2U11/13U624 A2
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(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date ft i 20 October 2011 (20.10.2011) 2U11/13U624 A2 (51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, C12N 5/10 (2006.01) CI2N 5/074 (2010.01) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, C12N 15/113 (2010.01) C07H 21/00 (2006.01) DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, (21) International Application Number: KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, PCT/US201 1/032679 ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (22) International Filing Date: NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, 15 April 201 1 (15.04.201 1) SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every (26) Publication Language: English kind of regional protection available): ARIPO (BW, GH, (30) Priority Data: GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, 61/325,003 16 April 2010 (16.04.2010) US ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, 61/387,220 28 September 2010 (28.09.2010) US TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, ΓΓ, LT, LU, (71) Applicant (for all designated States except US): IM¬ LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, MUNE DISEASE INSTITUTE, INC. [US/US]; 3 SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, Blackfan Circle, CLSB 3rd Floor, Boston, MA 021 15 GW, ML, MR, NE, SN, TD, TG). (US). Declarations under Rule 4.17 : (72) Inventors; and — as to applicant's entitlement to apply for and be granted (75) Inventors/Applicants (for US only): ROSSI, Derrick a patent (Rule 4.1 7(H)) [CA/US]; 162 Metropolitan Avenue, Roslindale, MA 0213 1 (US). WARREN, Luigi [GB/US]; 170 Brookline — as to the applicant's entitlement to claim the priority of Avenue, #6 12, Boston, MA 02215 (US). the earlier application (Rule 4.17(Hi)) (74) Agents: RESNICK, David et al; Nixon Peabody LLP, Published: 100 Summer Street, Boston, MA 021 10 (US). — without international search report and to be republished (81) Designated States (unless otherwise indicated, for every upon receipt of that report (Rule 48.2(g)) kind of national protection available): AE, AG, AL, AM, — with sequence listing part of description (Rule 5.2(a)) (54) Title: SUSTAINED POLYPEPTIDE EXPRESSION FROM SYNTHETIC, MODIFIED RNAS AND USES THEREOF < o (57) Abstract: Described herein are synthetic, modified RNAs for changing the phenotype of a cell, such as expressing a polypep o tide or altering the developmental potential. Accordingly, provided herein are compositions, methods, and kits comprising syn thetic, modified RNAs for changing the phenotype of a cell or cells. These methods, compositions, and kits comprising synthetic, modified RNAs can be used either to express a desired protein in a cell or tissue, or to change the differentiated phenotype of a cell to that of another, desired cell type. SUSTAINED POLYPEPTIDE EXPRESSION FROM SYNTHETIC, MODIFIED RNAS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Serial No.: 61/325,003 filed on April 16, 2010 and U.S. Provisional Patent Application Serial No.: 61/387,220 filed on September 28, 2010, the contents of which are incorporated herein by reference in their entirety. SEQUENCE LISTING [0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 8, 2011, is named 67442PCT.txt and is 7,196,077 bytes in size. FIELD OF THE INVENTION [0003] The field of the invention relates to synthetic, modified RNAs and uses thereof. BACKGROUND [0004] The ability to change the phenotype of a cell or cells, either to express a desired protein or to change the differentiated phenotype of the cell to that of another, desired cell type, has applications in both research and therapeutic settings. The phenotype of a cell is most commonly modified by expression of protein(s) from exogenous DNA or from recombinant viral vectors. These approaches have the potential for unintended mutagenic effects. [0005] One area of interest is the modification of cellular differentiation such that cells are directed to different developmental lineages. As one example, generating insulin-producing pancreatic β cells from acinar pancreatic cells or other somatic cell types, has the potential to treat diabetes. As but one other example, the ability to redifferentiate a tumor cell or tumor stem cell to a non-cancerous cell type can provide a therapy for cancer. Current protocols for altering cell fate tend to focus on the expression of factors, such as differentiation factors, dedifferentiation factors, transdifferentiation factors, and reprogramming factors, using viral- or DNA-mediated expression. [0006] An area of recent focus is the production of pluripotent or multipotent stem cells from non-embryonic sources. Induction of pluripotency was originally achieved by Yamanaka and colleagues using retroviral vectors to enforce expression of four transcription factors, KLF4, c-MYC, OCT4, and SOX2 (KMOS) (Takahashi, K. and S. Yamanaka, Cell, 2006. 126(4): p. 663-76 ; Takahashi, K., et al., Cell, 2007. 131(5): p. 861-72). Attempts to derive induced pluripotent stem (iPS) cells have also been made using excisable lentiviral and transposon vectors, or through repeated application of transient plasmid, episomal, and adenovirus vectors (Chang, C.-W., et al., Stem Cells, 2009. 27(5): p. 1042-1049; Kaji, K., et al., Nature, 2009. 458(7239): p. 771-5; Okita, K., et al., Science, 2008. 322(5903): p. 949-53; Stadtfeld, M., et al., Science, 2008. 322(5903): p. 945-9; Woltjen, K., et al., Nature, 2009; Yu, J., et al., Science, 2009: p. 1172482; Fusaki, N., et al., Proc Jpn Acad Ser B Phys Biol Sci, 2009. 85(8): p. 348-62). Human pluripotent cells have also been derived using two DNA-free methods: serial protein transduction with recombinant proteins incorporating cell-penetrating peptide moieties (Kim, D., et al., Cell Stem Cell, 2009. 4(6): p. 472-476; Zhou, H., et al., Cell Stem Cell, 2009. 4(5): p. 381-4), and infectious transgene delivery using the Sendai virus, which has a completely RNA-based reproductive cycle (Fusaki, N., et al., Proc Jpn Acad Ser B Phys Biol Sci, 2009. 85(8): p. 348-62). SUMMARY [0007] Provided herein are compositions, methods, and kits for changing the phenotype of a cell or cells. These methods, compositions, and kits can be used either to express a desired protein in a cell or tissue, or to change the differentiated phenotype of a cell to that of another, desired cell type. Significantly, the methods, compositions, and kits described herein do not utilize exogenous DNA or viral vector-based methods for the expression of protein(s), and thus, do not cause permanent modification of the genome or have the potential for unintended mutagenic effects. [0008] The compositions, methods, and kits described herein are based upon the direct introduction of synthetic RNAs into a cell, which, when translated, provide a desired protein or proteins. Higher eukaryotic cells have evolved cellular defenses against foreign, "non-self," RNA that ultimately result in the global inhibition of cellular protein synthesis, resulting in cellular toxicity. This response involves, in part, the production of Type I or Type II interferons, and is generally referred to as the "interferon response" or the "cellular innate immune response." The cellular defenses normally recognize synthetic RNAs as foreign, and induce this cellular innate immune response. The inventors have recognized that the ability to achieve sustained or repeated expression of an exogenously directed protein using synthetic RNA is hampered by the induction of this innate immune response. In the methods described herein, the effect of the cellular innate immune response is mitigated by using synthetic RNAs that are modified in a manner that avoids or reduces the response. Avoidance or reduction of the innate immune response permit sustained expression from exogenously introduced RNA necessary, for example, to modify the developmental phenotype of a cell. In one aspect, sustained expression is achieved by repeated introduction of synthetic, modified RNAs into a target cell or its progeny. [0009] The modified, synthetic RNAs described herein, in one aspect, can be introduced to a cell in order to induce exogenous expression of a protein of interest in a cell. The ability to direct exogenous expression of a protein of interest using the modified, synthetic RNAs described herein is useful, for example, in the treatment of disorders caused by an endogenous genetic defect in a cell or organism that impairs or prevents the ability of that cell or organism to produce the protein of interest. Accordingly, in some embodiments, compositions and methods comprising the modified, synthetic RNAs described herein can be used for the purposes of gene therapy. [0010] The modified, synthetic RNAs described herein can advantageously be used in the alteration of cellular fates and/or developmental potential. The ability to express a protein from an exogenous RNA permits both the alteration or reversal of the developmental potential of a cell, i.e., the reprogramming of the cell, and the directed differentiation of a cell to a more differentiated phenotype.