US 2012O322864A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0322864 A1 Rossi et al. (43) Pub. Date: Dec. 20, 2012 (54) SUSTAINED POLYPEPTIDE EXPRESSION (60) Provisional application No. 61/325,003, filed on Apr. FROM SYNTHETIC, MODIFIED RNAS AND 16, 2010, provisional application No. 61/387,220, USES THEREOF filed on Sep. 28, 2010. Publication Classification (75) Inventors: Derrick Rossi, Roslindale, MA (US); (51) Int. Cl. Luigi Warren, Cambridge, MA (US) CI2N5/07 (2010.01) A6IR 48/00 (2006.01) (73) Assignee: IMMUNE DISEASE INSTITUTE, C7H 2L/02 (2006.01) INC., Boston, MA (US) (52) U.S. Cl. ...................... 514/44 R; 536/23.5; 435/377 (57) ABSTRACT (21) Appl. No.: 13/590,880 Described herein are synthetic, modified RNAs for changing the phenotype of a cell. Such as expressing a polypeptide or altering the developmental potential. Accordingly, provided (22) Filed: Aug. 21, 2012 herein are compositions, methods, and kits comprising Syn thetic, modified RNAs for changing the phenotype of a cellor Related U.S. Application Data cells. These methods, compositions, and kits comprising Syn thetic, modified RNAs can be used either to express a desired (63) Continuation of application No. 13/088,009, filed on protein in a cell or tissue, or to change the differentiated Apr. 15, 2011. phenotype of a cell to that of another, desired cell type. Patent Application Publication Dec. 20, 2012 Sheet 1 of 45 US 2012/0322864 A1 s se xxx S s s nnnn s e sit. occa S. s Ra w s e s S. iiii is is iiiiSE SEE SS Patent Application Publication Dec. 20, 2012 Sheet 2 of 45 US 2012/0322864 A1 Patent Application Publication Dec. 20, 2012 Sheet 3 of 45 US 2012/0322864 A1 t S is is is is Patent Application Publication Dec. 20, 2012 Sheet 4 of 45 US 2012/0322864 A1 . S -- Patent Application Publication Dec. 20, 2012 Sheet 5 of 45 US 2012/0322864 A1 S. s s Unive -- Patent Application Publication Dec. 20, 2012 Sheet 6 of 45 US 2012/0322864 A1 S is Patent Application Publication Dec. 20, 2012 Sheet 7 of 45 US 2012/0322864 A1 Patent Application Publication Dec. 20, 2012 Sheet 8 of 45 US 2012/0322864 A1 Patent Application Publication Dec. 20, 2012 Sheet 9 of 45 US 2012/0322864 A1 waxes --- s as --ur VEHICLEvict- sa- i--1 -- awarr -21 --- in--si s 40 a-21 Patent Application Publication Dec. 20, 2012 Sheet 10 of 45 US 2012/0322864 A1 y/GEN/A GE/ Patent Application Publication Dec. 20, 2012 Sheet 11 of 45 US 2012/0322864 A1 Patent Application Publication Dec. 20, 2012 Sheet 12 of 45 US 2012/0322864 A1 ESSES Patent Application Publication Dec. 20, 2012 Sheet 13 of 45 US 2012/0322864 A1 & I FE : E 3 3 e s . R : 2e 18- , 75 / : : 8.5 - | r i s a 25 ------e.cc cc------8 ESSES Patent Application Publication Dec. 20, 2012 Sheet 14 of 45 US 2012/0322864 A1 s af s ar r sees i s S & & -: Elie :FSF C S: : C. SS cricEFSF Š 3. is S. S. is FEF, Patent Application Publication Dec. 20, 2012 Sheet 15 of 45 US 2012/0322864 A1 e R s co S itr s -S SE i- An s S X ŠE : Š t t c F3 Š"A. 88: ESF: i F: c :: : E. F8 88: ce i. Liz st Esfia. Š. it 3. ii Patent Application Publication Dec. 20, 2012 Sheet 16 of 45 US 2012/0322864 A1 Sii . iii. iii. i i i Patent Application Publication Dec. 20, 2012 Sheet 17 of 45 US 2012/0322864 A1 V,±01. S is Patent Application Publication Dec. 20, 2012 Sheet 18 of 45 US 2012/0322864 A1 f is is a SESSFEEE Patent Application Publication Dec. 20, 2012 Sheet 19 of 45 US 2012/0322864 A1 Šis stŠ Patent Application Publication Dec. 20, 2012 Sheet 20 of 45 US 2012/0322864 A1 SE --S ECS i sstss s s s s Patent Application Publication Dec. 20, 2012 Sheet 21 of 45 US 2012/0322864 A1 SE-i ECES E. sssi s (cont Patent Application Publication Dec. 20, 2012 Sheet 22 of 45 US 2012/0322864 A1 8 Patent Application Publication Dec. 20, 2012 Sheet 23 of 45 US 2012/0322864 A1 & S S & Š & ::::: s Patent Application Publication Dec. 20, 2012 Sheet 24 of 45 US 2012/0322864 A1 ES SS i. Patent Application Publication Dec. 20, 2012 Sheet 25 of 45 US 2012/0322864 A1 ES -- SS i. e Patent Application Publication Dec. 20, 2012 Sheet 26 of 45 US 2012/0322864 A1 ES SE s SS S. S S. S S. S SS S.S S S. S SS S. Patent Application Publication Dec. 20, 2012 Sheet 27 of 45 US 2012/0322864 A1 ES SS Patent Application Publication Dec. 20, 2012 Sheet 28 of 45 US 2012/0322864 A1 Patent Application Publication Dec. 20, 2012 Sheet 29 of 45 US 2012/0322864 A1 i 2 UNTRANSFEED as RNA Patent Application Publication Dec. 20, 2012 Sheet 31 of 45 US 2012/0322864 A1 SS S. C.S. 8 REY FRPS 8 Patent Application Publication Dec. 20, 2012 Sheet 32 of 45 US 2012/0322864 A1 : SS Patent Application Publication Dec. 20, 2012 Sheet 33 of 45 US 2012/0322864 A1 i. s ES Patent Application Publication Dec. 20, 2012 Sheet 34 of 45 US 2012/0322864 A1 Patent Application Publication Dec. 20, 2012 Sheet 35 of 45 US 2012/0322864 A1 ST ST SS ES TS 'S EST ST ST SS STSs TST cE--------. ST S. Seii. St. i Seii. "Set" Seii: S8. for-o-o-o-o-Too Patent Application Publication Dec. 20, 2012 Sheet 36 of 45 US 2012/0322864 A1 s s s s s s s s t s &S se s Patent Application Publication Dec. 20, 2012 Sheet 37 of 45 US 2012/0322864 A1 / if: Patent Application Publication Dec. 20, 2012 Sheet 38 of 45 US 2012/0322864 A1 Sir S: Patent Application Publication Dec. 20, 2012 Sheet 39 of 45 US 2012/0322864 A1 i SS s as Patent Application Publication Dec. 20, 2012 Sheet 40 of 45 US 2012/0322864 A1 8 s E ste see E3road is 489 ... sis 8a st w a s ocw. Patent Application Publication Dec. 20, 2012 Sheet 41 of 45 US 2012/0322864 A1 Patent Application Publication Dec. 20, 2012 Sheet 42 of 45 US 2012/0322864 A1 s See sts sis do is ses e e S. at ... - Ese E.ses s: s s s E se e.g. E es s s se s is - s s se se sixx 8ws w Yw x wwwe $8 *or S. s SS - rrrr s E. es is . do s Patent Application Publication Dec. 20, 2012 Sheet 43 of 45 US 2012/0322864 A1 . 3 4. E. s 32 24 s S is is a e S S S Patent Application Publication Dec. 20, 2012 Sheet 44 of 45 US 2012/0322864 A1 ############### $######### #####$#ffff; ######3 ~,,• ####### --- $###$ ~,,•••• ################################# ############# ############ --??• '$######### ############### ########################~### Patent Application Publication Dec. 20, 2012 Sheet 45 of 45 US 2012/0322864 A1 e yer i US 2012/0322864 A1 Dec. 20, 2012 SUSTAINED POLYPEPTIDE EXPRESSION N., et al., Proc.Jpn Acad Ser B Phy's Biol Sci, 2009.85(8): p. FROM SYNTHETIC, MODIFIED RNAS AND 348-62). Human pluripotent cells have also been derived USES THEREOF using two DNA-free methods: serial protein transduction with recombinant proteins incorporating cell-penetrating CROSS-REFERENCE TO RELATED peptide moieties (Kim, D., et al., Cell Stem Cell, 2009. 4(6): APPLICATIONS p. 472-476: Zhou, H., et al., Cell Stem Cell, 2009. 4(5): p. 0001. This application is a continuation application under 381-4), and infectious transgene delivery using the Sendai 35 U.S.C. S 120 of a co-pending U.S. application Ser. No. virus, which has a completely RNA-based reproductive cycle 13/088,009 filed on Apr. 15, 2011, which claims benefit under (Fusaki, N., et al., Proc.Jpn. Acad Ser B Phy's BiolSci, 2009. 35 U.S.C. S 119(e) of U.S. Provisional Patent Application Ser. 85(8): p. 348-62). No. 61/325,003 filed on Apr. 16, 2010 and U.S. Provisional Patent Application Ser. No. 61/387,220 filed on Sep. 28, SUMMARY 2010, the contents of which are incorporated herein by refer 0007 Provided herein are compositions, methods, and kits ence in their entirety. for changing the phenotype of a cell or cells. These methods, compositions, and kits can be used either to express a desired SEQUENCE LISTING protein in a cell or tissue, or to change the differentiated 0002 The instant application contains a Sequence Listing phenotype of a cell to that of another, desired cell type. which has been submitted in ASCII format via EFS-Web and Significantly, the methods, compositions, and kits described is hereby incorporated by reference in its entirety. Said ASCII herein do not utilize exogenous DNA or viral vector-based methods for the expression of protein(s), and thus, do not copy, created on Apr. 8, 2011, is named 67443US1.txt and is cause permanent modification of the genome or have the 7,196,077 bytes in size. potential for unintended mutagenic effects. FIELD OF THE INVENTION 0008. The compositions, methods, and kits described herein are based upon the direct introduction of synthetic 0003. The field of the invention relates to synthetic, modi RNAs into a cell, which, when translated, provide a desired fied RNAs and uses thereof.