Ribonucleoprotein transfection agents

Abstract

Provided herein are compositions and methods useful, inter alia, for the delivery of ribonucleoprotein complexes (e.g., Cas9/guide RNA complexes) into cells. The compositions and methods provided herein are particularly useful for the delivery of ribonucleoprotein complexes into pluripotent cells and lymphatic cells.

1 - 19 . (canceled) 
     
     
         20 . A method of forming a cell transfection composition, the method comprising:
 (i) contacting a ribonucleic acid, an endonuclease and an enhancer element, thereby forming a ribonucleoprotein-enhancer element complex; and   (ii) contacting a lipid aggregate-forming cationic lipid with said ribonucleoprotein-enhancer element complex, thereby forming a cell transfection composition.   
     
     
         21 . The method of  claim 20 , wherein said contacting of step (i) comprises contacting said ribonucleic acid with said endonuclease, thereby forming a ribonucleoprotein. 
     
     
         22 . The method of  claim 21 , wherein said ribonucleoprotein is contacted with said enhancer element, thereby forming said ribonucleoprotein-enhancer element complex. 
     
     
         23 . The method of  claim 20 , wherein said contacting of step (i) comprises:
 (ia) contacting said ribonucleic acid with said endonuclease, thereby forming a ribonucleoprotein; and   (ib) contacting said ribonucleoprotein with said enhancer element, thereby forming said ribonucleoprotein-enhancer element complex.   
     
     
         24 . The method of one of claims  claim 20 , wherein said ribonucleoprotein-enhancer element complex is in a first vessel and said lipid aggregate-forming cationic lipid is a in a second vessel. 
     
     
         25 . The method of  claim 24 , wherein said ribonucleoprotein-enhancer element complex is contacted with said lipid aggregate-forming cationic lipid in said second vessel. 
     
     
         26 . The method of  claim 24 , wherein said ribonucleoprotein-enhancer element complex is contacted with said lipid aggregate-forming cationic lipid in said first vessel. 
     
     
         27 . The method of one of  claim 20 , wherein said contacting of step (i) is in a physiologically acceptable solution. 
     
     
         28 . The method of  claim 20 , wherein said contacting of step (ii) is in a physiologically acceptable solution. 
     
     
         29 . (canceled) 
     
     
         30 . The method of  claim 20 , wherein said endonuclease is CRISPR associated protein 9 (Cas9). 
     
     
         31 . The method of one of  claims 20   30   claim 20 , wherein said ribonucleic acid is a guide RNA. 
     
     
         32 . The method of  claim 20 , wherein said lipid aggregate-forming cationic lipid has the structure of formula: 
       wherein 
       
         
           
           
               
               
           
         
           
         R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8  and R 9  are independently hydrogen, halogen, —CY 3 , —CN, —C(O)OH, —CH 2 C(O)OH, —C(O)NH 2 , —OH, —SH, —SO 2 Cl, —SO 3 H, —SO 4 H, —SO 2 NH 2 , —NO 2 , —NH 2 , —NHNH 2 , —ONH 2 , —NHC═(O)NHNH 2 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; 
         L 1  and L 2  are independently —C(O)—, —C(O)O—, —OC(O)—, —C(O)NH—, —NH—, —NHC(O)—, —O—, —S—, —S(O)—, —S(O) 2 NH—, —NHS(O) 2 —, substituted or unsubstituted alkylene or substituted or unsubstituted heteroalkylene; 
         1 is an integer from 1-8; 
         X is an anion; and 
         Y is —F, —Cl, —Br, or —I. 
       
     
     
         33 . The method of one of  claims 20   claim 32 , wherein said lipid aggregate-forming cationic lipid has the structure of formula: 
       
         
           
           
               
               
           
         
       
       wherein
 n is an integer from 1-6. 
 
     
     
         34 . The method of  claim 32 , wherein said lipid aggregate-forming cationic lipid has the structure of formula: 
       
         
           
           
               
               
           
         
       
       wherein
 m is an integer from 1-10; and 
 o and p are independently integers from 8-30 
 
     
     
         35 . (canceled) 
     
     
         36 . The method of  claim 20 , wherein said enhancer element comprises a polyamine moiety covalently attached to a nuclear localization signal sequence. 
     
     
         37 . The method of  claim 36 , wherein said polyamine moiety is a spermine moiety. 
     
     
         38 . (canceled) 
     
     
         39 . The method of  claim 36 , wherein said polyamine moiety is attached to the N-terminus of said enhancer element. 
     
     
         40 . The method of  claim 20 , wherein said enhancer element comprises the sequence of SEQ ID NO:1-SEQ ID NO:46. 
     
     
         41 . The method of  claim 20 , wherein said enhancer element comprises at least two nuclear localization signal (NLS) sequences. 
     
     
         42 . The method of  claim 20 , further comprising prior to said contacting in (ii) contacting said ribonucleoprotein-enhancer element complex with a donor nucleic acid. 
     
     
         43 - 66 . (canceled) 
     
     
         67 . A method of forming a cell transfection composition, the method comprising:
 (i) contacting a lipid aggregate-forming cationic lipid with a cell culture medium, thereby forming a lipid aggregate-forming cationic lipid medium;   (ii) contacting a ribonucleic acid, an endonuclease and an enhancer element, thereby forming a ribonucleoprotein-enhancer element complex;   (iii) contacting said lipid aggregate-forming cationic lipid medium with said ribonucleoprotein-enhancer element complex, thereby forming a cell transfection composition.   
     
     
         68 . A method of transfecting a ribonucleoprotein complex into a cell, the method comprising:
 contacting a cell with a composition comprising a ribonucleoprotein complex, a lipid aggregate-forming cationic lipid and an enhancer element,   wherein said lipid aggregate forming cationic lipid has the structure of formula:   
       
         
           
           
               
               
           
         
       
       wherein
 R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8  and R 9  are independently hydrogen, halogen, —CY 3 , —CN, —C(O)OH, —CH 2 C(O)OH, —C(O)NH 2 , —OH, —SH, —SO 2 Cl, —SO 3 H, —SO 4 H, 
 —SO 2 NH 2 , —NO 2 , —NH 2 , —NHNH 2 , —ONH 2 , —NHC═(O)NHNH 2 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; 
 L 1  and L 2  are independently —C(O)—, —C(O)O—, —OC(O)—, —C(O)NH—, —NH—, —NHC(O)—, —O—, —S—, —S(O)—, —S(O) 2 NH—, —NHS(O) 2 —, substituted or unsubstituted alkylene or substituted or unsubstituted heteroalkylene; 
 1 is an integer from 1-8; 
 X is an anion; and 
 Y is —F, —Cl, —Br, or —I.