Delivery of negatively charged proteins using cationic lipids

Abstract

Compositions, methods, strategies, kits, and systems for the delivery of negatively charged proteins, protein complexes, and fusion proteins, 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 delivered using the compositions, methods, strategies, kits, and systems provided herein include, without limitation, enzymes, transcription factors, genome editing proteins, Cas9 proteins, TALEs, TALENs, nucleases, binding proteins (e.g., ligands, receptors, antibodies, antibody fragments; nucleic acid binding proteins, etc.), structural proteins, and therapeutic proteins (e.g., tumor suppressor proteins, therapeutic enzymes, growth factors, growth factor receptors, transcription factors, proteases, etc.), as well as variants and fusions of such proteins.

1 - 49 . (canceled) 
     
     
         50 . A composition for delivering a protein into a cell, comprising
 (a) a Cas9 protein;   (b) a negatively charged molecule associated with the Cas9 protein resulting in a complex that is negatively charged; and   (c) a cationic polymer or a cationic lipid.   
     
     
         51 . The composition of  claim 50 , wherein the negatively charged molecule associated with the Cas9 protein is a guide RNA (gRNA). 
     
     
         52 . The composition of  claim 50 , wherein the Cas9 protein is a catalytically inactive Cas9 protein (dCas9). 
     
     
         53 . The composition of  claim 50 , wherein the composition comprises a net negative charge. 
     
     
         54 . The composition of  claim 51 , wherein the Cas9 protein and the guide RNA (gRNA) nucleic acid complex has a net charge of less than −5. 
     
     
         55 . The composition of  claim 51 , wherein the Cas9 protein and the guide RNA (gRNA) nucleic acid complex has a charge:molecular weight ratio of less than −0.03. 
     
     
         56 . A composition for delivering a protein into a cell, the composition comprising:
 (a) a protein associated with a supernegatively charged protein resulting in a protein:supernegatively charged protein complex that is negatively charged; and   (b) a cationic lipid,   wherein the complex has a net negative charge.   
     
     
         57 . The composition of  claim 56 , wherein the net negative charge of the complex is about −5 to about −50. 
     
     
         58 . The composition of  claim 56 , wherein the net negative charge of the complex is less than about −5. 
     
     
         59 . The composition of  claim 56 , wherein the complex has a charge:molecular weight ratio of less than −0.03. 
     
     
         60 . The composition of  claim 56 , wherein the protein retains its biological function after delivery into the cell and cellular uptake. 
     
     
         61 . The composition of  claim 56 , wherein the protein is a naturally-occurring negatively charged protein, or a negatively charged fragment thereof. 
     
     
         62 . The composition of  claim 56 , wherein the protein is a supernegatively charged protein or is engineered to be supernegatively charged, or a negatively charged fragment thereof. 
     
     
         63 . The composition of  claim 56 , wherein the protein:supernegatively charged protein complex has a net charge of less than −5. 
     
     
         64 . The composition of  claim 56 , wherein the protein is not negatively charged or supernegatively charged. 
     
     
         65 . The composition of  claim 56 , wherein the protein is fused to the supernegatively charged protein to form a fusion protein. 
     
     
         66 . The composition of  claim 56 , wherein the protein:supernegatively charged protein complex has a charge:molecular weight ratio of less than −0.03. 
     
     
         67 . The composition of  claim 56 , wherein the cationic lipid is selected from the group consisting of Lipofectamine® 2000, Lipofectamine® 3000, Lipofectamine® RNAiMAX, and Lipofectamine® LTX. 
     
     
         68 . The composition of  claim 50 , wherein the composition exhibits low toxicity when administered to a population of cells. 
     
     
         69 . The composition of  claim 68 , wherein at least 60% of the cells of the population are viable 24 hours after administration of an amount of the composition effective for delivery of the protein to be delivered into at least 1% of the cells. 
     
     
         70 . The composition of  claim 56 , wherein the composition exhibits low toxicity when administered to a population of cells. 
     
     
         71 . The composition of  claim 70 , wherein at least 60% of the cells of the population are viable 24 hours after administration of an amount of the composition effective for delivery of the protein to be delivered into at least 1% of the cells. 
     
     
         72 . The composition of  claim 50  further comprising a pharmaceutically acceptable excipient. 
     
     
         73 . The composition of  claim 56  further comprising a pharmaceutically acceptable excipient. 
     
     
         74 . The composition of  claim 56 , wherein the protein is implicated in a disease or disorder. 
     
     
         75 . The composition of  claim 74 , wherein the protein is a tumor suppressor protein, therapeutic enzyme, growth factor, growth factor receptor, or transcription factor. 
     
     
         76 . The composition of  claim 74 , wherein the protein is selected from Sirt1, PPARγ, PRDM16, PGC1a, TP53BP1, Utrophin, Dystrophin, Bik, IκBα, Von Hippel-Lindau disease tumor suppressor, and an E3 ubiquitin ligase. 
     
     
         77 . The composition of  claim 74 , wherein the protein is selected from TACC2, TP53BP1, DMBT1, BRCA1, ANP32A, UBR5, EPB41L3, KANK1, PHLPP2, CHD5, ZBTB7C, URIl, FBLN1, DLEC1, LRP12, LGR6, PHLPP1, MCC, BIN1, TSC1, E4F1, ATM, FOCAD, TP53INP1, REB3, SASH1, MN1, ARHGAP35, PDCD4, VHL, CADM1, MTUS2, ZGPAT, GLTSCR1, BCL11B, TP53BP2, DFNA5, FANCG, FLCN, NDRG1, NDRG2, ST13, CHFR, NBL1, SIRT2, DAB2, EAF2, EXTL3, FRMD3, PIDD1, and PTCH1. 
     
     
         78 . A composition comprising:
 (a) a protein that is associated with a negatively charged or a supernegatively charged protein resulting in a complex that is negatively charged; and   (b) a cationic polymer or a cationic lipid, wherein the composition comprises a net negative charge.   
     
     
         79 . The composition of  claim 78 , wherein the supernegatively charged protein comprises −7 GFP or −20 GFP, or a negatively charged fragment thereof. 
     
     
         80 . The composition of  claim 78 , wherein the protein is associated with the negatively charged or supernegatively charged protein via a non-covalent interaction. 
     
     
         81 . A method for delivering a protein to a cell, the method comprising contacting the cell with the composition of  claim 50 . 
     
     
         82 . The method of  claim 81 , wherein the contacting is performed in vivo, in vitro, or ex vivo. 
     
     
         83 . A method for delivering a protein to a cell, the method comprising contacting the cell with the composition of  claim 56 . 
     
     
         84 . The method of  claim 83 , wherein the contacting is performed in vivo, in vitro, or ex vivo. 
     
     
         85 . A method for delivering a protein to a cell, the method comprising contacting the cell with the composition of  claim 78 . 
     
     
         86 . The method of  claim 85 , wherein the contacting is performed in vivo, in vitro, or ex vivo.