Supercharged proteins for cell penetration

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 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 need thereof. The inventive complexes or pharmaceutical compositions thereof may be used to treat proliferative diseases, infectious diseases, cardiovascular diseases, inborn errors in metabolism, genetic diseases, etc.

1 - 131 . (canceled) 
     
     
         132 . A supercharged protein associated with a functional peptide or protein comprising:
 a supercharged protein having a theoretical net charge of at least +5 at physiological pH, wherein the supercharged protein is a supercharged protein variant of a wild-type protein and comprises a modified primary amino acid sequence as compared to the wild-type sequence; and   a functional peptide or protein selected from the group consisting of enzymes, DNA-binding proteins, histones, cytoskeletal proteins, receptor proteins, chaperone proteins, transcription factors, tumor suppressors, developmental regulators, growth factors, metastasis suppressors, pro-apoptotic proteins, and reprogramming factors,   wherein the supercharged protein associated with the functional peptide or protein is able to penetrate a cell and deliver the functional peptide or protein into the cell.   
     
     
         133 . The supercharged protein associated with a functional peptide or protein of  claim 132 , wherein the supercharged protein is covalently bound to the functional peptide or protein. 
     
     
         134 . The supercharged protein associated with a functional peptide or protein of  claim 132 , wherein the supercharged protein is covalently bound to the functional peptide or protein via a linker. 
     
     
         135 . The supercharged protein associated with a functional peptide or protein of  claim 134 , wherein the supercharged protein or the linker can be cleaved by a cellular enzyme. 
     
     
         136 . The supercharged protein associated with a functional peptide or protein of  claim 134 , wherein the linker comprises an amino acid sequence selected from the group consisting of: X-AGVF-X(SEQ ID NO: 136), X-GFLG-X (SEQ ID NO: 137), X-FK-X (SEQ ID NO: 138), X-AL-X (SEQ ID NO: 139), X-ALAL-X (SEQ ID NO: 140), and X-ALALA-X (SEQ ID NO: 141), wherein X denotes the supercharged protein or the functional peptide or protein. 
     
     
         137 . The supercharged protein associated with a functional peptide or protein of  claim 132 , wherein the supercharged protein is an immunoglobulin, a fragment of an immunoglobulin, or a immunoglobulin variant. 
     
     
         138 . The supercharged protein associated with a functional peptide or protein of  claim 132 , wherein the theoretical net charge of the supercharged protein is at least +10 at physiological pH. 
     
     
         139 . The supercharged protein associated with a functional peptide or protein of  claim 132 , wherein the supercharged protein has a charge to molecular weight ratio of at least 0.8. 
     
     
         140 . The supercharged protein associated with a functional peptide or protein of  claim 132 , wherein the functional protein is a protein chosen from the group consisting of histone acetyltransferases, histone deacetylases, DNA methyltransferases, kinases, phosphatases, proteases, oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, nucleases, zinc finger nucleases, and recombinases. 
     
     
         141 . The supercharged protein associated with a functional peptide or protein of  claim 142 , wherein the functional protein is a protein selected from the group consisting of: p53, Rb (retinoblastoma protein), BRCA1, BRCA2, PTEN, APC, CD95, ST7, ST14, BRMS1, CRSP3, DRG1, KAI1, KISS1, NM23, EGF, EPO, FGF, G-CSF, GM-CSF, HGF, HDGF, IGF, PDGF, TPO, TGF-α, TGF-β, VEGF, Cre recombinase, Dre recombinase, and FLP recombinase. 
     
     
         142 . The supercharged protein associated with a functional peptide or protein of  claim 132 , wherein the functional protein is a protein selected from the group consisting of BCL-2 family proteins, caspases, TIMP-family proteins, BMP-family growth factors, GDF-family growth factors, and zinc finger nucleases targeting a site within the human CCR5 gene. 
     
     
         143 . A supercharged immunoglobulin associated with a functional peptide or protein comprising:
 a supercharged immunoglobulin having a theoretical net charge of at least +5 at physiological pH, wherein the supercharged protein is a supercharged immunoglobulin variant of a wild-type immunoglobulin and comprises a modified primary amino acid sequence as compared to the wild-type sequence; and   a functional peptide or protein selected from the group consisting of enzymes, DNA-binding proteins, histones, cytoskeletal proteins, receptor proteins, chaperone proteins, transcription factors, tumor suppressors, developmental regulators, growth factors, metastasis suppressors, pro-apoptotic proteins, and reprogramming factors,   wherein the supercharged immunoglobulin associated with the functional peptide or protein is able to penetrate a cell and deliver the functional peptide or protein into the cell.   
     
     
         144 . A method of delivering a functional peptide or protein to a cell, comprising:
 contacting the cell with a supercharged protein associated with the functional peptide or protein of  claim 132 , under conditions sufficient for the functional peptide or protein to enter the cell.   
     
     
         145 . The method of  claim 144 , wherein the functional protein or peptide delivered to the cell is a transcription factor or a reprogramming factor. 
     
     
         146 . The method of  claim 144 , wherein the cell is a somatic cell from a subject diagnosed with a disease and is contacted with a supercharged protein associated with a reprogramming factor in an amount, for a time, and under conditions sufficient to induce reprogramming of the somatic cell to a pluripotent state. 
     
     
         147 . The method of  claim 146 , further comprising:
 isolating a pluripotent cell generated from the somatic cell;   differentiating the isolated pluripotent cell, or progeny thereof, into a differentiated cell type; and/or   using the pluripotent cell, or differentiated progeny thereof, in a cell replacement therapeutic approach.   
     
     
         148 . The method of  claim 144 , wherein the cell is a cell carrying a genomic allele associated with a disease, and the supercharged protein is associated with a nuclease specifically targeting the allele. 
     
     
         149 . The method of  claim 144 , wherein the functional protein is a recombinase, and the cell comprises a recombination site recognized by the recombinase in its genome. 
     
     
         150 . The method of  claim 149 , wherein the cell comprises a plurality of recombination sites recognized by the recombinase, and recombinase-mediated recombination of the plurality of recombination sites results in deletion of a genomic region. 
     
     
         151 . The method of  claim 144 , wherein the cell is a tumor cell, and the functional protein is a tumor suppressor protein, a metastasis suppressor protein, a cytostatic protein, or a cytotoxic protein.