(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) CORRECTED VERSION (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2010/129023 A9 11 November 2010 (11.11.2010) PCT (51) International Patent Classification: (74) Agent: BAKER, C , Hunter; Wolf, Greenfield & Sacks, C07K 14/00 (2006.01) P.C., 600 Atlantic Avenue, Boston, MA 02210-2206 (US). (21) International Application Number: PCT/US20 10/00 1250 (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (22) International Filing Date: AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, 28 April 2010 (28.04.2010) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, (25) Filing Language: English 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, (26) Publication Langi English KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, (30) Priority Data: ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, 61/173,430 28 April 2009 (28.04.2009) US NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, 61/321,428 6 April 2010 (06.04.2010) US SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicant (for all designated States except US): PRESI¬ DENT AND FELLOWS OF HARVARD COLLEGE (84) Designated States (unless otherwise indicated, for every [US/US]; 17 Quincy Street, Cambridge, MA 02138 (US). kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, (72) Inventors; and ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, (75) Inventors/Applicants (for US only): LIU, David, R. TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, [US/US]; 3 Whitman Circle, Lexington, MA 02420 (US). ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MCNAUGHTON, Brian, R. [US/US]; 1146 Cambridge MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, SM, Street, Apt. 2, Cambridge, MA 021 39 (US). CRONI- TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, CAN, James, Joseph [US/US]; 70 Park Street, #3 1, ML, MR, NE, SN, TD, TG). Somerville, MA 02138 (US). THOMPSON, David, B. Published: [US/US]; 50 Follen Street, # 11, Cambridge, MA 02138 (US). — without international search report and to be republished upon receipt of that report (Rule 48.2(g)) [Continued on next page] (54) Title: SUPERCHARGED PROTEINS FOR CELL PENETRATION FIGURE 44B < o (57) Abstract: Compositions, preparations, systems, and related methods for delivering a supercharged protein, or a complex of a supercharged protein and an agent (e.g., nucleic acids, peptides, proteins, small molecules) to cells are provided. Such systems and methods include the use of supercharged proteins. For example, superpositively charged proteins may be associated with nucleic acids (which typically have a net negative charge) via electrostatic interactions. In some embodiments, such systems and methods o involve altering the primary sequence of a protein in order to "supercharge" the protein (e.g., to generate a superpositively-charged protein). In some embodiments, complexes comprising supercharged proteins and one or more agents to be delivered are useful as therapeutic agents. In some embodiments, complexes and/or pharmaceutical compositions thereof are administered to a subject in o need thereof. The inventive complexes or pharmaceutical compositions thereof may be used to treat proliferative diseases, infec tious diseases, cardiovascular diseases, inborn errors in metabolism, genetic diseases, etc. w o 2010/129023 A9 1I 11 II I 1 I1 II I II III II I III III III II (48) Date of publication of this corrected version: (15) Information about Correction: 5 May 20 11 see Notice of 5 May 201 1 SUPERCHARGED PROTEINS FOR CELL PENETRATION Government Support [0001] This invention was made with U.S. Government support under contract number R01 GM 065400 awarded by the National Institutes of Health/NIGMS. The U.S. Government has certain rights in the invention. Background of the Invention [0002] The effectiveness of an agent intended for use as a therapeutic, diagnostic, or other application is often highly dependent on its ability to penetrate cellular membranes or tissue to induce a desired change in biological activity. Although many therapeutic drugs, diagnostic or other product candidates, whether protein, nucleic acid, organic small molecule, or inorganic small molecule, show promising biological activity in vitro, many fail to reach or penetrate target cells to achieve the desired effect, often due to physiochemical properties that result in inadequate biodistribution in vivo. [0003] In particular, nucleic acids have great potential as effective therapeutic agents and as research tools. The generality and sequence-specificity of siRNA-mediated gene regulation has raised the possibility of using siRNAs as gene-specific therapeutic agents (Bumcrot et al., 2006, Nat. Chem. Biol., 2:71 1-19; incorporated herein by reference). The suppression of gene expression by short interfering RNA (siRNA) has also emerged as a valuable tool for studying gene and protein function (Dorsett et al., 2004, Nat. Rev. Drug Discov., 3:318-29; Dykxhoorn et al., 2003, Nat. Rev. Mol. Cell. Biol., 4:457-67; Elbashir al., 2001, Nature, 4 11:494-98; each of which is incorporated herein by reference). However, the delivery of nucleic acids such as siRNAs to cells has been found to be unpredictable and is typically inefficient. One obstacle to effective delivery of nucleic acids to cells is inducing cells to take up the nucleic acid. Much work has been done to identify agents that can aid in the delivery of nucleic acids to cells. Commercially available cationic lipid reagents are typically used to transfect siRNA in cell culture. The effectiveness of cationic lipid-based siRNA delivery, however, varies greatly by cell type. Also, a number of cell lines including some primary neuron, T-cell, fibroblast, and epithelial cell lines have demonstrated resistance to common cationic lipid transfection techniques (Carlotti et al., 2004, Mol. Ther., 9:209-17; Ma et al, 2002, Neuroscience, 112:1-5; McManus et al., 2002, J. Immunol., 169:5754-60; Strait et al., 2007, Am. J. Physiol. Renal Physiol., 293:F601-06; each of which is incorporated herein by reference). Alternative transfection approaches including electroporation (Jantsch et ai, 2008, J. Immunol. Methods, 337:71-77; incorporated herein by reference) and virus- mediated siRNA delivery (Brummelkamp et ai, 2002, Cancer Cell, 2:243-47; Stewart et ai, 2003, RNA, 9:493-501; each of which is incorporated herein by reference) have also been used; however, these methods can be cytotoxic or perturb cellular function in unpredictable ways and have limited value for the delivery of nucleic acids (e.g., siRNA) as therapeutic agents in a subject. [0004] Recent efforts to address the challenges of nucleic acid delivery have resulted in a variety of new nucleic acid delivery platforms. These methods include lipidoids (Akinc et a , 2008, Nat. Biotechnoi, 26:561-69; incorporated herein by reference), cationic polymers (Segura and Hubbell, 2007, Bioconjug. Chem., 18:736-45; incorporated herein by reference), inorganic nanoparticles (Sokolova and Epple, Angew Chem. Int. Ed. Engl., 47:1382-95; incorporated herein by reference), carbon nanotubes (Liu et ai, 2007, Angew Chem. Int. Ed. Engl., 46:2023-27; incorporated herein by reference), cell-penetrating peptides (Deshayes et al, 2005, Cell Moi Life Sci., 62: 1839-49; and Meade and Dowdy, 2008, Adv. Drug Deliv. Rev., 60: 530-36; both of which are incorporated herein by reference), and chemically modified siRNA (Krutzfeldt et ai, 2005, Nature 438: 685-89; incorporated herein by reference). Each of these delivery systems offers benefits for particular applications; in most cases, however, questions regarding cytotoxicity, ease of preparation, stability, or generality remain. Easily prepared reagents capable of effectively delivering nucleic acids {e.g., siRNA) to a variety of cell lines without significant cytotoxicity therefore remain of considerable interest. [0005] Given the current interest in RNAi therapies and other nucleic acid-based therapies, there remains a need in the art for reagents and systems that can be used to deliver nucleic acids as well as other agents (e.g., peptides, proteins, small molecules) to a wide variety of cell types predictably and efficiently. [0006] Similarly, the inability of most proteins to spontaneously enter mammalian cells limits their usefulness as research tools and their potential as therapeutic agents. Proteins have demonstrated great potential as research tools (including hormones, cytokines, and antibodies) and as human therapeutics (including erythropoietin, insulin, and interferons). Due to the inability of most proteins to spontaneously enter cells, however, exogenous proteins are largely restricted to interacting with extracellular targets. Over the past decade, techniques for the delivery of proteins into mammalian cells have been developed to address intracellular targets. These techniques include lipid-based reagents (Zelphati et al, J. Biol. Chem. 276, 35103-351 10, 2001), nanoparticles (Hasadsri et al, J. Biol. Chem., 2009), vault ribonucleoprotein particles (Lai et al., ACSNano 3, 691-699, 2009), and genetic or chemical fusion to receptor ligands (Gabel et al, J. Cell Biol. 103, 1817-1827, 1986; Rizk et al, Proc. Natl. Acad. Sci. U.S.A. 106, 1101 1-1 1015, 2009) or cell-penetrating peptides (Wadia et al, Curr. Protein Pept. Sci. 4, 97-104, 2003; Zhou et al, Cell Stem Cell 4, 381-384, 2009). Perhaps the most common method for protein delivery is genetic fusion to protein transduction domains (PTDs) including the HIV-1 transactivator of transcription (Tat) peptide and polyarginine peptides.