(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 WO 2016/070129 Al 6 May 2016 (06.05.2016) W P O P C T (51) International Patent Classification: (74) Agent: BAKER, C , Hunter; Wolf, Greenfield & Sacks, A61K 9/00 (2006.01) C07K 14/435 (2006.01) P.C., 600 Atlantic Avenue, Boston, MA 02210-2206 (US). (21) International Application Number: (81) Designated States (unless otherwise indicated, for every PCT/US20 15/058479 kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (22) International Filing Date: BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, 30 October 2015 (30.10.201 5) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (25) Filing Language: English HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, (26) Publication Language: English MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, (30) Priority Data: PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, 14/529,010 30 October 2014 (30. 10.2014) US SD, 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: PRESIDENT AND FELLOWS OF HAR¬ VARD COLLEGE [US/US]; 17 Quincy Street, Cam (84) Designated States (unless otherwise indicated, for every bridge, MA 02138 (US). kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, (72) Inventors: LIU, David, R.; 3 Whitman Circle, Lexington, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, MA 02420 (US). THOMPSON, David, B.; 50 Follen TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, Street, # 11, Cambridge, MA 02138 (US). ZURIS, John, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, Anthony; 60 Wadsworth St., Apt. 22B, Cambridge, MA LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, 02142 (US). SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). [Continued on next page] (54) Title: DELIVERY OF NEGATIVELY CHARGED PROTEINS USING CATIONIC LIPIDS (57) Abstract: Compositions, methods, strategies, kits, and systems for the delivery of negatively charged proteins, protein complexes, and fusion pro teins, using cationic polymers or lipids are provided. Delivery of proteins into cells can be effected in vivo, ex vivo, or in vitro. Proteins that can be de Net negatively Cationic lipids livered using the compositions, methods, strategies, kits, and systems charged protein provided herein include, without limitation, enzymes, transcription factors, genome editing proteins, Cas9 proteins, TALEs, TALENs, nucleases, binding Treatment of proteins (e.g., ligands, receptors, antibodies, antibody fragments; nucleic acid mammalian cells with cationic lipidanionic binding proteins, etc.), structural proteins, and therapeutic proteins (e.g., tu - protein complexes mor suppressor proteins, therapeutic enzymes, growth factors, growth factor receptors, transcription factors, proteases, etc.), as well as variants and fu sions of such proteins. Extracellularspace Cytoplasm \ \ Liposome endoc tosis Endosomai escape Endosome o FIG. 27B © v o o w o 2016/070129 Al II 11 II I 1 Illlll I II II III II III III II I II Published: DELIVERY OF NEGATIVELY CHARGED PROTEINS USING CATIONIC LIPIDS RELATED APPLICATION [0001] This application is claims priority to U.S. patent application, U.S.S.N. 14/529,010, filed October 30, 2014, which is a continuation-in-part of and claims priority under 35 U.S.C. § 120 to U.S. patent application, U.S.S.N. 14/462,189, filed August 18, 2014, to U.S. patent application, U.S.S.N. 14/462,163, filed August 18, 2014, and to International Application, PCT/US2014/05424735, filed September 5, 2014, which claims priority under U.S.C. § 365(c) to U.S. patent application, U.S.S.N. 14/462,189, filed August 18, 2014, and to U.S. patent application, U.S.S.N. 14/462,163, filed August 18, 2014, and also claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent application, U.S.S.N. 61/874,746, filed September 6, 2013, the entire contents of each of which are incorporated herein by reference. BACKGROUND OF THE INVENTION [0002] Macromolecular delivery into mammalian cells is an attractive approach for cell manipulation, as it would allow modulation of gene expression and modification of the genome, which, in turn, would open new avenues for research and enable the therapeutic targeting of molecules currently viewed as "undruggable" by small molecules. In particular, recombinant nucleases targeting genes or alleles associated with disease have great potential as therapeutic agents. The current methods of macromolecular delivery include viral delivery of nucleic acid molecules, receptor-mediated delivery of nucleic acids or proteins, and the use of protein fusions with cell-penetrating peptides such as TAT, Arg9, or Penetratin for the delivery of proteins. Each of these delivery systems offers benefits for particular applications; in most cases, however, questions regarding efficacy, cytotoxicity, and ease of preparation remain. Easily prepared reagents capable of effectively delivering macromolecules (e.g. , functional effector proteins) to a variety of cell lines without significant cytotoxicity or other adverse side effect remain of considerable concern. [0003] Most proteins do not spontaneously enter mammalian cells and are thus naturally limited in their use as research tools and their potential as therapeutic agents. Techniques for the delivery of proteins into mammalian cells have been developed recently to address intracellular targets. These techniques include the use of lipid-based reagents (Zelphati et a , J. Biol. Chem. 276, 35103-351 10, 2001), nanoparticles (Hasadsri et al., J. Biol. Chem., 2009), vault ribonucleoprotein particles (Lai et al, ACS Nano 3, 691-699, 2009); 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); and fusion to 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. These cationic PTDs promote association with negatively charged cell-surface structures and subsequent endocytosis of exogenous proteins. Both Tat and polyarginine have been used to deliver a variety of macromolecules into cells both in vitro and in vivo (Wadia et al, Curr. Protein Pept. Sci. 4, 97-104, 2003; Zhou et al, Cell Stem Cell 4, 381-384, 2009; Myou et al, J. Immunol. 169, 2670-2676, 2002; Bae et al, Clin. Exp. Immunol. 157, 128-138, 2009; Schwarze et al, Science 285, 1569-1572, 1999). Despite these advances, intracellular targets remain difficult to affect using exogenous proteins, and even modest success can require toxic concentrations of the respective transduction agent due to the low efficiency with which proteins are functionally delivered into cells (Zhou et al, Cell Stem Cell 4, 381-384, 2009; Wang et al, Nat. Biotechnol. 26, 901-908, 2008). Therefore, there remains a need for better delivery systems for getting functional effector proteins into cells to target intracellular biomolecules. SUMMARY OF THE INVENTION [0004] The present disclosure provides systems, compositions, preparations, kits, and related methods for delivering proteins into cells using cationic polymers or cationic lipids. In some embodiments, the proteins to be delivered are negatively charged proteins, also referred to herein as anionic proteins, such as, for example, naturally occurring or engineered negatively charged proteins. In some embodiments, the proteins to be delivered are associated with a negatively charged protein, e.g., via covalent or non-covalent interactions, to form a protein complex. In some such embodiments, the complex comprising the protein to be delivered associated with the negatively charged proteins, e.g., a supernegatively charged protein or a naturally occurring negatively charged protein, has a net negative charge. In some embodiments, the proteins to be delivered bind a nucleic acid thus forming a protein:nucleic acid complex having a net negative charge. [0005] Some aspects of this disclosure are based on the discovery that negatively charged proteins can be associated with cationic polymers or cationic lipids and that such protein:lipid complexes are efficiently delivered into cells. This technology can be applied to naturally negatively charged proteins (e.g., the proteins listed in tables 3-6, Sirtl (-59, 86 kDa), PPARg (- 13, 54 kDa), PRDM16 (-23, 140 kDa), PGCla (-15, 9 1 kDa), TP53BP1 (-148, 213 kDa), Utrophin (-142, 394 kDa), Dystrophin (-89, 426 kDa), Bik (-17, 18 kDa), ΙκΒα (-29, 35 kDa), Von Hippel-Lindau disease tumor suppressor (-18, 24 kDa), E3 ubiquitin ligases, metal-binding proteins, VP64 transcriptional activators, the anionic 3xFLAG peptide tag, and fusions thereof), to engineered supernegatively charged proteins (e.g., supernegatively charged GFP or streptavidin variants), to proteins that bind to nucleic acids and form negatively charged protein:nucleic acid complexes (e.g., Cas9 proteins, and variants and fusions thereof), or to protein fusions in which a protein to be delivered is associated with a negatively