Delivery system for functional nucleases

Abstract

Compositions, methods, strategies, kits, and systems for the supercharged protein-mediated delivery of functional effector proteins into cells in vivo, ex vivo, or in vitro are provided. Compositions, methods, strategies, kits, and systems for delivery of functional effector proteins using cationic lipids and cationic polymers are also provided. Functional effector proteins include, without limitation, transcriptional modulators (e.g., repressors or activators), recombinases, nucleases (e.g., RNA-programmable nucleases, such as Cas9 proteins; TALE nuclease, and zinc finger nucleases), deaminases, and other gene modifying/editing enzymes. Functional effector proteins include TALE effector proteins, e.g., TALE transcriptional activators and repressors, as well as TALE nucleases. Compositions, methods, strategies, and systems for the delivery of functional effector proteins into cells is useful for therapeutic and research purposes, including, but not limited to, the targeted manipulation of a gene associated with disease, the modulation of the expression level of a gene associated with disease, and the programming of cell fate.

1 - 62 . (canceled) 
     
     
         63 . A composition comprising a Cas9 protein and a cationic lipid a cationic polymer, or both a cationic lipid and cationic polymer, wherein the Cas9 protein is associated with a gRNA, and the composition is capable of delivering the Cas9 protein to the interior of a cell. 
     
     
         64 . The composition of  claim 63 , wherein the composition exhibits low toxicity when administered to a population of cells. 
     
     
         65 . The composition of  claim 63 , wherein at least 60% of the cells are viable following administration of the composition. 
     
     
         66 . The composition of  claim 63 , wherein the cationic lipids are selected from the group consisting of Lipofectamine® 2000, Lipofectamine® 3000, Lipofectamine® RNAiMAX, and Lipofectamine® LTX. 
     
     
         67 . The composition of  claim 63 , wherein the Cas9 protein comprises a wild type Cas9 protein or a variant thereof, a Cas9 nickase, or a nuclease-inactivated Cas9 (dCas9) or a variant thereof. 
     
     
         68 . The composition of  claim 67 , wherein dCas9 is fused to a transcriptional activator or a transcriptional repressor. 
     
     
         69 . The composition of  claim 68 , wherein the transcriptional activator is selected from the group consisting of VP16, VP64, and p65. 
     
     
         70 . The composition of  claim 67 , wherein dCas9 is fused to a nuclease domain. 
     
     
         71 . The composition of  claim 70 , wherein nuclease domain comprises a FokI nuclease domain. 
     
     
         72 . The composition of  claim 67 , wherein the dCas9 is fused to a recombinase catalytic domain. 
     
     
         73 . The composition of  claim 72 , wherein the recombinase catalytic domain comprises a Hin recombinase catalytic domain, a Gin recombinase catalytic domain, or a Tn3 recombinase catalytic domain. 
     
     
         74 - 75 . (canceled) 
     
     
         76 . The composition of  claim 67 , wherein dCas9 is fused to an epigenetic modifier. 
     
     
         77 . The composition of  claim 76 , wherein the epigenetic modifier is selected from the group consisting of histone demethylase, histone methyltransferase, hydroxylase, histone deacetylase, and histone acetyltransferase. 
     
     
         78 . The composition of  claim 77 , wherein the epigenetic modifier comprises the LSD1 histone demethylase or TET1 hydroxylase. 
     
     
         79 - 90 . (canceled) 
     
     
         91 . A method comprising administering the pharmaceutical composition of  claim 63 , to a subject in need thereof. 
     
     
         92 . A method of introducing a Cas9 protein into a cell, the method comprising contacting the cell with the composition of  claim 63 , under conditions suitable for the Cas9 protein to enter the cell, thereby introducing the Cas9 protein into the cell. 
     
     
         93 . The composition of  claim 68 , wherein the transcriptional repressor is a KRAB protein or a SID protein. 
     
     
         94 . The composition of  claim 63 , wherein the Cas9 protein comprises a wild type Cas9 protein or a Cas9 variant that comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 44, a Cas9 nickase, or a nuclease-inactivated Cas9 (dCas9) that comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 45 and comprises a D10A and/or a H840A substitution as compared to the amino acid sequence of a wild type Cas9. 
     
     
         95 . The composition of  claim 63  further comprising a nuclear localization signal (NLS).