The existence of "off target" binding indicates a dsRNA is capable of specifically binding to one or 20 more non-target gene messages. In certain embodiments, a dsRNA as provided herein (see, e.g., sequences in the Sequence Listing identified herein) applicable to therapeutic use will exhibit a greater stability, minimal interferon response, and little or no "off target" binding. Still further embodiments provide methods for selecting more efficacious dsRNA 25 by using one or more reporter gene constructs comprising a constitutive promoter, such as a cytomegalovirus (CMV) or phosphoglycerate kinase (PGK) promoter, operably fused to, and capable of altering the expression of one or more reporter genes, such as a luciferase, chloramphenicol (CAT), or p-galactosidase, which, in turn, is operably fused in-frame with a dsRNA (such as one having a length between about 15 base-pairs and 30 about 40 base-pairs or from about 5 nucleotides to about 24 nucleotides, or about 25 nucleotides to about 40 nucleotides) that contains a target sequence, as provided herein. Individual reporter gene expression constructs may be co-transfected with one or more dsRNA or analog thereof. The capacity of a given dsRNA to reduce the expression 77 Attorney Docket No. U8-U9PCI Customer No.: 36,814 level of target may be determined by comparing the measured reporter gene activity in cells transfected with or without a dsRNA molecule of interest. Certain embodiments disclosed herein provide methods for selecting one or more modified dsRNA molecule(s) that employ the step of predicting the stability of a dsRNA 5 duplex. In some embodiments, such a prediction is achieved by employing a theoretical melting curve wherein a higher theoretical melting curve indicates an increase in dsRNA duplex stability and a concomitant decrease in cytotoxic effects. Alternatively, stability of a dsRNA duplex may be determined empirically by measuring the hybridization of a single RNA analog strand as described herein to a complementary target gene within, for 10 example, a polynucleotide array. The melting temperature (i.e., the T, value) for each modified RNA and complementary RNA immobilized on the array can be determined and, from this Tm value, the relative stability of the modified RNA pairing with a complementary RNA molecule determined. For example, Kawase et al. (Nucleic Acids Res. 14:7727, 1986) have described an 15 analysis of the nucleotide-pairing properties of Di (inosine) to A, C, G , and T, which was achieved by measuring the hybridization of oligonucleotides (ODNs) with Di in various positions to complementary sets of ODNs made as an array. The relative strength of nucleotide-pairing is I-C > I-A > I-G ~ I-T. Generally, Di containing duplexes showed lower Tm values when compared to the corresponding wild type (WT) nucleotide pair. 20 The stabilization of Di by pairing was in order of Dc > Da > Dg > Dt > Du. As a person of skill in the art would understand, although universal-binding nucleotides are used herein as an example of determining duplex stability (i.e., the Tm value), other nucleotide substitutions (e.g., 5-methyluridine for uridine) or further modifications (e.g., a ribose modification at the 2'-position) can also be evaluated by these or similar methods. 25 In still further embodiments of the presently disclosed methods, one or more anti-codon within an antisense strand of a dsRNA molecule or analog thereof is substituted with a universal-binding nucleotide in a second or third position in the anti-codon of the antisense strand. By substituting a universal-binding nucleotide for a first or second position, the one or more first or second position nucleotide-pair 30 substitution allows the substituted dsRNA molecule to specifically bind to mRNA wherein a first or a second position nucleotide-pair substitution has occurred, wherein the one or more nucleotide-pair substitution results in an amino acid change in the corresponding gene product. 78 Attorney UocKet No. U0-U9PCTI Customer No.: 36,814 Any of these methods of identifying dsRNA of interest can also be used to examine a dsRNA that decreases expression of a target gene by RNA interference, comprising a first strand that is complementary to a target T mRNA and a second and third strand that have non-overlapping complementarity to the first strand, wherein the 5 first and at least one of the second or third strand form a double-stranded region of about 5 to about 13 base pairs; wherein at least one pyrimidine of the dsRNA comprises a pyrimidine nucleoside according to Formula I or II: R 0 O RI NH 2 6 3NH N (I) R 4 5' N R (R5 N 4' 'R R 8 3' 2' R3 R2 R3 R2 wherein R 1 and R 2 are each independently a -H, -OH, -OCH 3 , -OCH 2

OCH

2

CH

3 , 10 -OCH 2

CH

2 0CH 3 , halogen, substituted or unsubstituted C 1 -Cio alkyl, alkoxy, alkoxyalkyl, hydroxyalkyl, carboxyalkyl, alkylsulfonylamino, aminoalkyl, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, substituted or unsubstituted C 2

-C

10 alkenyl, substituted or unsubstituted -0-allyl, -O-CH 2

CH=CH

2 , -0-CH=CHCH 3 , substituted or unsubstituted C 2 -Cio alkynyl, 15 carbamoyl, carbamyl, carboxy, carbonylamino, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, -NH 2 , -NO 2 , -C-N, or heterocyclo group; R 3 and R 4 are each independently a hydroxyl, a protected hydroxyl, or an internucleoside linking group; and R 5 and R 8 are independently 0 or S. In certain embodiments, at least one nucleoside is according to Formula I in which R 1 is methyl and R 2 is -OH, or R1 is 20 methyl, R2 is -OH, and R 8 is S. In certain embodiments, at least one nucleoside is according to Formula I in which R' is methyl and R 2 is -0-methyl, or R' is methyl, R 2 is -0-methyl, and R 8 is 0. In other embodiments, the internucleoside linking group covalently links from about 5 to about 40 nucleosides. 79 Attorney Docket No. U-09FUF Customer No.: 36,814 Compositions and Methods of Use As set forth herein, dsRNA of the instant disclosure are designed to target a target gene (including one or more mRNA splice variant thereof) that is expressed at an elevated level or continues to be expressed when it should not, and is a causal or contributing 5 factor associated with, for example, atherosclerosis, diabetes mellitus, and cerebrovascular disease, state, or adverse condition. In this context, a dsRNA or analog thereof of this disclosure will effectively downregulate expression of a target gene to levels that prevent, alleviate, or reduce the severity or recurrence of one or more associated disease symptoms. Alternatively, for various distinct disease models in which 10 expression of a target gene is not necessarily elevated as a consequence or sequel of disease or other adverse condition, down regulation of a target gene will nonetheless result in a therapeutic result by lowering gene expression (i.e., to reduce levels of a selected mRNA or protein product of a target gene). Furthermore, dsRNAs of this disclosure may be targeted to lower expression of target, which can result in upregulation 15 of a "downstream" gene whose expression is negatively regulated, directly or indirectly, by a target protein. The dsRNA molecules of the instant disclosure comprise useful reagents and can be used in methods for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications. In certain embodiments, aqueous suspensions contain dsRNA of this disclosure in 20 admixture with suitable excipients, such as suspending agents or dispersing or wetting agents. Exemplary suspending agents include sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia. Representative dispersing or wetting agents include naturally-occurring phosphatides (e.g., lecithin), condensation products of an alkylene 25 oxide with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., 30 polyethylene sorbitan monooleate). In certain embodiments, the aqueous suspensions can optionally contain one or more preservatives (e.g., ethyl or n-propyl-p-hydroxybenzoate), one or more coloring agents, one or more flavoring agents, or one or more sweetening agents (e.g., sucrose, saccharin). In additional embodiments, dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water 80 Auorney VOCKet iNo. u6-uvru i Customer No.: 36,814 provide dsRNA of this disclosure in admixture with a dispersing or wetting agent, suspending agent and optionally one or more preservative, coloring agent, flavoring agent, or sweetening agent. The present disclosure includes dsRNA compositions prepared for storage or 5 administration that include a pharmaceutically effective amount of a desired compound in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., A.R. Gennaro edit., 1985, hereby incorporated by reference herein. In certain embodiments, pharmaceutical 10 compositions of this disclosure can optionally include preservatives, antioxidants, stabilizers, dyes, flavoring agents, or any combination thereof. Exemplary preservatives include sodium benzoate, sorbic acid, chlorobutanol, and esters of p-hydroxybenzoic acid. The dsRNA compositions of the instant disclosure can be effectively employed as pharmaceutically-acceptable formulations. Pharmaceutically-acceptable formulations 15 prevent, alter the occurrence or severity of, or treat (alleviate one or more symptom(s) to a detectable or measurable extent) of a disease state or other adverse condition in a subject. A pharmaceutically acceptable formulation includes salts of the above compounds, e.g., acid addition salts, such as salts of hydrochloric acid, hydrobromic acid, acetic acid, or benzene sulfonic acid. A pharmaceutical composition or formulation refers 20 to a composition or formulation in a form suitable for administration into a cell, or a subject such as a human (e.g., systemic administration). The formulations of the present disclosure, having an amount of dsRNA sufficient to treat or prevent a disorder associated with target gene expression are, for example, suitable for topical (e.g., creams, ointments, skin patches, eye drops, ear drops) application or administration. Other routes of 25 administration include oral, parenteral, sublingual, bladder wash-out, vaginal, rectal, enteric, suppository, nasal, and inhalation. The term parenteral, as used herein, includes subcutaneous, intravenous, intramuscular, intraarterial, intraabdominal, intraperitoneal, intraarticular, intraocular or retrobulbar, intraaural, intrathecal, intracavitary, intracelial, intraspinal, intrapulmonary or transpulmonary, intrasynovial, and intraurethral injection 30 or infusion techniques. The pharmaceutical compositions of the present disclosure are formulated to allow the dsRNA contained therein to be bioavailable upon administration to a subject. In further embodiments, dsRNA of this disclosure can be formulated as oily suspensions or emulsions (e.g., oil-in-water) by suspending dsRNA in, for example, a 81 Attomey 1)OcKet No. Uz-U91u i Customer No.: 36,814 vegetable oil (e.g., arachis oil, olive oil, sesame oil or coconut oil) or a mineral oil (e.g., liquid paraffin). Suitable emulsifying agents can be naturally-occurring gums (e.g., gum acacia or gum tragacanth), naturally-occurring phosphatides (e.g., soy bean, lecithin, esters or partial esters derived from fatty acids and hexitol), anhydrides (e.g., sorbitan 5 monooleate), or condensation products of partial esters with ethylene oxide (e.g., polyoxyethylene sorbitan monooleate). In certain embodiments, the oily suspensions or emulsions can optionally contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. In related embodiments, sweetening agents and flavoring agents can optionally be added to provide palatable oral preparations. In yet other embodiments, 10 these compositions can be preserved by optionally adding an anti-oxidant, such as ascorbic acid. In further embodiments, dsRNA of this disclosure can be formulated as syrups and elixirs with sweetening agents (e.g., glycerol, propylene glycol, sorbitol, glucose or sucrose). Such formulations can also contain a demulcent, preservative, flavoring, 15 coloring agent, or any combination thereof. In other embodiments, pharmaceutical compositions comprising dsRNA of this disclosure can be in the form of a sterile, injectable aqueous or oleaginous suspension. The sterile injectable preparation can also be a sterile, injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (e.g., as a solution in 1,3-butanediol). Among the exemplary acceptable 20 vehicles and solvents useful in the compositions of this disclosure is water, Ringer's solution, or isotonic sodium chloride solution. In addition, sterile, fixed oils may be employed as a solvent or suspending medium for the dsRNA of this disclosure. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of parenteral 25 formulations. Within certain embodiments of this disclosure, pharmaceutical compositions and methods are provided that feature the presence or administration of one or more dsRNA or analogs thereof of this disclosure, combined, complexed, or conjugated with a polypeptide, optionally formulated with a pharmaceutically-acceptable carrier, such as a 30 diluent, stabilizer, buffer, or the like. The negatively charged dsRNA molecules of this disclosure may be administered to a patient by any standard means, with or without stabilizers, buffers, or the like, to form a composition suitable for treatment. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present disclosure may also be 82 Attorney Docket No. 08-09PCT Customer No.: 36,814 formulated and used as a tablet, capsule or elixir for oral administration, suppository for rectal administration, sterile solution, or suspension for injectable administration, either with or without other compounds known in the art. Thus, dsRNAs of the present disclosure may be administered in any form, such as nasally, transdermally, parenterally, 5 or by local injection. In accordance with this disclosure, dsRNA molecules (optionally substituted or modified or conjugated), compositions thereof, and methods for inhibiting expression of a target gene in a cell or organism are provided. In certain embodiments, this disclosure provides methods and dsRNA compositions for treating a subject, including a human cell, 10 tissue or individual, having a disease or at risk of developing a disease caused by or associated with the expression of a target gene. In one embodiment, the method includes administering a dsRNA of this disclosure or a pharmaceutical composition containing the dsRNA to a cell or an organism, such as a mammal, such that expression of the target gene is silenced. Subjects (e.g., mammalian, human) amendable for treatment using the 15 dsRNA molecules (optionally substituted or modified or conjugated), compositions thereof, and methods of the present disclosure include those suffering from one or more disease or condition mediated, at least in part, by overexpression or inappropriate expression of a target gene, or which are amenable to treatment by reducing expression of a target protein, including coronary artery disease (i.e., coronary heart disease, ischaemic 20 heart disease), atherosclerosis, diabetes mellitus, dyslipidemia (e.g., hyperlipidemia), peripheral vascular and ischemic cerebrovascular disease, and risk of ischemic stroke (cerebral thrombosis and cerebral embolisms) and hemorrhagic stroke (cerebral hemorrhage and subarachnoid hemorrhage), cancer (e.g., lung cancer, heptacellular carcinoma, bladder cancer, and pancreatic cancer) . Within exemplary embodiments, the 25 compositions and methods of this disclosure are also useful as therapeutic tools to regulate expression of target to treat or prevent symptoms of, for example, the conditions listed herein. In any of the methods disclosed herein there may be used with one or more dsRNA, or substituted or modified dsRNA, as described herein, comprising a first strand 30 that is complementary to a human target mRNA and a second strand and a third strand that is each complementary to non-overlapping regions of the first strand, wherein the second strand and third strands can anneal with the first strand to form at least two double-stranded regions spaced apart by up to 10 nucleotides and thereby forming a gap between the second and third strands, and wherein the mdRNA molecule optionally 83 Attorney Docket No. 08-09PCT Customer No.: 36,814 includes at least one double-stranded region of 5 base pairs to 13 base pairs. In other embodiments, subjects can be effectively treated, prophylactically or therapeutically, by administering an effective amount of one or more dsRNA having a first strand that is complementary to a human target mRNA and a second strand and a third strand that is 5 each complementary to non-overlapping regions of the first strand, wherein the second strand and third strands can anneal with the first strand to form at least two double stranded regions spaced apart by up to 10 nucleotides and thereby forming a gap between the second and third strands, and wherein the mdRNA molecule optionally includes at least one double-stranded region of 5 base pairs to 13 base pairs and at least one 10 pyrimidine of the mdRNA is substituted with a pyrimidine nucleoside according to Formula I or II: R1 O RI NH 2 6 3NH (I) R 4 ' R5 2 R4 5 N 4' 1' R8 R8 3' 2'

R

3 R R wherein R' and R 2 are each independently a -H, -OH, -OCH 3 , -OCH 2 0CH 2

CH

3 ,

-OCH

2

CH

2 0CH 3 , halogen, substituted or unsubstituted C 1 -Cio alkyl, alkoxy, alkoxyalkyl, 15 hydroxyalkyl, carboxyalkyl, alkylsulfonylamino, aminoalkyl, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, substituted or unsubstituted C 2 -Cio alkenyl, substituted or unsubstituted -0-allyl, -O-CH 2

CH=CH

2 , -O-CH=CHCH 3 , substituted or unsubstituted C 2

-C

10 alkynyl, carbamoyl, carbamyl, carboxy, carbonylamino, substituted or unsubstituted aryl, 20 substituted or unsubstituted aralkyl, -NH 2 , -NO 2 , -C=N, or heterocyclo group; R 3 and R4 are each independently a hydroxyl, a protected hydroxyl, or an internucleoside linking group; and R 5 and R8 are independently 0 or S. In certain embodiments, at least one nucleoside is according to Formula I in which R 1 is methyl and R 2 is -OH, or R1 is methyl, R 2 is -OH, and R 8 is S. In other embodiments, the internucleoside linking group 25 covalently links from about 5 to about 40 nucleosides. 84 Attorney pocket No. us-uv1' i Customer No.: 36,814 In any of the methods described herein, the dsRNA used may include multiple modifications. For example, a dsRNA can have at least one 5-methyluridine, 2'-0 methyl-5-methyluridine, LNA, 2'-methoxy, 2'-fluoro, 2'-deoxy, phosphorothioate linkage, inverted base terminal cap, or any combination thereof. In certain exemplary methods, a 5 dsRNA will have from one to all 5-methyluridines and have up to about 75% LNA. In other exemplary methods, a dsRNA will have from one to all 5-methyluridines and have up to about 75% 2'-methoxy provided the 2'-methoxy are not at the Argonaute cleavage site. In still other exemplary methods, a dsRNA will have from one to all 5 methyluridines and have up to about 100% 2'-fluoro substitutions. In further exemplary 10 methods, a dsRNA will have from one to all 5-methyluridines and have up to about 75% 2'-deoxy. In further exemplary methods, a dsRNA will have up to about 75% LNA and have up to about 75% 2'-methoxy. In still other embodiments, a dsRNA will have up to about 75% LNA and have up to about 100% 2'-fluoro. In further exemplary methods, a dsRNA will have up to about 75% LNA and have up to about 75% 2'-deoxy. In further 15 exemplary methods, a dsRNA will have up to about 75% 2'-methoxy and have up to about 100% 2'-fluoro. In further exemplary methods, a dsRNA will have up to about 75% 2'-methoxy and have up to about 75% 2'-deoxy. In further embodiments, a dsRNA will have up to about 100% 2'-fluoro and have up to about 75% 2'-deoxy. In other exemplary methods for using multiply modified dsRNA, a dsRNA will 20 have from one to all uridines substituted with 5-methyluridine, up to about 75% LNA, and up to about 75% 2'-methoxy. In still further exemplary methods, a dsRNA will have from one to all 5-methyluridines, up to about 75% LNA, and up to about 100% 2'-fluoro. In further exemplary methods, a dsRNA will have from one to all 5-methyluridines, up to about 75% LNA, and up to about 75% 2'-deoxy. In further exemplary methods, a dsRNA 25 will have from one to all 5-methyluridines, up to about 75% 2'-methoxy, and up to about 75% 2'-fluoro. In further exemplary methods, a dsRNA will have from one to all 5 methyluridines, up to about 75% 2'-methoxy, and up to about 75% 2'-deoxy. In more exemplary methods, a dsRNA will have from one to all 5-methyluridines, up to about 100% 2'-fluoro, and up to about 75% 2'-deoxy. In yet other exemplary methods, a 30 dsRNA will have from one to all 5-methyluridines, up to about 75% LNA, up to about 75% 2'-methoxy, up to about 100% 2'-fluoro, and up to about 75% 2'-deoxy. In other exemplary methods, a dsRNA will have up to about 75% LNA, up to about 75% 2'-methoxy, and up to about 100% 2'-fluoro. In further exemplary methods, a dsRNA will have up to about 75% LNA, up to about 75% 2'-methoxy, and up to about 75% 85 Attorney Docket No. 08-09PCT Customer No.: 36,814 2'-deoxy. In more exemplary methods, a dsRNA will have up to about 75% LNA, up to about 100% 2'-fluoro, and up to about 75% 2'-deoxy. In still further exemplary methods, a dsRNA will have up to about 75% 2'-methoxy, up to about 100% 2'-fluoro, and up to about 75% 2'-deoxy. 5 In any of these exemplary methods for using multiply modified dsRNA, the dsRNA may further comprise up to 100% phosphorothioate internucleoside linkages, from one to ten or more inverted base terminal caps, or any combination thereof. Additionally, any of these dsRNA may have these multiple modifications on one strand, two strands, three strands, a plurality of strands, or all strands, or on the same or different 10 nucleoside within a dsRNA molecule. Finally, in any of these multiple modification dsRNA, the dsRNA must have gene silencing activity. In further embodiments, subjects can be effectively treated, prophylactically or therapeutically, by administering an effective amount of one or more dsRNA, or substituted or modified dsRNA as described herein, having a first strand that is complementary to a 15 target mRNA and a second strand and a third strand that is each complementary to non-overlapping regions of the first strand, wherein the second strand and third strands can anneal with the first strand to form at least two double-stranded regions spaced apart by up to 10 nucleotides and thereby forming a gap between the second and third strands, and wherein the combined double-stranded regions total about 15 base pairs to about 20 40 base pairs and the mdRNA molecule optinally has blunt ends. In still further embodiments, methods disclosed herein there may be used with one or more dsRNA that comprises a first strand that is complementary to a target mRNA and a second strand and a third strand that is each complementary to non-overlapping regions of the first strand, wherein the second strand and third strands can anneal with the first strand to form at 25 least two double-stranded regions spaced apart by up to 10 nucleotides and thereby forming a gap between the second and third strands, and wherein the mdRNA molecule optionally includes at least one double-stranded region of 5 base pairs to 13 base pairs, the mdRNA molecule optinally has blunt ends, and at least one pyrimidine of the mdRNA is substituted with a pyrimidine nucleoside according to Formula I or II: 30 R O R1 NH 2 6 3NH N (I) R 4 5 R 5 NRR5 N 4' R 8

R

8 3' 2' 86 Attorney Docket No. 08-U9PCT Customer No.: 36,814 wherein R1 and R 2 are each independently a -H, -OH, -OCH 3 , -OCH 2

OCH

2

CH

3 ,

-OCH

2

CH

2 0CH 3 , halogen, substituted or unsubstituted C1-C 10 alkyl, alkoxy, alkoxyalkyl, hydroxyalkyl, carboxyalkyl, alkylsulfonylamino, aminoalkyl, dialkylamino, 5 alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, substituted or unsubstituted C 2 -C1o alkenyl, substituted or unsubstituted -0-allyl, -O-CH 2

CH=CH

2 , -O-CH=CHCH 3 , substituted or unsubstituted C 2 -C1o alkynyl, carbamoyl, carbamyl, carboxy, carbonylamino, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, -NH 2 , -NO 2 , -C-N, or heterocyclo group; R 3 and R 4 10 are each independently a hydroxyl, a protected hydroxyl, or an internucleoside linking group; and R 5 and R 8 are independently 0 or S. In certain embodiments, at least one nucleoside is according to Formula I in which R' is methyl and R 2 is -OH, or R1 is methyl, R 2 is -OH, and R 8 is S. In certain embodiments, at least one nucleoside is according to Formula I in which R' is methyl and R 2 is -0-methyl, or R 1 is methyl, R 2 is 15 0-methyl, and R 8 is 0. In other embodiments, the internucleoside linking group covalently links from about 5 to about 40 nucleosides. Within additional aspects of this disclosure, combination formulations and methods are provided comprising an effective amount of one or more dsRNA of the present disclosure in combination with one or more secondary or adjunctive active agents that are 20 formulated together or administered coordinately with the dsRNA of this disclosure to control a target gene-associated disease or condition. Useful adjunctive therapeutic agents in these combinatorial formulations and coordinate treatment methods include, for example, dsRNAs that target and decrease the expression of other genes whose abbarent expression is related to a disease or condition described herein (e.g., bladder cancer 25 and/liver cancer), enzymatic nucleic acid molecules, allosteric nucleic acid molecules, antisense, decoy, or aptamer nucleic acid molecules, antibodies such as monoclonal antibodies, small molecules and other organic or inorganic compounds including metals, salts and ions, and other drugs and active agents indicated for treating a target gene 87 Attorney Docket No. 08-09PCT Customer No.: 36,814 associated disease or condition, including chemotherapeutic agents used to treat cancer, steroids, non-steroidal anti-inflammatory drugs (NSAIDs), tyrosine kinase inhibitors, or the like. Exemplary chemotherapeutic agents include alkylating agents (e.g., cisplatin, 5 oxaliplatin, carboplatin, busulfan, nitrosoureas, nitrogen mustards, uramustine, temozolomide), antimetabolites (e.g., aminopterin, methotrexate, mercaptopurine, fluorouracil, cytarabine), taxanes (e.g., paclitaxel, docetaxel), anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idaruicin, mitoxantrone, valrubicin), bleomycin, mytomycin, actinomycin, hydroxyurea, topoisomerase inhibitors (e.g., camptothecin, 10 topotecan, irinotecan, etoposide, teniposide), monoclonal antibodies (e.g., alemtuzumab, bevacizumab, cetuximab, gemtuzumab, panitumumab, rituximab, tositumomab, trastuzumab), vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinorelbine), cyclophosphamide, prednisone, leucovorin, oxaliplatin. Some adjunctive therapies may be directed at targets that interact or associate with 15 the target gene or affect specific target gene biological activities. Adjunctive therapies include statins (e.g., rosuvastatin, lovastatin, atorvastatin, cerivastatin, fluvastatin, mevastatin, pitavastatin, pravastatin, simvastatin), bile acid-binding resins, stanol and sterol esters from plants, and inhibitors of cholesterol absorption, fibrates (e.g., fenofibrate, bezafibrate, ciprofibrate, clofibrate, gemfibrozil), niacin, fish-oils, ezetimibe, 20 amlodipine, other lipid-altering agents, additional small molecules, rationally designed peptides, and antibodies or fragments thereof. Genes may be targeted via the RNAi pathway by way of a dsRNA and used in combination with two or more dsRNAs of this disclosure To practice the coordinate administration methods of this disclosure, a dsRNA is 25 administered, simultaneously or sequentially, in a coordinated treatment protocol with one or more of the secondary or adjunctive therapeutic agents contemplated herein. The coordinate administration may be done in any order, and there may be a time period while only one or both (or all) active therapeutic agents, individually or collectively, exert their biological activities. A distinguishing aspect of all such coordinate treatment methods is 30 that the dsRNA present in a composition elicits some favorable clinical response, which may or may not be in conjunction with a secondary clinical response provided by the secondary therapeutic agent. For example, the coordinate administration of the dsRNA with a secondary therapeutic agent as contemplated herein can yield an enhanced 88 Attorney vocKet No. uzs-uvru i Customer No.: 36,814 (synergistic) therapeutic response beyond the therapeutic response elicited by either or both the purified dsRNA or secondary therapeutic agent alone. In another embodiment, a dsRNA of this disclosure can include a conjugate member on one or more of the terminal nucleotides of a dsRNA. The conjugate member 5 can be, for example, a lipophile, a terpene, a protein binding agent, a vitamin, a carbohydrate, or a peptide. For example, the conjugate member can be naproxen, nitroindole (or another conjugate that contributes to stacking interactions), folate, ibuprofen, or a C5 pyrimidine linker. In other embodiments, the conjugate member is a glyceride lipid conjugate (e.g., a dialkyl glyceride derivatives), vitamin E conjugates, or 10 thio-cholesterols. Additional conjugate members include peptides that function, when conjugated to a modified dsRNA of this disclosure, to facilitate delivery of the dsRNA into a target cell, or otherwise enhance delivery, stability, or activity of the dsRNA when contacted with a biological sample (e.g., a target cell expressing the target gene). Exemplary peptide conjugate members for use within these aspects of this disclosure, 15 include peptides PN27, PN28, PN29, PN58, PN61, PN73, PN1 58, PN159, PN173, PN182, PN183, PN202, PN204, PN250, PN361, PN365, PN404, PN453, PN509, and PN963, described, for example, in U.S. Patent Application Publication Nos. 2006/0040882 and 2006/0014289, and U.S. Provisional Patent Application Nos. 60/822,896 and 60/939,578; and PCT Application PCT/US2007/075744, which are all 20 incorporated herein by reference. In certain embodiments, when peptide conjugate partners are used to enhance delivery of dsRNA of this disclosure, the resulting dsRNA formulations and methods will often exhibit further reduction of an interferon response in target cells as compared to dsRNAs delivered in combination with alternate delivery vehicles, such as lipid delivery vehicles (e.g., LipofectamineTM). 25 In still another embodiment, a dsRNA or analog thereof of this disclosure may be conjugated to the polypeptide and admixed with one or more non-cationic lipids or a combination of a non-cationic lipid and a cationic lipid to form a composition that enhances intracellular delivery of the dsRNA as compared to delivery resulting from contacting the target cells with a naked dsRNA. In more detailed aspects of this 30 disclosure, the mixture, complex or conjugate comprising a dsRNA and a polypeptide can be optionally combined with (e.g., admixed or complexed with) a cationic lipid, such as Lipofectine T. To produce these compositions comprised of a polypeptide, dsRNA and a cationic lipid, the dsRNA and peptide may be mixed together first in a suitable medium such as a cell culture medium, after which the cationic lipid is added to the mixture to 89 Attorney Docket No. 08-09PCT Customer No.: 36,814 form a dsRNA/delivery peptide/cationic lipid composition. Optionally, the peptide and cationic lipid can be mixed together first in a suitable medium such as a cell culture medium, followed by the addition of the dsRNA to form the dsRNA/delivery peptide/cationic lipid composition. 5 This disclosure also features the use of dsRNA compositions comprising surface-modified liposomes containing, for example, poly(ethylene glycol) lipids (PEG modified, or long-circulating liposomes or stealth liposomes) (Lasic et al., Chem. Rev. 95:2601, 1995; Ishiwata et al., Chem. Pharm. Bull. 43:1005, 1995; Lasic et al., Science 267:1275, 1995; Oku et al., Biochim. Biophys. Acta 1238:86, 1995; Liu et al., J. Biol. 10 Chem. 42:24864, 1995; PCT Publication Nos. WO 96/10391; WO 96/10390; WO 96/10392). In another embodiment, compositions are provided for targeting dsRNA molecules of this disclosure to specific cell types, such as hepatocytes. For example, dsRNA can be complexed or conjugated glycoproteins or synthetic glycoconjugates 15 glycoproteins or synthetic glycoconjugates having branched galactose (e.g., asialoorosomucoid), N-acetyl-D-galactosamine, or mannose (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429, 1987; Baenziger and Fiete, Cell 22: 611, 1980; Connolly et al., J. Biol. Chem. 257:939, 1982; Lee and Lee, Glycoconjugate J. 4:317, 1987; Ponpipom et al., J. Med. Chem. 24:1388, 1981) for a targeted delivery to, for example, the liver. 20 A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence of, or treat (alleviate a symptom to some extent, preferably all of the symptoms) a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of subject being treated, the physical characteristics of the specific subject under consideration for 25 treatment, concurrent medication, and other factors that those skilled in the medical arts will recognize. For example, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients may be administered depending on the potency of a dsRNA of this disclosure. A specific dose level for any particular patient depends upon a variety of factors 30 including the activity of the specific compound employed, age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy. Following administration of dsRNA compositions as disclosed herein, test subjects will exhibit about a 10% up to about a 99% reduction in one or more symptoms associated with the 90 Attorney DoceKe iNo. u6-uyr' i Customer No.: 36,814 disease or disorder being treated, as compared to placebo-treated or other suitable control subjects. Dosage levels in the order of about 0.1 mg to about 140 mg per kilogram of body weight per day can be useful in the treatment of the above-indicated conditions (about 5 0.5 mg to about 7 g per patient per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient. A dosage form of a dsRNA or composition thereof of this disclosure can be liquid, 10 an emulsion, or a micelle, or in the form of an aerosol or droplets. A dosage form of a dsRNA or composition thereof of this disclosure can be solid, which can be reconstituted in a liquid prior to administration. The solid can be administered as a powder. The solid can be in the form of a capsule, tablet, or gel. In addition to in vivo gene inhibition, a skilled artisan will appreciate that the dsRNA and analogs thereof of the present 15 disclosure are useful in a wide variety of in vitro applications, such as scientific and commercial research (e.g., elucidation of physiological pathways, drug discovery and development), and medical and veterinary diagnostics. Nucleic acid molecules and polypeptides can be administered to cells by a variety of methods known to those of skill in the art, including administration within 20 formulations that comprise a dsRNA alone, a dsRNA and a polypeptide complex / conjugate alone, or that further comprise one or more additional components, such as a pharmaceutically acceptable carrier, diluent, excipient, adjuvant, emulsifier, stabilizer, preservative, or the like. Other exemplary substances used to approximate physiological conditions include pH adjusting and buffering agents, tonicity adjusting agents, and 25 wetting agents, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and mixtures thereof For solid compositions, conventional nontoxic pharmaceutically acceptable carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, 30 sucrose, magnesium carbonate, and the like. In certain embodiments, the dsRNA and compositions thereof can be encapsulated in liposomes, administered by iontophoresis, or incorporated into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors (see, e.g., PCT Publication No. WO 00/53722). In certain 91 Attorney DoCket No. U0-U9PCT Customer No.: 36,814 embodiments of this disclosure, the dsRNA may be administered in a time release formulation, for example, in a composition that includes a slow release polymer. The dsRNA can be prepared with carriers that will protect against rapid release, for example, a controlled release vehicle such as a polymer, microencapsulated delivery system, or 5 bioadhesive gel. Prolonged delivery of the dsRNA, in various compositions of this disclosure can be brought about by including in the composition agents that delay absorption, for example, aluminum monosterate hydrogels and gelatin. Alternatively, a dsRNA composition of this disclosure can be locally delivered by direct injection or by use of, for example, an infusion pump. Direct injection of dsRNAs 10 of this disclosure, whether subcutaneous, intramuscular, or intradermal, can be done by using standard needle and syringe methodologies or by needle-free technologies, such as those described in Conry et al., Clin. Cancer Res. 5:2330, 1999 and PCT Publication No. WO 99/31262. The dsRNA of this disclosure and compositions thereof may be administered to 15 subjects by a variety of mucosal administration modes, including oral, rectal, vaginal, intranasal, intrapulmonary, or transdermal delivery, or by topical delivery to the eyes, ears, skin, or other mucosal surfaces. In one embodiment, the mucosal tissue layer includes an epithelial cell layer, which can be pulmonary, tracheal, bronchial, alveolar, nasal, buccal, epidermal, or gastrointestinal. Compositions of this disclosure can be 20 administered using conventional actuators, such as mechanical spray devices, as well as pressurized, electrically activated, or other types of actuators. The dsRNAs can also be administered in the form of suppositories, e.g., for rectal administration. For example, these compositions can be mixed with an excipient that is solid at room temperature but liquid at the rectal temperature so that the dsRNA is released. Such materials include, for 25 example, cocoa butter and polyethylene glycols. Further methods for delivery of nucleic acid molecules, such as the dsRNAs of this disclosure, are described, for example, in Boado et al., J. Pharm. Sci. 87:1308, 1998; Tyler et al., FEBS Lett. 421:280, 1999; Pardridge et al., Proc. Nat'l Acad. Sci. USA 92:5592, 1995; Boado, Adv. Drug Delivery Rev. 15:73, 1995; Aldrian-Herrada et al., 30 Nucleic Acids Res. 26:4910, 1998; Tyler et al., Proc. Nat'l Acad. Sci. USA 96:7053-7058, 1999; Akhtar et al., Trends Cell Bio. 2:139, 1992; "Delivery Strategies for Antisense Oligonucleotide Therapeutics," ed. Akhtar, 1995, Maurer et al., Mol. Membr. Biol. 16:129, 1999; Hofland and Huang, Handb. Exp. Pharmacol 13 7:165, 1999; and Lee et al., A CS Symp. Ser. 752:184, 2000; PCT Publication No. WO 94/02595. 92 Attorney Docket No. 08-09PCT Customer No.: 36,814 All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, non-patent publications, figures, tables, and websites referred to in this specification are expressly incorporated herein by reference, in their entirety. 93 Attorney Docket No. 08-09PCT Customer No.: 36,814 EXAMPLES EXAMPLE 1 KNOCKDOWN OF GENE EXPRESSION BY MDRNA The gene silencing activity of dsRNA as compared to nicked or gapped versions 5 of the same dsRNA was examined using a dual fluorescence assay. A total of 22 different genes were targeted at ten different sites each (see Table 1). A Dicer substrate dsRNA molecule was used, which has a 25 nucleotide sense strand, a 27 nucleotide antisense strand, and a two deoxynucleotide overhang at the 3'-end of the antisense strand (referred to as a 25/27 dsRNA). The nicked version of each 10 dsRNA Dicer substrate has a nick at one of positions 9 to 16 on the sense strand as measured from the 5'-end of the sense strand. For example, an ndsRNA having a nick at position 11 has three strands - a 5'-sense strand of 11 nucleotides, a 3'-sense strand of 14 nucleotides, and an antisense strand of 27 nucleotides (which is also referred to as an N1 1-14/27 mdRNA). In addition, each of the sense strands of the ndsRNA have three 15 locked nucleic acids (LNAs) evenly distributed along each sense fragment. If the nick is at position 9, then the LNAs can be found at positions 2, 6, and 9 of the 5' sense strand fragment and at positions 11, 18, and 23 of the 3' sense strand fragment. If the nick is at position 10, then the LNAs can be found at positions 2, 6, and 10 of the 5' sense strand fragment and at positions 12, 18, and 23 of the 3' sense strand fragment. If the nick is at 20 position 11, then the LNAs can be found at positions 2, 6, and 11 of the 5' sense strand fragment and at positions 13, 18, and 23 of the 3' sense strand fragment. If the nick is at position 12, then the LNAs can be found at positions 2, 6, and 12 of the 5' sense strand fragment and at positions 14, 18, and 23 of the 3' sense strand fragment. If the nick is at position 13, then the LNAs can be found at positions 2, 7, and 13 of the 5' sense strand 25 fragment and at positions 15, 18, and 23 of the 3' sense strand fragment. If the nick is at position 14, then the LNAs can be found at positions 2, 7, and 14 of the 5' sense strand fragment and at positions 16, 18, and 23 of the 3' sense strand fragment. If the nick is at position 15, then the LNAs can be found at positions 2, 8, and 15 of the 5' sense strand fragment and at positions 17, 19, and 23 of the 3' sense strand fragment. If the nick is at 30 position 16, then the LNAs can be found at positions 2, 8, and 16 of the 5' sense strand fragment and at positions 18, 19, and 23 of the 3' sense strand fragment. Similarly, a gapped version of each dsRNA Dicer substrate has a single nucleotide missing at one of 94 Attorney Uocket No. UZ-U9C I Customer No.: 36,814 positions 10 to 17 on the sense strand as measured from the 5'-end of the sense strand. For example, a gdsRNA having a gap at position 11 has three strands - a 5'-sense strand of 11 nucleotides, a 3'-sense strand of 13 nucleotides, and an antisense strand of 27 nucleotides (which is also referred to as G1-(1)-13/27 mdRNA). In addition, each of the 5 sense strands of the gdsRNA contain three locked nucleic acids (LNAs) evenly distributed along each sense fragment (as described for the nicked counterparts). In sum, three dsRNA were tested at each of the ten different sites per gene - an unmodified dsRNA, a nicked mdRNA with three LNAs per sense strand fragment, and a single nucleotide gapped mdRNA with three LNAs per sense strand fragment. In other 10 words, 660 different dsRNA were examined. Briefly, multiwell plates were seeded with about 7-8 x 10 5 HeLa cells/well in DMEM having 10% fetal bovine serum, and incubated overnight at 37"C / 5% CO 2 . The HeLa cell medium was changed to serum-free DMEM just prior to transfection. The psiCHECKTM-2 vector, containing about a 1,000 basepair insert of a target gene, diluted 15 in serum-free DMEM was mixed with diluted GenJetT" transfection reagent (SignalDT Biosystems, Hayward, California) according to the manufacturer's instructions and then incubated at room temperature for 10 minutes. The GenJet/ psiCHECKTM_2_target gene solution was added to the HeLa cells and then incubated at 37*C, 5% CO 2 for 4.5 hours. After the vector transfection, cells were trypsinized and suspended in antibiotic-free 20 DMEM containing 10% FBS at a concentration of 105 cells per mL. To transfect the dsRNA, the dsRNA was formulated in OPTI-MEM I reduced serum medium (Gibco@ Invitrogen, Carlsbad, California) and placed in multiwell plates. Then Lipofectamine TM RNAiMAX (Invitrogen) was mixed with OPTI-MEM per manufacture's specifications, added to each well containing dsRNA, mixed manually, and 25 incubated at room temperature for 10-20 minutes. Then 30 pL of vector-transfected HeLa cells at 105 cells per mL were added to each well (final dsRNA concentration of 25 nM), the plates were spun for 30 seconds at 1,000 rpm, and then incubated at 37 0 C / 5%

CO

2 for 2 days. The Cell Titer Blue (CTB) reagent (Promega, Madison, Wisconson) was used to assay for cell viability and proliferation - none of the dsRNA showed any 30 substantial toxicity. After transfecting, the media and CTB reagent were removed and the wells washed once with 100 PBS. Cells were assayed for firefly and Renilla luciferase reporter activity by first adding Dual-GloTM Luciferase Reagent (Promega, Madison, WI) for 10 minutes with shaking, and then quantitating the luminescent signal on a VICTORTM 95 Attorney Docket No. US-U9PIT Customer No.: 36,814 1420 Multilabel Counter (PerkinElmer). After measuring the firefly luminescence, Stop & Glo* Reagent (Promega, Madison, WI) was added for 10 minutes with shaking to simultaneously quench the firefly reaction and initiate the Renilla luciferase reaction, which was then quantitated on a VICTOR 3 TM 1420 Multilabel Counter (PerkinElmer). 5 The results are presented in Table 1. 96 n I 4 It 'I N C 4 - C N kn It. m. I - eN M I Cl1 Cl4 Cl1 m~ m m ClON~. 0 N . ~r - - l C N Cl 0 0 0 - N 00O oR m - O o -o - - - - - - - - - - - - - - - - - - S'N N Cl o. ' ON o - rN '( (I (I oI '. N ' 00o C N oI 4 '.0 '( o' e '(n 0 ' ' N 0 ON 0-Cl I * 'o 0 N 0 O -Cl I * N '0 N 00 00000000000000 ON ON ON ON ON ON ON ON ON O 000000000 C C C oo i In oo'D i -4 m w oo o m to 'N 'n '.0 '.0 '.0 %0 '. ~ ~ ON ON ONN ON O ON O N O N O N O N O N O N O N O N O N O "R n qo OR OR Ni m -,* '.6 ,c r o6oR 5' c -~ c4 m4 ,, v){ '.6 N- 00 ON 0) tn cl t- oo( . 00 00 0 000-- - w oll 0* ON - al CD Cl CD C C) CD C> C> Cl 4 (N NN m o m m m m m ' ' .NO C 0 * Cl(10 C\ 01 0 0 a% l N N (I N 0 'N C(N N N CN . o - - - o6- 6 - o ( - - - - I S00000 ON ON ON ON ON ON ON O O tn~ oo" %C IC c r- 0 n C4. 6 r: .6i o - C l N l C Cl CD w %C C) a, CD w ( ' rj' Mj Cl Cl Cl en Cl Cl Cl C14 NI NI 'IT mI I ' ' I m oo o .- < - N rn <D \m ,* w) , - m 0%C NO n - 0 . N N N N N N N N N N N N N N N ZO , ~ 'N .0 N 00 ON 0 l 1010N '. 00 ON 0 - l (N 'N '01N00 r- .000 c-4 - - r- Cl ClC-lClCtlC Cl e0 : 00 V\ oD rn N '.0 M '0 I ON '.0 O r - 0 o N in ~~~ ~ ~ ~ r- m r 4 ; i 6O ms o m\ O' ~ N ' ~ N Cl r~ - I 0 .~~.~ -~( '0 WN & ON N . .N N .C '. ON '( o> a-, N) r-t r- Cl c9 c -t CDON 0N Cl ON '(N ON ON o 000 000 000 ONON4N N IN N N N M M ON O Cln Cl Cl Cl Cl Cl C CCl Cl Cl D Cl Cl C lCl CD W'N (IN ('N wI ' I I 00 (IN 00 'I 0 00 00 ON0 - Cl o N C7, (N 0.0 C\ 0 0 ON 0 5 - Cl C> c c '.0 N) 00, C' ~~ cl cq CA es oo^ q eq ~^ " 1 ' n M n c f \C '0 '.C \C0 '.0 r- r-- r- rt r' r0 rN N rN rNN 00 0000000000000000 m C% e -D C l ClC lClC)lC 0. ~ ~ ~ ,- N N N Cl CllCC "'l'C~ 7lClC C Cl o l C l Cs \ Cl td ( ' -q n1 4 m1 to -n m M e - C I (N '. N 0 N N N Cl (N N 'N '0 N 00 ON 0 l rN ( . d4 4 s - - - - - o - - - C os 'Ar s r s s a o o -~~ ~ ~ N " ts M ~t N ~' ' N N N, N N , - - - - - - - - - -J -N - - 0 00 - - Cl 06 IClC C)en N m N C I ,0N r 00 0 00 C4 \.6 00= Cl - 06 r.: C - \C ~ C4 O A Cl 00 C4 Cl tN 't C4 '. - ON 00 ~ . NN - l - C - Nl SIN "r c C4 MN '0 Nl N U~ON 0) - Clc - 'N '0 N 0 O l c N 00 ON 0) t- wl C c I N o Co - - - -- - - - - cl Nl Nl N Cl4 Nl Nl Nl ml mCl)e n. WSIC \C0 '.o \C0 '0 - N N - N r- N N - N N N 00 00 w 00 W0 W 00 W 00 00 (ON ON o% ON O N ON O N O ON O0N ON (ON all ON ON O7N O (ON ON ON 6.O o6 6' c c4 r, zff '.o R6 r-.7 ofo 6 r, 100 C.l 1.0 1.0 -. 0 Cl O (I 160 6 R 0 - C r, ~ O N 0 ~ N S NCC r0N 0 IN O Cl r) ONl 'I Ni 6 -r -6 \p -) w- C C -W)r C N 11 CD - -0 U~ ON - Cl ~ ~ ' N '.0 N 00 7N 0 N Cl "t / . 0 N 0 C I o :0 - - - - - - - - - - Nl N C4 Cl N Cl4 Cl Cl Ml Cl MI m/ en f fnf fn- M- c!N f/N "IN fN cN /n I CN '! c! f m c!N e/ n en c!N C/N CN &/N f/N f/N fN C/N 0/ /N fN tK 060 o c c n 4 i , - 6 a c r TC r- ,:N ux CD - m c'!l ' C! q OR M1 000 l/:0 - W CIN SN C 0 i SIN '0 Cl N - N O tn~ / C 0 0 0 - 0 0) C) Cl CD C) 't 4 Cli C/; Cl Cl- Ci 0.' ' CD 0 N m 0 Ct W '.0 1-, w. - -) \.0 Cl (. l '~'N 0 C> ~ 'r ' C:) - - -/ 0 - - - - u oo Ce f/N o/N C\ CN 0 C ON 0-C C/N CD CIN '0 N> 0 0> 0 0 0 0 0 - - - - - 00 C C14 -> SIN I ~ r I '0 0 N 00 00 1- SIN - SN 0: M/ 00 Cl Nt Nl-r4 00 Cl Cl C/ l C l N m / m F- N o '. 00 N tn W)*j / / I I SN (N '~0 N O - - - - o6ClC C Q_4 P4. e. P 40 4P40 4 - --- - - "4~ 44H H H H "44 - - F - - 0

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N 00 cN - w \c r- I/N '0 Nc0)N - c4 mN 'IT N W' .0 N- w ON O c \.0 '.0 '. \.'0 \.0 '.0 N- N- N- N- N N N N N N 00 00 00 00 00 00 00 00 00 00 C0 0 0 00C)C C D >- -- - - - - - N Nl NlCl Cl4 "l Nl N N 00000000000000 00000000000000 000000 = 00 000000mw0000 00 oo 00 00 00 00 00 00 0 , ON N ON ON ON ON ON ON ON ON 0. 0) 0 0 D 0> 0 D 0 0 C0 &n iN I/N I/N /N I/N I)N /N i/n /N I/n IN W/N I/N W)N W)N IN l '.C \.o 'D \0 '.0 \.O '.C '.0 '.C \.0 00 00 ON r'N- 0 r0 N - 00 NC> - C4 9, 00 r In I/n ON ' I N 00 ON'R ' r O (-j Cl 0 00 N- Ii nN cl l ON 4 ON 00 ON mN 00 - -wlIN ~~~~~ \- C 0- C C / N '0 ClN ON- I\ Cl - oz *N 't 4/ '0 N- 00 ON 0n - C VN W)N '.0 N ) \c ON 0 o - o Z I \N '.0 N 00 o u - - - - - - - - - - - - - - - - - - - - - - - - - - aN Cl~000 Cl' \0 - - 'O N - Cl NO WO 0 - ClN ' NN/ 0 C m Z Cl 'IT CN 'tN 'ITN N m0 Cl IN W) W/N \/N \CN 'O 'ON ON - mN 'I rN 00 00 ON -, - -n W - r- r 06z u 0000 0 00 0 0000 0000 \C \'0 IO ' ' O 'D 'O 0 NO ' Cl C Cl 4 Cl C E. - -I - 4 4 4 - -4 w0 00 0 w0 0 00 00 00 00 ON ON ON ON ON ONl ON ON ON ON -) - -CD- - N N N N c ml N -N N- N T - - - - - - - - - - - - - - - - - - - - - - - - 0 0 ~ m o N- N Cl f V-f 00 N e n - Cl Ct - - -lN 'D m -n N C; e v l l Cl 00 : C' 00 q 0 - N 7 C 0: 0 t N- 11 rl C 17, 00 "T Cl N O Om O= N t O O \- ON O 0 0 0 0 0 0 Q0 - - ~ - - - - - - - - - - - - - - - --- - - ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - -~ ~ ~ - ~ 0C l * t 1 - w olC m~N 'I '0 0 O N - ln 'I-' r- N 0N Cl cq n - n '. - -N -\ ol - - -l - - CD Cl (D C> CD CD Cl Cl Cl CD ~e ~ 0 me m 4 m ma mi s& m mmm 444 T 1-4 N N N N N N IN N N M I M M 0 00 'n 0, c, 0 '. m F.0 \c~ "D ' l Cl0 Cl - 0 0 e' IN C l 0 0 O - Cl en - - N - - - - - C N m - Cl c o r w '. 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N 00T .4 0 .- "t 4 O\ 0 0D 0 0 0 0 0D 0D 0 0D - - - - - - - - - - Nl "l Nl Nl N CD - - - - - - -- - - .- - - - - - - - -N - - - - - - - - - - - - - - - - - - - \C0 '. C . o '0 I-- '0 N - N ' N - N - N - N - N- NW 0 0 0 0 00 00 00 00 00 00 ON N~l~ l~l 00C~~ NO '(N '(N 0'IN00 0 0 r, '.0 -0 rN '.0 C)N \C( - 0 '. 0 00 kn Cl IN ON C) r-, - W in N O O4 N o 0 0 C"4 'N- On C1 N- .4 06 rN 6 00 C3 - r-N 4' r- oo ~N N cl - w- I f r-N ci 4. ml 4- w. t f '0 <D 0) 0 'n 00 T t'N '.0 N 00 O;N 0> - Cl e 'N . Nz 00 ON 0- w Cl C, M WIN W. 00 N o- - 'n 'In 'N 'WN WN V) 'V) 'In (n '(N \.c \c0 '0 '.C I'D '.C '0 \D0 '.0 '.0 N 000 00 000 00 00 O\ ON ON ON ON ON1 ON ON ON ON 0 0 0 0 0 0 0 00D C C C C m 00000 00 000000000000000000000 00000000 ON, ON oN ON\ ON C3N O\ ON, ON '. '0 .0 '. '0 .0 N N N N N N N 0 0 00 00 00 00 00 00 00 O N ~ ~ ~ ~ ~ - 000 '.00 w m- 00 CD N cN O . IN N 'N ~ 'N 0 ' N wi " o00 C ONN - ) o C 0 D '0 00 w 'N 000w000 m ~ m. m. . - - Cl~ l N l ' ~~j m/ C- Nl el Nl '.0D o. 'IN W. N , 00 ON 0 -T Cl c/N W( '.0 NC0 D Cl4 mN MI a)0 N(0 D l NlClC C 4 Cl 4 N N~ C,4 N~~ 00 C)+- 0 - 'In Cl m/ -) r/N 'I N C 0 I O / C 0 C/n 'I O Cl Cl Cl 00 0 1 .0 - - 0> - l T3 00 ON 0) '.D 'In 'In WN '( " Cl'.0 100 0 0 \ . '. N 0> m/ m/ '(N '/n '.0 '0 N N 00 00 - "l - '- 00 ON ON O O Z NlC lC Cl4 ml m/ m/ / - - - - - Cl Nl - - - - - - -C 0 co l cN~ ( . 00 N 0 -w N 'i IN '0 N 0 N 0 - C cN 00 wI '0N 00 00 - -- - - - - ------ - - - - - - - - n N " N Nq N N,4 - oD -M4-- N N 0 N N N Cl M M 00 M * N -1 - - - l - - i - - - - - - - - - -0 -~ -N -. -~ ON N I , 7 00 -1-a,00 m cc kn -- -T -q -D -D cN N4=~~'0 L~N f f ( N l ~ '0 S N N N N ~ N ~0 00 ~ 0 ~ ~ ~ ~ ~ ~ N O ~ & 0 m 0 ON 0.c-I CN -n C> '6 I= 0 0 n -nc- I" - c '.0 N , V0 ON 0 T V Cl Cl -i c- c o~ C C,6 r ; w~ 00 C; 6N 06 ,6 4( wN Co M 'I - n %D - - w - D - , m - t - n - , - W - C- N m 'I V- o - - - - - - - - - - - - - - - - - - - - - - - - - - - ON ON ON ON ON ON ON ON - 0 0 0 0 0 0 0 0 0 0 - - - - - - - ON all '.0 '.\ '.0 '.0 '.0 '0 ' N N ND N> Nl ND N> N ) N , N O0NN ~ oo 0D C0 00wt - - m r a 1 0 m o- m;- - 44 C\ ON N 0 w' In "D m O I -t .- m. e ,r 0 ON m N m m C, N. -I C r - C N M '(N '0 ND 00 ON 0 - Nl M( t (N \.o N- 00 ON\ 0 - Nl m( 'IN -n 0 Nc r w ,a ON 0 C,00 O 0 Cl 0'0 0 - - CD M - - 0 -- C D - Cl CD C4 ml "T 'n -C r- W C ) -~ "( M~ 'I O ON O ONO ON O N N O N O O O O O O O ON ON ON ON ON ON ON ON ' - C--i Ci' Cf C .6 0 6 ON - C l (N O( ON0 C C \ O 'N 0' O z ONOvNO NO NO N - - - - '.0 ON\ 0 'N ON0 C\ ' (1 00 ND CN ND (N C, ) C CD Cl -C -C - -. -C -c -C 1. 1 - r- r- R r - r - r, r ON - ~ ~ ~ ~ ~ ~ ~ ~ ~~l 00 Clrl- C ~ N ~ 0 0 ( 4 - ( N ' ~ l 0 .- 0r( l 'N- 00 b . 00 - CD r4 00 m 00 r '. w o06a s r4 or-4s sCi d as C N- = - ON 00, 'IN W) N Cl N Nl lCl~ - Cl1 - - - - o - 'N ' N o N 0 - o o w a\ 0 0 - Co o ot ' (N-'~ N N N N N N N N N 0 , -000 000 00 ON ON ON ON O NON ON '(N (N '(N '(N 'n I 'N WN W)N '.0 '.0 '.0 '.0 '.0 '.0 \.C '.0 '.D '0 N- N - N - N - N N N S C l l l C C C C C C C l Cl C l C 00 ~n 00 C> C m m kn " CD 0 a e m o N e U00 ~ _ ON Cl '(N e~N o -0 N00 - 0 '.0 N '( Cl O 0-~ ~ ~ ~ ~ ~ ~ ON ~ ~ ~ N0 N N N 'N N.0 0N '. - m- o '. o. N .2 ClON Cl O '.O000\0O0 ON ON O - - -N ON ON - - - - - \ n I- r- o N ON 0 l ~~- 'N . N 00 O 0 - Clc ~'N '0 N 00 N 0 N C IN '( 00N 0 N 0 0 N - - - N - - - -N - - - - CD l C C l C C> l C l C l C l C l C <0 cl) rqCA 0q N k n kn 0 - : r. - >, -C -- 0t ke)~ co- 00 HV 00 to0- l N NNN N ~~ 0--0 14) -i 0 0 0 0 cu 0 - c)( l4 0 C C ~~ C) c)CDC V~ En 'M. 0) ~~-~ 0 4.0 0 4~ 0 r. U 0~~~~~ -g - lCl 0) N0's~ kn C lCiCi Cl C C mf 0 C. 10 N 0 4 t 0 -+ - -= 00 a, Cl CD Cl Cl)csC Attorney Docket No. 08-09P1 Customer No.: 36,814 EXAMPLE 2 KNOCKDOWN OF p-GALACTOSIDASE ACTIVITY BY GAPPED DSRNA DICER SUBSTRATE The activity of a Dicer substrate dsRNA containing a gap in the double-stranded structure in 5 silencing LacZ mRNA as compared to the normal Dicer substrate dsRNA (i.e., not having a gap) was examined. Nucleotide Sequences of dsRNA and mdRNA Targeting LacZ mRNA The nucleic acid sequence of the one or more sense strands, and the antisense strand of the dsRNA and gapped dsRNA (also referred to herein as a meroduplex or mdRNA) are shown below 10 and were synthesized using standard techniques. The RISC activator LacZ dsRNA comprises a 21 nucleotide sense strand and a 21 nucleotide antisense strand, which can anneal to form a double stranded region of 19 base pairs with a two deoxythymidine overhang on each strand (referred to as 21/21 dsRNA). LacZ dsRNA (21/21) - RISC Activator 15 Sense 5'-CUACACAAAUCAGCGAUUUdTdT-3' (SEQ ID NO:1) Antisense 3'-dTdTGAUGUGUUUAGUCGCUAAA-5' (SEQ ID NO:2) The Dicer substrate LacZ dsRNA comprises a 25 nucleotide sense strand and a 27 nucleotide antisense strand, which can anneal to form a double-stranded region of 25 base pairs with one blunt end and a cytidine and uridine overhang on the other end (referred to as 25/27 20 dsRNA). LacZ dsRNA (25/27) - Dicer Substrate Sense 5'-CUACACAAAUCAGCGAUUUCCAUdGdT-3' (SEQ ID NO:3) Antisense 3'-CUGAUGUGUUUAGUCGCUAAAGGUA C A - 5' (SEQ ID NO:4) The LacZ mdRNA comprises two sense strands of 13 nucleotides (5'-portion) and 11 nucleotides 25 (3'-portion) and a 27 nucleotide antisense strand, which three strands can anneal to form two double-stranded regions of 13 and 11 base pairs separated by a single nucleotide gap (referred to as a 13, 11/27 mdRNA). The 5'-end of the 11 nucleotide sense strand fragment may be optionally 106 Attorney Docket No. 08-09PCT Customer No.: 36,814 phosphorylated. The "*" indicates a gap - in this case, a single nucleotide gap (i.e., a cytidine is missing). LacZ mdRNA (13, 11/27) - Dicer Substrate Sense 5'-CUACACAAAUCAG*GAUUUCCAUdGdT-3' (SEQ ID NOS:5, 6) 5 Antisense 3'-CUGAUGUGUUUAGUCGCUAAAGGUA CA -5' (SEQ ID NO:4) Each of the LacZ dsRNA or mdRNA was used to transfect 91acZ/R cells. Transfection Six well collagen-coated plates were seeded with 5 x 105 91acZ/R cells/well in a 2 ml volume per well, and incubated overnight at 37"C / 5% CO 2 in DMEM/high glucose media. 10 Preparation for transfection: 250 pl of OPTIMEM media without serum was mixed with 5 pLl of 20 pmol/pl dsRNA and 5pl of HIPERFECT transfection solution (Qiagen) was mixed with another 250 pl OPTIMEM media. After both mixtures were allowed to equilibrate for 5 minutes, the RNA and transfection solutions were combined and left at room temperature for 20 minutes to form transfection complexes. The final concentration of HIPERFECT was 50 pM, and the dsRNAs were 15 tested at 0.05nM, 0.lnM, 0.2nM, 0.5nM, lnM, 2nM, 5nM, and 1OnM, while the mdRNA was tested at 0.2nM, 0.5nM, InM, 2nM, 5nM, l0nM, 20nM, and 50nM. Complete media was removed, the cells were washed with incomplete OPTIMEM, and then 500 pl transfection mixture was applied to the cells, which were incubated with gentle shaking at 37"C for 4 hours. After transfecting, the transfection media was removed, cells were washed once with complete DMEM/high glucose 20 media, fresh media added, and the cells were then incubated for 48 hours at 37 0 C, 5% CO 2 . B-Galactosidase Assay Transfected cells were washed with PBS, and then detached with 0.5 ml trypsin/EDTA. The detached cells were suspended in 1 ml complete DMEM/high glucose and transferred to a clean tube. The cells were harvested by centrifugation at 250 x g for 5 minutes, and then resuspended in 25 50 ptl 1x lysis buffer at 4 0 C. The lysed cells were subjected to two freeze-thaw cycles on dry ice and a 37 0 C water bath. The lysed samples were centrifuged for 5 minutes at 4 0 C and the supernatant was recovered. For each sample, 1.5 pl and 10 pl of lysate was transferred to a clean tube and sterile water added to a final volume of 30 pl followed by the addition of 70 pl o-nitrophenyl-p-D-galactopyranose (ONPG) and 200 pl lx cleavage buffer with B-mercaptoethanol. 30 The samples were mixed briefly, incubated for 30 minutes at 37 0 C, and then 500 pl stop buffer was 107 Attorney Docket No. 08-09PCT Customer No.: 36,814 added (final volume 800 pl). B-Galactosidase activity for each sample was measured in disposable cuvettes at 420 nm. Protein concentration was determined by the BCA (bicinchoninic acid) method. For the purpose of the instant example, the level of measured LacZ activity was correlated with the quantity of LacZ transcript within 9L/LacZ cells. Thus, a reduction in B-galactosidase 5 activity after dsRNA transfection, absent a negative impact on cell viability, was attributed to a reduction in the quantity of LacZ transcripts resulting from targeted degradation mediated by the LacZ dsRNA. Results Knockdown activity in transfected and untransfected cells was normalized to a Qneg control 10 dsRNA and presented as a normalized value of the Qneg control (i.e., Qneg represented 100% or normala" gene expression levels). Both the lacZ RISC activator and Dicer substrate dsRNAs molecule showed good knockdown of B-galactosidase activity at concentration as low as 0.1 nM (Figure 2), while the Dicer substrate antisense strand alone (single stranded 27mer) had no silencing effect. A gapped mdRNA showed good knockdown although somewhat lower than that of intact 15 RISC activator and Dicer substrate dsRNAs (Figure 2). The presence of the gapmer cytidine (i.e., the missing nucleotide) at various concentrations (0.1 gM to 50 pM) had no effect on the activity of the mdRNA (data not shown). None of the dsRNA or mdRNA solutions showed any detectable toxicity in the transfected 9L/LacZ cells. The IC 50 of the lacZ mdRNA was calculated to be 3.74 nM, which is about 10 fold lower than what had been previously measured for lacZ dsRNA 21/21 20 (data not shown). These results show that a meroduplex (gapped dsRNA) is capable of inducing gene silencing. EXAMPLE 3 KNOCKDOWN OF INFLUENZA GENE EXPRESSION BY NICKED DSRNA The activity of a nicked dsRNA (21/21) in silencing influenza gene expression as compared 25 to a normal dsRNA (i.e., not having a nick) was examined. Nucleotide Sequences of dsRNA and mdRNA Targeting Influenza mRNA The dsRNA and nicked dsRNA (another form of meroduplex, referred to herein as ndsRNA) are shown below and were synthesized using standard techniques. The RISC activator influenza G1498 dsRNA comprises a 21 nucleotide sense strand and a 21 nucleotide antisense strand, which 108 Attorney Docket No. 08-09PCT Customer No.: 36,814 can anneal to form a double-stranded region of 19 base pairs with a two deoxythymidine overhang on each strand. G1498-wt dsRNA (21/21) Sense 5'-GGAUCUUAUUUCUUCGGAGdTdT-3' (SEQ ID NO:7) 5 Antisense 3'-dTdTCCUAGAAUAAAGAAGCCUC-5' (SEQ ID NO:8) The RISC activator influenza G1498 dsRNA was nicked on the sense strand after nucleotide 11 to produce a ndsRNA having two sense strands of 11 nucleotides (5'-portion, italic) and 10 nucleotides (3'-portion) and a 21 nucleotide antisense strand, which three strands can anneal to form two double-stranded regions of 11 (shown in italics) and 10 base pairs separated by a one nucleotide 10 gap (which may be referred to as G1498 11, 10/21 ndsRNA-wt). The 5'-end of the 10 nucleotide sense strand fragment may be optionally phosphorylated, as depicted by a "p" preceding the nucleotide (e.g., pC). G1498 ndsRNA-wt (11, 10/21) Sense 5'-GGAUCUUAUUUCUUCGGAGdTdT-3' (SEQ ID NO:9, 10) 15 Antisense 3'-dTdTCCUAGAAUAAAGAAGCCUC-5' (SEQ ID NO:8) G1498 ndsRNA-wt (11, 10/21) Sense 5'- GGA UCUUA UUUpCUUCGGAGdTdT-3' (SEQ ID NOS:9, 10) Antisense 3'-dTdTCCUAGAAUAAA GAAGCCUC-5' (SEQ ID NO:8) In addition, each of these G1498 dsRNAs were made with each U substituted with a 20 5-methyluridine (ribothymidine) and are referred to as G1498 dsRNA-rT. Each of the G1498 dsRNA or ndsRNA (meroduplex), with or without the 5-methyluridine substitution, was used to transfect HeLa S3 cells having an influenza target sequence associated with a luciferase gene. Also, the G1498 antisense strand alone or the antisense strand annealed to the 11 nucleotide sense strand portion alone or the 10 nucleotide sense strand portion alone were examined for activity. 25 Transfection and Dual Luciferase Assay The reporter plasmid psiCHECK T M -2 (Promega, Madison, WI), which constitutively expresses both firefly luc2 (Photinus pyralis) and Renilla (Renilla reniformis, also known as sea pansy) luciferases, was used to clone in a portion of the influenza NP gene downstream of the 109 Attorney Docket No. 08-09PCT Customer No.: 36,814 Renilla translational stop codon that results in a Renilla-influenza NP fusion mRNA. The firefly luciferase in the psiCHECKTM-2 vector is used to normalize Renilla luciferase expression and serves as a control for transfection efficiency. Multi-well plates were seeded with HeLa S3 cells/well in 100 pil Ham's F 12 medium and 5 10% fetal bovine serum, and incubated overnight at 37 0 C / 5% CO 2 . The HeLa S3 cells were transfected with the psiCHECK T M -influenza plasmid (75 ng) and G1498 dsRNA or ndsRNA (final concentration of 10 nM or 100 nM) formulated in Lipofectamine T M 2000 and OPTIMEM reduced serum medium. The transfection mixture was incubated with the HeLa S3 cells with gentle shaking at 37"C for about 18 to 20 hours. 10 After transfecting, firefly luciferase reporter activity was measured first by adding Dual GloTM Luciferase Reagent (Promega, Madison, WI) for 10 minutes with shaking, and then quantitating the luminescent signal using a VICTOR 3 TM 1420 Multilabel Counter (PerkinElmer, Waltham, MA). After measuring the firefly luminescence, Stop & Glo* Reagent (Promega, Madison, WI) was added for 10 minutes with shaking to simultaneously quench the firefly reaction 15 and initiate the Renilla luciferase reaction, and then the Renilla luciferase luminescent signal was quantitated VICTOR 3 TM 1420 Multilabel Counter (PerkinElmer, Waltham, MA). Results Knockdown activity in transfected and untransfected cells was normalized to a Qneg control dsRNA and presented as a normalized value of the Qneg control (i.e., Qneg represented 100% or 20 "normal" gene expression levels). Thus, a smaller value indicates a greater knockdown effect. The G1498 dsRNA-wt and dsRNA-rT showed similar good knockdown at a 100 nM concentration (Figure 3). The G1498 ndsRNA-rT, whether phosphorylated or not, showed good knockdown although somewhat lower than the G1498 dsRNA-wt (Figure 3). Similar results were obtained with dsRNA or ndsRNA at 10 nM (data not shown). None of the G1498 dsRNA or ndsRNA solutions 25 showed any detectable toxicity in HeLa S3 cells at either 10 nM or 100 nM. Even the presence of only half a nicked sense strand (an 11 nucleotide or 10 nucleotide strand alone) with a G 1498 antisense strand showed some detectable activity. These results show that a nicked-type meroduplex dsRNA molecule is unexpectedly capable of promoting gene silencing. 110 Attorney Docket No. 08-09PCT Customer No.: 36,814 EXAMPLE 4 KNOCKDOWN ACTIVITY OF NICKED MDRNA In this example, the activity of a dicer substrate LacZ dsRNA of Example 1 having a sense strand with a nick at various positions was examined. In addition, a dideoxy nucleotide (i.e., ddG) 5 was incorporated at the 5'-end of the 3'-most strand of a sense sequence having a nick or a single nucleotide gap to determine whether the in vivo ligation of the nicked sense strand is "rescuing" activity. The ddG is not a substrate for ligation. Also examined was the influenza dicer substrate dsRNA of Example 7 having a sense strand with a nick at one of positions 8 to 14. The "p" designation indicates that the 5'-end of the 3'-most strand of the nicked sense influenza sequence 10 was phosphorylated. The "L" designation indicates that the G at position 2 of the 5'-most strand of the nicked sense influenza sequence was substituted for a locked nucleic acid G. The Qneg is a negative control dsRNA. The dual fluorescence assay of Example 3 was used to measure knockdown activity with 5 nM of the LacZ sequences and 0.5 nM of the influenza sequences. The lacZ dicer substrate (25/27, 15 LacZ-DS) and lacZ RISC activator (21/21, LacZ) are equally active, and the LacZ-DS can be nicked in any position between 8 and 14 without affecting activity (Figure 3). In addition, the inclusion of a ddG on the 5'-end of the 3'-most LacZ sense sequence having a nick (LacZ:DSNkd13-3'dd) or a one nucleotide gap (LacZ:DSNkd13D1-3'dd) was essentially as active as the unsubstituted sequence (Figure 4). The influenza dicer substrate (G1498DS) nicked at any 20 one of positions 8 to 14 was also highly active (Figure 5). Phosphorylation of the 5'-end of the 3' most strand of the nicked sense influenza sequence had essentially no effect on activity, but addition of a locked nucleic acid appears to improve activity. EXAMPLE 5 MEAN INHIBITORY CONCENTRATION OF MDRNA 25 In this example, a dose response assay was performed to measure the mean inhibitory concentration (IC 5 o) of the influenza dicer substrate dsRNA of Example 8 having a sense strand with a nick at position 12, 13, or 14, including or not a locked nucleic acid. The dual luciferase assay of Example 2 was used. The influenza dicer substrate dsRNA (G1498DS) was tested at 0.0004 nM, 0.002 nM, 0.005 nM, 0.019 nM, 0.067 nM, 0.233 nM, 0.816 nM, 2.8 nM, and l0nM, 30 while the mdRNA with a nick at position 13 (G1498DS:Nkdl3) was tested at 0.001 nM, 0.048 nM, 111 Attorney Docket No. 08-09PCT Customer No.: 36,814 0.167 nM, 1 nM, 2 nM, 7 nM, and 25 nM (see Figure 6). Also tested were RISC activator molecules (21/21) with or without a nick at various positions (including G1498DS:Nkdl 1, G1498DS:Nkdl2, and G1498DS:Nkdl4), each of the nicked versions with a locked nucleic acid as described above (data not shown). The Qneg is a negative control dsRNA. 5 The IC 5 o of the RISC activator G1498 was calculated to be about 22 pM, while the dicer substrate G1498DS ICso was calculated to be about 6 pM. The IC 50 of RISC and Dicer mdRNAs range from about 200 pM to about 15 nM. The inclusion of a single locked nucleic acid reduced the

IC

50 of Dicer mdRNAs by up 4 fold (data not shown). These results show that a meroduplex dsRNA having a nick or gap in any position is capable of inducing gene silencing. 10 EXAMPLE 6 KNOCKDOWN ACTIVITY OF GAPPED MDRNA The activity of an influenza dicer substrate dsRNA having a sense strand with a gap of differing sizes and positions was examined. The influenza dicer substrate dsRNA of Example 8 was generated with a sense strand having a gap of 0 to 6 nucleotides at position 8, a gap of 4 15 nucleotides at position 9, a gap of 3 nucleotides at position 10, a gap of 2 nucleotides at position 11, and a gap of 1 nucleotide at position 12 (see Table 2). The Qneg is a negative control dsRNA. Each of the mdRNAs was tested at a concentration of 5 nM (data not shown) and 10 nM. The mdRNAs have the following antisense strand 5'-CAUUGUCUCCGAAGAAAUAAGAUCCUU (SEQ ID NO: 11), and nicked or gapped sense strands as shown in Table 2. 20 Table 2. mdRNA 5' Sense* (SEQ ID NO.) 3' Sense (SEQ ID NO.) P aSze I t G1498:DSNkd8 GGAUCUUA (12) UUUCUUCGGAGACAAdTdG (13) 8 0 67.8 G1498:DSNkd8D1 GGAUCUUA (12) UUCUUCGGAGACAAdTdG (14) 8 1 60.9 G1498:DSNkd8D2 GGAUCUUA (12) UCUUCGGAGACAAdTdG (15) 8 2 48.2 G1498:DSNkd8D3 GGAUCUUA (12) CUUCGGAGACAAdTdG (16) 8 3 44.1 G1498:DSNkd8D4 GGAUCUUA (12) UUCGGAGACAAdTdG (17) 8 4 30.8 G1498:DSNkd8D5 GGAUCUUA (12) UCGGAGACAAdTdG (18) 8 5 10.8 G1498:DSNkd8D6 GGAUCUUA (12) CGGAGACAAdTdG (19) 8 6 17.9 G1498:DSNkd9D4 GGAUCUUAU (20) UCGGAGACAAdTdG (18) 9 4 38.9 G1498:DSNkd10D3 GGAUCUUAU (21) UCGGAGACAAdTdG (18) 10 3 38.4 112 Attorney Docket No. 08-09PCT Customer No.: 36,814 mdRNA 5' Sense* (SEQ ID NO.) 3' Sense (SEQ ID NO.) p Siz % G1498:DSNkd11D2 GGAUCUUAUUU (22) UCGGAGACAAdTdG (18) 11 2 46.2 G1498:DSNkd12D1 GGAUCUUAUUUC (23) UCGGAGACAAdTdG (18) 12 1 49.6 Plasmid -- - 5.3 * G indicates a locked nucleic acid G in the 5' sense strand. t % KD means percent knockdown activity. The dual fluorescence assay of Example 2 was used to measure knockdown activity. Similar results were obtained at both the 5 nM and 10 nM concentrations. These data show that an 5 mdRNA having a gap of up to 6 nucleotides still has activity, although having four or fewer missing nucleotides shows the best activity (see, also, Figure 7). Thus, mdRNA having various sizes gaps that are in various different positions have knockdown activity. To examine the general applicability of a sequence having a sense strand with a gap of differing sizes and positions, a different dsRNA sequence was tested. The lacZ RISC dsRNA of 10 Example 1 was generated with a sense strand having a gap of 0 to 6 nucleotides at position 8, a gap of 5 nucleotides at position 9, a gap of 4 nucleotides at position 10, a gap of 3 nucleotides at position 11, a gap of 2 nucleotides at position 12, a gap of 1 nucleotide at position 12, and a nick (gap of 0) at position 14 (see Table 3). The Qneg is a negative control dsRNA. Each of the mdRNAs was tested at a concentration of 5 nM (data not shown) and 25 nM. The lacZ mdRNAs 15 have the following antisense strand 5'-AAAUCGCUGAUUUGUGUAGdTdTUAAA (SEQ ID NO:2) and nicked or gapped sense strands as shown in Table 3. Table 3. mdRNA 5' Sense* (SEQ ID NO.) 3' Sense* (SEQ ID NO.) Gap Gap Pos Size LacZ:Nkd8 CUACACAA (24) AUCAGCGAUUUdTdT (25) 8 0 LacZ:Nkd8D2 CUACACAA (24) UCAGCGAUUUdTdT (26) 8 1 LacZ:Nkd8D2 CUACACAA (24) CAG CGAUUUdTdT (27) 8 2 LacZ:Nkd8D3 CUACACAA (24) AGCGAUUUdTdT (28) 8 3 LacZ:Nkd8D4 CUACACAA (24) GC GAUUUdTdT (29) 8 4 LacZ:Nkd8D5 CUACACAA (24) CGAUUUdTdT (30) 8 5 LacZ:Nkd8D6 CUACACAA (24) GAUUUdTdT (31) 8 6 LacZ:Nkd9D5 CUACACAAA (32) GAUUUdTdT (31) 9 5 LacZ:NkdlOD4 CUACACAAAU (33) GAUJIUdTdT (3 1) 10 4 LacZ:Nkdl 1D3 CUACACAAAUC (34) GAUUUdTdT (31) 11 3 113 Attorney Docket No. 08-09PCT Customer No.: 36,814 mdRNA 5' Sense* (SEQ ID NO.) 3' Sense* (SEQ ID NO.) Gap Gap Pos Size LacZ:Nkdl2D2 CUACACAAAUCA (35) GAUUUdTdT (31) 12 2 LacZ:Nkdl3D CUACACAAAUCAG (36) GAUUUdTdT (31) 13 1 LacZ:Nkdl4 CUACACAAAUCAGC (37) GAUUUdTdT (1 14 0 * A indicates a locked nucleic acid A in each sense strand. The dual fluorescence assay of Example 3 was used to measure knockdown activity. Figure 8 shows that an mdRNA having a gap of up to 6 nucleotides has substantial activity and the position of the gap may affect the potency of knockdown. Thus, mdRNA having various sizes gaps that are 5 in various different positions and in different mdRNA sequences have knockdown activity. EXAMPLE 7 KNOCKDOWN ACTIVITY OF SUBSTITUTED MDRNA The activity of an influenza dsRNA RISC sequences having a nicked sense strand and the sense strands having locked nucleic acid substitutions were examined. The influenza RISC 10 sequence G 1498 of Example 3 was generated with a sense strand having a nick at positions 8 to 14 counting from the 5'-end. Each sense strand was substituted with one or two locked nucleic acids as shown in Table 4. The Qneg and Plasmid are negative controls. Each of the mdRNAs was tested at a concentration of 5 nM. The antisense strand used was 5'- CUCCGAAGAAAUAAGAUCCdTdT (SEQ ID NO:8). 114 Attorney Docket No. 08-09PCT Customer No.: 36,814 Table 4. mdRNA 5' Sense* (SEQ ID NO.) 3' Sense* (SEQ ID NO.) Nick % Pos KD G1498-wt GGAUCUUAUUUCUUCGGAGdTdT (7) - 85.8 G1498-L GGAUCUUAUUUCUUCGGAGdTdT (61) - 86.8 G1498:Nkd8-1 GGAUCUUA (12) UUUCUUCGGAGdTdT (47) 8 36.0 G1498:Nkd8-2 GGAUCUIJA (40) UJIUCUJCGGAGdTdT (54) 8 66.2 G1498:Nkd9-1 GGAUCUUAU (20) UUCUUCGGAGdTdT (48) 9 60.9 G1498:Nkd9-2 GGAUCUUAU (41) UUCUUCGGAGdTdT (55) 9 64.4 G1498:NkdlO-1 GGAUCUUAUU (21) UCUUCGGAGdTdT (49) 10 58.2 G1498:NkdlO-2 GGAUCUUAUU (42) UCUUCGGAGdTdT (56) 10 68.5 G1498:Nkd11-1 GGAUCUUAUUU (22) CUUCGGAGdTdT (50) 11 75.9 G1498:Nkdl 1-2 GGAUCUUAUUU (43) CUUCGGAGdTdT (57) 11 67.1 G1498:Nkdl2-1 GGAUCUUAUUIUC (23) UUCGGAGdTdT (51) 12 59.9 G1498:Nkdl2-2 GGAUCUUAUUUC (44) UUCGGAGdTdT (58) 12 72.8 G1498:Nkdl3-1 GGAUCUUAUUUCU (38) UCGGAGdTdT (52) 13 37.1 G1498:Nkdl3-2 GGAUCUUAUUUCU (45) UCGGAGdTdT (59) 13 74.3 G1498:Nkdl4-1 GGAUCUUAUUUCUU (39) CGGAGdTdT (53) 14 29.0 G1498:Nkdl4-2 GGAUCUUAUUUCUU (46) CGGAGdTdT (60) 14 60.2 Qneg - 0 Plasmid - 3.6 * Nucleotides that are bold and underlined are locked nucleic acids. The dual fluorescence assay of Example 3 was used to measure knockdown activity. These data show that increasing the number of locked nucleic acid substitutions tends to increase activity 5 of an mdRNA having a nick at any of a number of positions. The single locked nucleic acid per sense strand appears to be most active when the nick is at position 11 (see Figure 9). But, multiple locked nucleic acids on each sense strand make mdRNA having a nick at any position as active as the most optimal nick position with a single substitution (i.e., position 11) (Figure 9). Thus, mdRNA having duplex stabilizing modifications make mdRNA essentially equally active regardless 10 of the nick position. Similar results were observed when locked nucleic acid substitutions were made in the LacZ dicer substrate mdRNA of Example 2 (SEQ ID NOS:3 and 4). The lacZ dicer was nicked at positions 8 to 14, and a duplicate set of nicked LacZ dicer molecules were made with the exception that the A at position 3 (from the 5'-end) of the 5' sense strand was substituted for a locked nucleic 115 Attorney Docket No. 08-09PCT Customer No.: 36,814 acid A (LNA-A). As is evident from Figure 10, most of the nicked lacZ dicer molecules containing LNA-A were as potent in knockdown activity as the unsubstituted lacZ dicer. EXAMPLE 8 MDRNA KNOCKDOWN OF INFLUENZA VIRUS TITER 5 The activity of a dicer substrate nicked dsRNA in reducing influenza virus titer as compared to a wild-type dsRNA (i.e., not having a nick) was examined. The influenza dicer substrate sequence (25/27) is as follows: Sense 5'-GGAUCUUAUUUCUUCGGAGACAAdTdG (SEQ ID NO:62) Antisense 5'-CAUUGUCUCCGAAGAAAUAAGAUCCUU (SEQ ID NO: 11) 10 The mdRNA sequences have a nicked sense strand after position 12, 13, and 14, respectively, as counted from the 5'-end, and the G at position 2 is substituted with locked nucleic acid G. For the viral infectivity assay, Vero cells were seeded at 6.5 x 104 cells/well the day before transfection in 500 pl 10% FBS/DMEM media per well. Samples of 100, 10, 1, 0.1, and 0.01 nM stock of each dsRNA were complexed with 1.0 pl (1 mg/ml stock) of Lipofectamine T 2000 15 (Invitrogen, Carlsbad, CA) and incubated for 20 minutes at room temperature in 150 ptl OPTIMEM (total volume) (Gibco, Carlsbad, CA). Vero cells were washed with OPTIMEM, and 150 pl of the transfection complex in OPTIMEM was then added to each well containing 150 pl of OPTIMEM media. Triplicate wells were tested for each condition. An additional control well with no transfection condition was prepared. Three hours post transfection, the media was removed. Each 20 well was washed once with 200 pl PBS containing 0.3% BSA and 10 mM HEPES/PS. Cells in each well were infected with WSN strain of influenza virus at an MOI 0.01 in 200 pl of infection media containing 0.3% BSA/10 mM HEPES/PS and 4 tg/ml trypsin. The plate was incubated for 1 hour at 37*C. Unadsorbed virus was washed off with the 200 pl of infection media and discarded, then 400 ptl DMEM containing 0.3% BSA/10 mM HEPES/PS and 4 tg/ml trypsin was added to 25 each well. The plate was incubated at 37'C, 5% CO 2 for 48 hours, then 50 pl supernatant from each well was tested in duplicate by TCID 50 assays (50% Tissue-Culture Infective Dose, WHO protocol) in MDCK cells and titers were estimated using the Spearman and Karber formula. The results show that these mdRNAs show about a 50% to 60% viral titer knockdown, even at a concentration as low as10 pM (Figure 11). 116 Attorney Docket No. 08-09PCT Customer No.: 36,814 An in vivo influenza mouse model was also used to examine the activity of a dicer substrate nicked dsRNA in reducing influenza virus titer as compared to a wild-type dsRNA (i.e., not having a nick). Female BALB/c mice (age 8-10 weeks with 5-10 mice per group) were dosed intranasally with 120 nmol/kg/day dsRNA (formulated in C12-norArg(NH 3 +Cl~)-C12/DSPE 5 PEG2000/DSPC/cholesterol at a ratio of 30:1:20:49) for three consecutive days before intranasal challenge with influenza strain PR8 (20 PFU/mouse). Two days after infection, whole lungs are harvested from each mouse and placed in a solution of PBS/0.3% BSA with antibiotics, homogenize, and measure the viral titer (TCIDso). Doses were well tolerated by the mice, indicated by less than 2% body weight reduction in any of the dose groups. The mdRNAs tested exhibit 10 similar, if not slightly greater, virus reduction in vivo as compared to unmodified and unnicked G1498 dicer substrate (see Figure 12). Hence, mdRNA are active in vivo. EXAMPLE 9 EFFECT OF MDRNA ON CYTOKINE INDUCTION The effect of the mdRNA structure on cytokine induction in vivo was examined. Female 15 BALB/c mice (age 7-9 weeks) were dosed intranasally with about 50 pIM dsRNA (formulated in C12-norArg(NH 3 +Cl-)-C12/DSPE-PEG2000/DSPC/cholesterol at a ratio of 30:1:20:49) or with 605 nmol/kg/day naked dsRNA for three consecutive days. About four hours after the final dose is administered, the mice were sacrificed to collect bronchoalveolar fluid (BALF), and collected blood is processed to serum for evaluation of the cytokine response. Bronchial lavage was performed 20 with 0.5 mL ice-cold 0.3% BSA in saline two times for a total of 1 mL. BALF was spun and supernatants collected and frozen until cytokine analysis. Blood was collected from the vena cava immediately following euthanasia, placed into serum separator tubes, and allowed to clot at room temperature for at least 20 minutes. The samples were processed to serum, aliquoted into Millipore ULTRAFREE 0.22 tm filter tubes, spun at 12,000 rpm, frozen on dry ice, and then stored at -70'C 25 until analysis. Cytokine analysis of BALF and plasma were performed using the Procartalm mouse 1 0-Plex Cytokine Assay Kit (Panomics, Fremont, CA) on a Bio-PlexTM array reader. Toxicity parameters were also measured, including body weights, prior to the first dose on day 0 and again on day 3 (just prior to euthanasia). Spleens were harvested and weighed (normalized to final body weight). The results are provided in Table 5. 30 117 Attorney Docket No. 08-09PCT Customer No.: 36,814 Table 5. In vivo Cytokine Induction by Naked mdRNA Cytokine G1498 G1498:Nkd G1498:DS G1498:DSNkd G1498:DSNkd G1498:DSNkd 11-1 12-1 13-1 14-1 IL-6 (gmL 90.68 10.07 77.35 17.17 18.21 38.59 Fold decrease - 9 - 5 4 2 IL40) (g/m) 661.48 20.32 1403.61 25.07 37.70 57.02 Fold decrease - 33 - 56 37 25 TNFa pgn) 264.49 25.59 112.95 20.52 29.00 64.93 Fold decrease - 10 - 6 4 2 The mdRNA (RISC or dicer sized) induced cytokines to lesser extent than the intact (i.e., not nicked) parent molecules. The decrease in cytokine induction was greatest when looking at IL 12(p40), the cytokine with consistently the highest levels of induction of the 10 cytokine multiplex 5 assay. For the mdRNA, the decrease in IL-12 (p40) ranges from 25- to 56-fold, while the reduction in either IL-6 or TNFa induction was more modest (the decrease in these two cytokines ranges from 2- to 10-fold). Thus, the mdRNA structure appears to provide an advantage in vivo in that cytokine induction is minimized compared to unmodified dsRNA. Similar results were obtained with the formulated mdRNA, although the reduction in 10 induction was not as prominent. In addition, the presence or absence of a locked nucleic acid has no effect on cytokine induction. These results are shown in Table 6. Table 6. In vivo Cytokine Induction by Formulated mdRNA Cytokine G1498:DS G1498:Nkd G1498:Nkd G1498:DSNkd G1498:DSNkd 12-1 13-1 14-1 13 IL-6 Conc (pg/mL) 29.04 52.95 10.28 7.79 44.29 Fold decrease - -1.8 3 4 -1.5 IL-12 (p40) Conc (pg/mL) 298.93 604.24 136.45 126.71 551.49 Fold decrease - 0 2 2 1 TNFa Conc (pg/mL) 13.49 21.35 3.15 3.15 18.69 Fold decrease - -1.6 4 4 1.4 15 118 Attorney Docket No. 08-09PCT Customer No.: 36,814 EXAMPLE 10 Survivin siRNA Induce Caspase Activation Induction of caspase activity and cell death by transfection of Survivin siRNA in a KU-7 bladder cancer cell line was examined. 5 Both Caspase 3 and 7 are effector caspases that mediate programmed cell death (i.e., apoptosis), which plays an important role in preventing cancer. Therefore, the ability of a drug, for example an siRNA, to induce the activity of caspases in a cancer or pre-cancer cell and consequently induce apoptosis to prevent cancer or treat cancer in a human subject is highly advantageous. In this Example, Survivin siRNA transfected into a bladder cancer cell line induced 10 Caspase 3 and Caspase 7 activity, and further induced cell morphology changes observed via microscopy analysis that were highly indicative of apopotsis. Accordingly, the data indicate that Survivin siRNA may induce apoptosis in bladder cancer cells. Briefly, KU-7 cells were plated at a density of 7,500 cells/well on a 96-well plate. Twenty fours later, 25 ptL mixture containing 25 nM or 5 nM siRNA and RNAi MAX diluted 1/50 in 15 optiMEM media was added to each well containing 75 tL cell medium with 10% fetal bovine serum. The transfection was performed in triplacate. The transfection mixture was incubated with the cells for 24 hours. Following the incubation, cells were lysed, RNA extracted and qRT-PCR was performed to determine gene expression levels. The Qneg siRNA served as the negative control. Separately, the transfected cells of the duplicate plate were lysed, and CASPASE-GLO 20 reagent was added (PROMEGA) to measure caspase activity per the manufacturer's protocol. Caspase activity was measured with a Wallac Plate reader. Cell viability was measured with the CELLTITER 96 assay kit (PROMEGA) per the manufacturer's protocol. The sequence specific Survivin siRNA and a negative control siRNA (i.e., a scrambled sequence of a Survivin siRNA) used in this Example are shown below. Knockdown activity in 25 transfected and untransfected cells was normalized to a Qneg control dsRNA (QIAGEN) and presented as a normalized value of the Qneg control (i.e., Qneg represented 100% or "normal" gene expression levels). Survivin- 11 (DX9792): 30 Sense Strand: 5'- CCAGUGUUUCUUCUGCUUCTT - 3' (SEQ ID NO: 1896) Antisense Strand: 5'- GAAGCAGAAGAAACACUGGTT - 3' (SEQ ID NO: 1897) 119 Attorney Docket No. 08-09PCT Customer No.: 36,814 Survivin- 11UNA (DX9794): Sense Strand: 5'- CCAGUGUUUCUUCUGCUUCunaUunaU - 3' (SEQ ID NO: 1898) 5 Antisense Strand: 5'- GAAGCAGAAGAAACACUGGunaUunaU - 3' (SEQ ID NO: 1899) Survivin-11UNA-SCR (DX9794) - negative control: Sense Strand: 5'- UCCCGUUCUAGUGUUUCCUunaUunaU - 3' (SEQ ID NO: 10 1900) Antisense Strand: 5'- AGGAAACACUAGAACGGGAunaUunaU - 3' (SEQ ID NO: 1901) Hydroxymethyl substituted monomer(s) in the sequences of the table below are identified 15 as "unaX" where X is the one letter code for the nucleomonomer (e.g., "unaU" indicates that the uracil comprises a hydroxymethyl substituted monomer). In this Example, the Survivin:286UJNA siRNA is a double-stranded RNA having a 19 base pair region with two blunt ends and two hydroxymethyl substituted monomers were covalently linked to the 3'-end of both the sense strand and the antisense strand. 20 The results for the percent survivin gene knockdown, percent cell death induction, and the fold caspase induction are shown below in Table 10. The percent knockdown (%KD) is relative to Qneg siRNA, percent cell death induction (%CD) is relative to untransfected cells; and fold caspase induction (Ca) is also relative to untransfected cells. 25 Table 10 siRNA KU-7 Cells siRNA Identifier Conc. % KD % CD Ca Survivin-11 25 nM 80 19 7.1 (DX9792) 5 nM 85 17 5.2 Survivin-11UNA 25 nM 79 23 6.6 (DX9794) 5 nM 84 23 5.2 Survivin-11UNA- 25 nM 0 9 1 SCR (DX9794) 5 nM 15 8 1.1 120 Attorney Docket No. 08-09PCT Customer No.: 36,814 The results show that Survivin siRNA reducted target gene expression levels relative to the Qneg siRNA negative control in the KU-7 bladder cancer cell line. Further, Survivin siRNA induced over a five-fold increase in caspase activity relative to untransfected cells. Lastly, these 5 results show that there is a correlation between siRNA mediated knockdown of Survivin gene expression and both cell death induction and caspase activity induction. In other words, upon a reduction in Survivin gene expression mediated by the Survivin siRNA, both caspase activity increases and cell death induction increases. 10 The sequences referred to herein (e.g., by way of SEQ ID NOs.) are also disclosed in a sequence listing, an entire printable copy of which accompanies the specification as one text file recorded on a single compact disk (CD). The CD (labeled CD#2) is also labelled with (i) the applicant's name, (ii) the title of invention, (iii) a reference number, (iv) the date on which the data was recorded on the CD and (v) the relevant computer operating system. 15 Throughout the description and the claims of this specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps. 20 The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention before the priority date of each claim of this application. 121

Claims (25)

1. An RNA molecule that down regulates the expression of a target mRNA, the RNA molecules comprising a first strand that is complementary to a human target mRNA and a second strand complementary to the first strand, wherein the first strand and second strands can anneal to form a double-stranded region having from about 15 base pairs to about 40 base pairs.

2. The RNA molecule of claim 1 wherein the first strand is from 19 to 24 nucleotides in length or from 25 to 29 nucleotides in length and is complementary to a human target gene selected from the group consisting of tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR), erythroblastic leukemia viral oncogene homolog (ERBB), platelet derived growth factor (PDGF), platelet derived growth factor receptor (PDGFR), breakpoint cluster region (BCR)-abelson murine leukemia viral oncogene homolog (ABL), steroid-5-alpha-reductase, alpha polypeptide 1 (SRD5A1), steroid-5-alpha-reductase, alpha polypeptide 2 (SRD5A2), phosphoinositide-3-kinase, catalytic (PIK3C), mitogen-activated protein kinase (MAPK), p38 MAPK family, hypoxia inducible factor 1 alpha (HIF 1A), protein kinase N3 (PKN3), interleukin 17A (IL17A), interleukin 6 (IL6), interleukin 18 (ILl 8), tumor necrosis factor (ligand) superfamily member 13b (TNFSF 13B), mitogen-activated protein kinase 1 (MAPK 1), v-raf- 1 murine leukemia viral oncogene homolog 1 (RAF 1), v-AKT murine thymoma viral oncogene (AKT), FK506 binding protein 12-rapamycin associated protein 1 (FRAP 1), mitogen-activated protein kinase 2 (MAPK2), cyclin-dependent kinase 2 (CDK2), ATP-binding cassette, subfamily B, member 1 (ABCB 1), B-cell CLL/lymphoma 2 (BCL2), angiopoietin 2 (ANGPT2), checkpoint kinase 1 homolog (CHEKI), insulin-like growth factor 1 receptor (IGF 1 R), signal transducer and activator of transcription 3 (STAT3), matrix metalloproteinase (MMP), folate hydrolase (prostate-specific membrane antigen) 1 (FOLH 1), v-myc myelocytomatosis viral oncogene homolog (avian) (MYC), telomerase RNA component (TERC), telomerase reverse transcriptase (TERT), protein kinase C, alpha (PRKCA), RAS viral (v-ras) oncogene homolog (RAS), chemokine (C-X-C motif) ligand or receptor (CXC), Wingless-Type MMTV (Murine Mammary Tumor Virus) Integration Site (WNT), toll-like receptor (TLR), Fc fragment of IgE, low affinity II, receptor for (CD23) (FCER2), FOS gene, (FOS, FOSB, FOSLI, OR FOSL2), hydroxysteroid (11-beta) dehydrogenase (HSDl 11B1), JUN gene (cJUN, JUNB, or JUND), thymidine phosphorylase (TYMP), early growth response (EGR), zeste homolog 122 Attorney Docket No. 08-09PCT Customer No.: 36,814 2 (EZH2), cyclin Dl (CCND1), Fas (TNF receptor superfamily, member 6) (FAS), proliferating cell nuclear antigen (PCNA), fibroblast growth factor 2 (FGF2), tumor growth factor-beta (TGF-p), tumor growth factor-beta receptor (TGF-pR), tumor-associated calcium signal transducer 1 (TACSTD 1), Mucin 1 (MUC 1), protein tyrosine phosphatase, non-receptor-Il (Noonan Syndrome 1) (PTPN1 1), neuregulin 1 (NRG1), membrane metallo-endopeptidase (MME), CD19 molecule (CD 19), CD40 molecule, TNF receptor superfamily member 5 (CD40), apolipoprotein B (including Ag(x) antigen) (ApoB), synuclein, alpha (non A4 component of amyloid precursor) (SNCA), silent mating type information regulation 2 homolog (SIRT2), histone deacetylase (HDAC), membrane-spanning 4-domains, subfamily A, member 1 (MS4A 1), CD22 molecule (CD22), diacylglycerol o-acyltransferase 1 (DGAT1), diacylglycerol o-acyltransferase 2 (DGAT2), CD3 molecule (CD3), proprotein convertase subtilisin-like kexin type 9 (PCSK9), MET (Mesenchymal epithelial transition factor) (c-Met proto-oncogene), catenin (cadherin-associated protein) (beta-catenin) (CTNNB 1), inhition of DNA binding proteins (Inhibition of Differentiation Proteins, Dominant Negative Helix-Loop-Helix Protein) (ID), protein tyrosine phosphatase, non receptor type 1 (PTPN 1), tie-I (TIE1; tyrosine kinase with immunoglobulin and EGF factor homology domains 1), tek tyrosine kinase (TEK), fibroblast growth factor receptor (FGFR), mitogen-activated protein kinase 3 (MAPK3), survivin (BIRC5), and polo-like kinase family genes (PLKI).

3. The RNA molecule of claim 1 or claim 2 wherein the RNA molecule has at least one blunt end.

4. The RNA molecule of any one of claims 1 to 3 wherein the RNA molecule has at least one 3'-overhang.

5. The RNA molecule of any one of claims I to 4 further comprising at least one acyclic nucleomonomer.

6. The RNA molecule of claim 5 wherein the acyclic nucleomonomer is selected from the group consisting of: 123 Attorney Docket No. 08-09PCT Customer No.: 36,814 0 Base O - Base o Base O Base OH 0 0 OH 0 O-R 0 S-R ~O O -F =OO-P=O -O-P=O Monomer D Monomer E Monomer F Monomer G 0 O Base O0 Base 0 O Base O NH O NH O N -O- =O P= - =O R N Monomer H Monomer I O'R Monomer J wherein, R is selected from the group consisting of hydrogen, a methyl group, C(1-10) alkyl, cholesterol, naturally or non-naturally occurring amino acid, sugar, vitamin, flurophore, polyamine and fatty acid.

7. The RNA molecule of claim 6 wherein at least one acyclic nucleomonomer is linked to the blunt end of the RNA molecule.

8. The RNA molecule of claim 6 or claim 7, wherein at least one acyclic nucleomonomer is in the double-stranded region of the RNA molecule.

9. A method for reducing the expression of a human target gene, comprising administering an RNA molecule of any one of claims 1 to 8 to a cell expressing the target gene, wherein the RNA molecule reduces expression of the target gene in the cell.

10. The method of claim 9 wherein the cell is a human cell.

11. A meroduplex ribonucleic acid (mdRNA) molecule that down regulates the expression of a target mRNA, the mdRNA molecules comprising a first strand of 15 to 40 124 Attorney Docket No. 08-09PCT Customer No.: 36,814 nucleotides in length that is complementary to a target mRNA and a second strand and a third strand that are each complementary to non-overlapping regions of the first strand, wherein the second strand and third strand can anneal with the first strand to form at least two double-stranded regions spaced apart by a nick or a gap.

12. The mdRNA molecule of claim 11 wherein the first strand is 15 to 25 nucleotides in length or 26 to 40 nucleotides in length.

13. The mdRNA molecule of claim 11 wherein the first strand is from 19 to 24 nucleotides in length or from 25 to 29 nucleotides in length and is complementary to a human target gene selected from the group consisting of tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR), erythroblastic leukemia viral oncogene homolog (ERBB), platelet derived growth factor (PDGF), platelet derived growth factor receptor (PDGFR), breakpoint cluster region (BCR) abelson murine leukemia viral oncogene homolog (ABL), steroid-5-alpha-reductase, alpha polypeptide 1 (SRD5A1), steroid-5-alpha-reductase, alpha polypeptide 2 (SRD5A2), phosphoinositide-3-kinase, catalytic (PIK3C), mitogen-activated protein kinase (MAPK), p38 MAPK family, hypoxia-inducible factor 1 alpha (HIF A), protein kinase N3 (PKN3), interleukin 17A (IL1 7A), interleukin 6 (IL6), interleukin 18 (IL1 8), tumor necrosis factor (ligand) superfamily member 13b (TNFSF13B), mitogen-activated protein kinase 1 (MAPK1), v-raf-1 murine leukemia viral oncogene homolog 1 (RAF1), v-AKT murine thymoma viral oncogene (AKT), FK506 binding protein 12-rapamycin associated protein 1 (FRAP 1), mitogen-activated protein kinase 2 (MAPK2), cyclin-dependent kinase 2 (CDK2), ATP-binding cassette, subfamily B, member 1 (ABCB1), B-cell CLL/lymphoma 2 (BCL2), angiopoietin 2 (ANGPT2), checkpoint kinase 1 homolog (CHEKI), insulin-like growth factor 1 receptor (IGF 1 R), signal transducer and activator of transcription 3 (STAT3), matrix metalloproteinase (MMP), folate hydrolase (prostate-specific membrane antigen) 1 (FOLH 1), v-myc myelocytomatosis viral oncogene homolog (avian) (MYC), telomerase RNA component (TERC), telomerase reverse transcriptase (TERT), protein kinase C, alpha (PRKCA), RAS viral (v-ras) oncogene homolog (RAS), chemokine (C-X-C motif) ligand or receptor (CXC), Wingless-Type MMTV (Murine Mammary Tumor Virus) Integration Site (WNT), toll-like receptor (TLR), Fc fragment of IgE, low affinity II, receptor for (CD23) (FCER2), FOS gene, (FOS, FOSB, FOSL1, OR FOSL2), hydroxysteroid (11-beta) dehydrogenase (HSD1 1B1), JUN gene (cJUN, JUNB, or JUND), thymidine phosphorylase (TYMP), early growth response (EGR), zeste homolog 125 Attorney Docket No. 08-09PCT Customer No.: 36,814 2 (EZH2), cyclin D1 (CCND 1), Fas (TNF receptor superfamily, member 6) (FAS), proliferating cell nuclear antigen (PCNA), fibroblast growth factor 2 (FGF2), tumor growth factor-beta (TGF-p), tumor growth factor-beta receptor (TGF-pR), tumor-associated calcium signal transducer 1 (TACSTD1), Mucin 1 (MUC 1), protein tyrosine phosphatase, non-receptor-Il (Noonan Syndrome 1) (PTPN 11), neuregulin 1 (NRG1), membrane metallo-endopeptidase (MME), CD19 molecule (CD 19), CD40 molecule, TNF receptor superfamily member 5 (CD40), apolipoprotein B (including Ag(x) antigen) (ApoB), synuclein, alpha (non A4 component of amyloid precursor) (SNCA), silent mating type information regulation 2 homolog (SIRT2), histone deacetylase (HDAC), membrane-spanning 4-domains, subfamily A, member 1 (MS4A1), CD22 molecule (CD22), diacylglycerol o-acyltransferase 1 (DGAT1), diacylglycerol o-acyltransferase 2 (DGAT2), CD3 molecule (CD3), proprotein convertase subtilisin-like kexin type 9 (PCSK9), MET (Mesenchymal epithelial transition factor) (c-Met proto-oncogene), catenin (cadherin-associated protein) (beta-catenin) (CTNNB 1), inhition of DNA binding proteins (Inhibition of Differentiation Proteins, Dominant Negative Helix-Loop-Helix Protein) (ID), protein tyrosine phosphatase, non receptor type 1(PTPN1), tie-i (TIE1; tyrosine kinase with immunoglobulin and EGF factor homology domains 1), tek tyrosine kinase (TEK), fibroblast growth factor receptor (FGFR), mitogen-activated protein kinase 3 (MAPK3), survivin (BIRC5), and polo-like kinase family genes (PLKI).

14. The mdRNA molecule of any one of claims 11 to 13 wherein the gap comprises from 1 to 10 unpaired nucleotides.

15. The mdRNA molecule of any one of claims 11 to 14 wherein the mdRNA molecule has at least one blunt end.

16. The mdRNA molecule of any one of claims 11 to 15 wherein the mdRNA molecule has at least one 3'-overhang comprising one to four nucleotides that are not part of the gap.

17. The mdRNA molecule of any one of claims II to 16 further comprising at least on acyclic nucleomonomer.

18. The mdRNA molecule of claim 17 wherein the at least one acyclic nucleomonomer selected from the group consisting of: 126 Attorney Docket No. 08-09PCT Customer No.: 36,814 0 O Base O o Base O O Base O 0 Base OH 0 OH 0 O-R 0 S-R 0 -o-p-oI Monomer D Monomer E Monomer F Monomer G 0 O Base 0 O Base 0 O Base 0 NH O NH 0 N II I I -O-P=O O= -OP=O R -- = k R N Monomer H Monomer 1 O R Monomer J wherein, R is selected from the group consisting of hydrogen, methyl group, C(1-10) alkyl, cholesterol, naturally or non-naturally occurring amino acid, sugar, vitamin, flurophore, polyamine and fatty acid.

19. The mdRNA molecule of claim 18 wherein at least one acyclic nucleomonomer is linked to the blunt end of the mdRNA molecule.

20. The mdRNA molecule of claim 18 or claim 19, wherein at least one acyclic nucleomonomer is in one of the double-stranded regions of the mdRNA molecule.

21. A method for reducing the expression of a human target gene, comprising administering an mdRNA molecule of any one of claims 11 to 20 to a cell expressing the target gene, wherein the mdRNA molecule reduces expression of the target gene in the cell.

22. The method of claim 21 wherein the cell is a human cell. 127 Attorney Docket No. 08-09PCT Customer No.: 36,814

23. Use of the mdRNA molecule of any one of claims 11 to 20 or the RNA molecule of any one of claim 1 to 8 for the manufacture of a medicament for use in the therapy of cancer, a metabolic disease or inflammatory disease.

24. The RNA molecule of claim 1, substantially as hereinbefore described and with reference to any of the Examples, Figures and/or Sequence Listing.

25. The mdRNA molecule of claim 11, substantially as hereinbefore described and with reference to any of the Examples, Figures and/or Sequence Listing. 128