Compositions and methods for nucleic acid and/or protein payload delivery

Abstract

Provided are methods and compositions for nanoparticle delivery of payloads (e.g., nucleic acid and/or protein payloads) to cells. In some embodiments, a subject nanoparticle includes a core and a sheddable layer encapsulating the core, where the core includes (i) an anionic polymer composition; (ii) a cationic polymer composition; (iii) a cationic polypeptide composition; and (iv) a nucleic acid and/or protein payload; and where: (a) the anionic polymer composition includes polymers of D-isomers of an anionic amino acid and polymers of L-isomers of an anionic amino acid, and/or (b) the cationic polymer composition comprises polymers of D-isomers of a cationic amino acid and polymers of L-isomers of a cationic amino acid. In some cases, the polymers of D-isomers of an anionic and/or cationic amino acid are present at a ratio, relative to the polymers of L-isomers, in a range of from 10:1 to 1:10.

What is claimed is: 
     
         1 . A nanoparticle, comprising a core and a sheddable layer encapsulating the core, wherein the core comprises:
 (i) an anionic polymer composition;   (ii) a cationic polymer composition;   (iii) a cationic polypeptide composition; and   (iv) a nucleic acid and/or protein payload,   wherein (a) said anionic polymer composition comprises polymers of D-isomers of an anionic amino acid and polymers of L-isomers of an anionic amino acid; and/or (b) said cationic polymer composition comprises polymers of D-isomers of a cationic amino acid and polymers of L-isomers of a cationic amino acid.   
     
     
         2 . The nanoparticle of  claim 1 , wherein said anionic polymer composition comprises a first anionic polymer selected from poly(D-glutamic acid) (PDEA) and poly(D-aspartic acid) (PDDA); and comprises a second anionic polymer selected from poly(L-glutamic acid) (PLEA) and poly(L-aspartic acid) (PLDA). 
     
     
         3 . The nanoparticle of  claim 1  or  claim 2 , wherein said cationic polymer composition comprises a first cationic polymer selected from poly(D-arginine), poly(D-lysine), poly(D-histidine), poly(D-ornithine), and poly(D-citrulline); and comprises a second cationic polymer selected from poly(L-arginine), poly(L-lysine), poly(L-histidine), poly(L-ornithine), and poly(L-citrulline). 
     
     
         4 . The nanoparticle of any one of  claims 1 - 3 , wherein said polymers of D-isomers of an anionic amino acid are present at a ratio, relative to said polymers of L-isomers of an anionic amino acid, in a range of from 10:1 to 1:10. 
     
     
         5 . The nanoparticle of any one of  claims 1 - 4 , wherein said polymers of D-isomers of a cationic amino acid are present at a ratio, relative to said polymers of L-isomers of a cationic amino acid, in a range of from 10:1 to 1:10. 
     
     
         6 . The nanoparticle of any one of  claims 1 - 5 , wherein the sheddable layer is an anionic coat. 
     
     
         7 . The nanoparticle of any one of  claims 1 - 6 , wherein the sheddable layer is pH and/or glutathione sensitive. 
     
     
         8 . The nanoparticle of any one of  claims 1 - 7 , wherein the sheddable layer comprises one or more of: silica, a peptoid, a polycysteine, calcium, calcium phosphate, calcium sulfate, manganese, manganese phosphate, manganese sulfate, magnesium, magnesium phosphate, magnesium sulfate, iron, iron phosphate, iron sulfate, lithium, lithium phosphate, and lithium sulfate. 
     
     
         9 . The nanoparticle of  claim 8 , wherein the sheddable layer is a silica coat. 
     
     
         10 . The nanoparticle of any one of  claims 1 - 9 , further comprising a surface coat surrounding the sheddable layer. 
     
     
         11 . The nanoparticle of  claim 10 , wherein the surface coat comprises a cationic component that interacts electrostatically with the sheddable layer. 
     
     
         12 . The nanoparticle of  claim 10  or  claim 11 , wherein the surface coat comprises one or more of: a polymer of a cationic amino acid, a poly(arginine), an anchoring domain, a cationic anchoring domain, a cell penetrating peptide, a viral glycoprotein, a heparin sulfate proteoglycan, and a targeting ligand. 
     
     
         13 . The nanoparticle of any one of  claims 10 - 12 , wherein the surface coat is zwitterionic and multivalent. 
     
     
         14 . The nanoparticle of any one of  claims 10 - 13 , wherein the surface coat comprises one or more targeting ligands. 
     
     
         15 . The nanoparticle of  claim 14 , wherein at least one of the one or more targeting ligands is conjugated to an anchoring domain that interacts with the sheddable layer. 
     
     
         16 . The nanoparticle of  claim 15 , wherein the anchoring domain is a cationic anchoring domain selected from RRRRRRRRR (SEQ ID NO: 15) and HHHHHH (SEQ ID NO: 16). 
     
     
         17 . The nanoparticle of  claim 15  or  claim 16 , wherein the anchoring domain is conjugated to the at least one of the one or more targeting ligands via a linker. 
     
     
         18 . The nanoparticle of  claim 17 , wherein the linker is not a polypeptide. 
     
     
         19 . The nanoparticle of  claim 17 , wherein the linker is a polypeptide. 
     
     
         20 . The nanoparticle of any one of  claims 17 - 19 , wherein the linker is conjugated to the targeting ligand via sulfhydryl or amine-reactive chemistry, and/or the linker is conjugated to the anchoring domain via sulfhydryl or amine-reactive chemistry. 
     
     
         21 . The nanoparticle of any one of  claims 17 - 20 , wherein said at least one of the one or more targeting ligands comprises a cysteine residue and is conjugated to the linker via the cysteine residue. 
     
     
         22 . The nanoparticle of any one of  claims 14 - 21 , wherein said one or more targeting ligands provides for targeted binding to a family B G-protein coupled receptor (GPCR). 
     
     
         23 . The nanoparticle of  claim 22 , wherein said targeting ligand comprises a cysteine substitution, at one or more internal amino acid positions, relative to a corresponding wild type amino acid sequence. 
     
     
         24 . The nanoparticle of  claim 22  or  claim 23 , wherein said targeting ligand comprises an amino acid sequence having 85% or more identity to the amino acid sequence HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 1). 
     
     
         25 . The nanoparticle of  claim 24 , wherein said targeting ligand comprises a cysteine substitution at one or more of positions L10, S11, and K12 of the amino acid sequence set forth in SEQ ID NO: 1). 
     
     
         26 . The nanoparticle of  claim 25 , wherein said targeting ligand comprises the amino acid sequence HGEGTFTSDL C KQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 2). 
     
     
         27 . The nanoparticle of any one of  claims 14 - 26 , wherein the surface coat comprises one or more targeting ligands that provides for targeted binding to a cell surface protein selected from c-Kit, CD27, and CD150. 
     
     
         28 . The nanoparticle of any one of  claims 14 - 27 , wherein the surface coat comprises one or more targeting ligands selected from the group consisting of: rabies virus glycoprotein (RVG) fragment, ApoE-transferrin, lactoferrin, melanoferritin, ovotransferritin, L-selectin, E-selectin, P-selectin, PSGL-1, ESL-1, CD44, death receptor-3 (DR3), LAMP1, LAMP2, Mac2-BP, stem cell factor (SCF), CD70, SH2 domain-containing protein 1A (SH2D1A), a exendin-4, GLP1, a targeting ligand that targets α5β1, RGD, a Transferrin ligand, an FGF fragment, succinic acid, a bisphosphonate, CD90, CD45f, CD34, a hematopoietic stem cell chemotactic lipid, sphingosine, ceramide, sphingosine-1-phosphate, ceramide-1-phosphate, and an active targeting fragment of any of the above. 
     
     
         29 . The nanoparticle of any one of  claims 14 - 28 , wherein the surface coat comprises stem cell factor (SCF) or a targeting fragment thereof, CD70 or a targeting fragment thereof, and SH2 domain-containing protein 1A (SH2D1A) or a targeting fragment thereof. 
     
     
         30 . The nanoparticle of any one of  claims 14 - 29 , wherein the surface coat comprises one or more targeting ligands that provides for targeted binding to target cells selected from: bone marrow cells, hematopoietic stem cells (HSCs), hematopoietic stem and progenitor cells (HSPCs), peripheral blood mononuclear cells (PBMCs), myeloid progenitor cells, lymphoid progenitor cells, T-cells, B-cells, NKT cells, NK cells, dendritic cells, monocytes, granulocytes, erythrocytes, megakaryocytes, mast cells, basophils, eosinophils, neutrophils, macrophages, erythroid progenitor cells, megakaryocyte-erythroid progenitor cells (MEPs), common myeloid progenitor cells (CMPs), multipotent progenitor cells (MPPs), hematopoietic stem cells (HSCs), short term HSCs (ST-HSCs), IT-HSCs, long term HSCs (LT-HSCs), endothelial cells, neurons, astrocytes, pancreatic cells, pancreatic β-islet cells, liver cells, muscle cells, skeletal muscle cells, cardiac muscle cells, hepatic cells, fat cells, intestinal cells, cells of the colon, and cells of the stomach. 
     
     
         31 . The nanoparticle of any one of  claims 14 - 29 , wherein the surface coat comprises a two or more targeting ligands, the combination of which provides for targeted binding to cells selected from: bone marrow cells, hematopoietic stem cells (HSCs), hematopoietic stem and progenitor cells (HSPCs), peripheral blood mononuclear cells (PBMCs), myeloid progenitor cells, lymphoid progenitor cells, T-cells, B-cells, NKT cells, NK cells, dendritic cells, monocytes, granulocytes, erythrocytes, megakaryocytes, mast cells, basophils, eosinophils, neutrophils, macrophages, erythroid progenitor cells, megakaryocyte-erythroid progenitor cells (MEPs), common myeloid progenitor cells (CMPs), multipotent progenitor cells (MPPs), hematopoietic stem cells (HSCs), short term HSCs (ST-HSCs), IT-HSCs, long term HSCs (LT-HSCs), endothelial cells, neurons, astrocytes, pancreatic cells, pancreatic β-islet cells, liver cells, muscle cells, skeletal muscle cells, cardiac muscle cells, hepatic cells, fat cells, intestinal cells, cells of the colon, and cells of the stomach. 
     
     
         32 . The nanoparticle of any one of  claims 1 - 31 , wherein the cationic polypeptide composition comprises a polypeptide that comprises a nuclear localization signal (NLS). 
     
     
         33 . The nanoparticle of  claim 32 , wherein the NLS comprises the amino acid sequence set forth in any one of SEQ ID NOs: 151-157 and 201-264. 
     
     
         34 . The nanoparticle of any one of  claims 1 - 33 , wherein the cationic polypeptide composition comprises a histone tail peptide (HTP). 
     
     
         35 . The nanoparticle of  claim 34 , wherein the HTP is conjugated to a cationic amino acid polymer. 
     
     
         36 . The nanoparticle of  claim 35 , wherein the HTP is conjugated to a cationic amino acid polymer via a cysteine residue. 
     
     
         37 . The nanoparticle of  claim 35  or  claim 36 , wherein the cationic amino acid polymer comprises poly(lysine). 
     
     
         38 . The nanoparticle of any one of  claims 33 - 37 , wherein said cationic polypeptide composition comprises histone peptides having a branched structure. 
     
     
         39 . The nanoparticle of any one of  claims 1 - 38 , wherein the payload comprises one or more of: (i) a CRISPR/Cas guide RNA, (ii) a DNA molecule encoding a CRISPR/Cas guide RNA, (iii) a nucleic acid molecule encoding a CRISPR/Cas RNA-guided polypeptide, (iv) a CRISPR/Cas RNA-guided polypeptide, (v) a CRISPR/Cas guide RNA complexed with a CRISPR/Cas RNA-guided polypeptide, (vi) a nucleic acid molecule encoding a zinc finger protein (ZFP), (vii) a ZFP, (viii) a nucleic acid molecule encoding a transcription activator-like effector (TALE) protein, (ix) a TALE protein, and (x) a DNA donor template. 
     
     
         40 . The nanoparticle of any one of  claims 1 - 39 , wherein the payload comprises (i) a CRISPR/Cas guide RNA and/or a DNA molecule encoding said CRISPR/Cas guide RNA; and (ii) a CRISPR/Cas RNA-guided polypeptide and/or a nucleic acid molecule encoding said CRISPR/Cas RNA-guided polypeptide. 
     
     
         41 . The nanoparticle of  claim 40 , wherein the payload further comprises a DNA donor template. 
     
     
         42 . A nanoparticle formulation, comprising:
 (a) a first nanoparticle according to any one of  claims 1 - 41 , wherein the payload comprises one or more of: (i) a CRISPR/Cas guide RNA, (ii) a DNA molecule encoding a CRISPR/Cas guide RNA, (iii) a nucleic acid molecule encoding a CRISPR/Cas RNA-guided polypeptide, (iv) a CRISPR/Cas RNA-guided polypeptide, (v) a CRISPR/Cas guide RNA complexed with a CRISPR/Cas RNA-guided polypeptide, (vi) a nucleic acid molecule encoding a zinc finger protein (ZFP), (vii) a ZFP, (viii) a nucleic acid molecule encoding a transcription activator-like effector (TALE) protein, and (ix) a TALE protein; and   (b) a second nanoparticle comprising a nucleic acid payload that comprises a DNA donor template.   
     
     
         43 . A multi-layered nanoparticle, comprising:
 (a) an inner core comprising a payload comprising a DNA donor template;   (b) a first sheddable layer surrounding the inner core;   (c) an intermediate core surrounding the first sheddable layer, wherein the intermediate core comprises one or more of: (i) a CRISPR/Cas guide RNA, (ii) a DNA molecule encoding a CRISPR/Cas guide RNA, (iii) a nucleic acid molecule encoding a CRISPR/Cas RNA-guided polypeptide, (iv) a CRISPR/Cas RNA-guided polypeptide, (v) a CRISPR/Cas guide RNA complexed with a CRISPR/Cas RNA-guided polypeptide, (vi) a zinc finger protein (ZFP), (vii) a DNA molecule encoding a ZFP, (viii) a transcription activator-like effector (TALE) protein, and (ix) a DNA molecule encoding a TALE protein; and   (d) a second sheddable layer surrounding the intermediate core.   
     
     
         44 . The multi-layered nanoparticle of  claim 43 , wherein the first and/or second sheddable layer comprises one or more of: silica, a peptoid, a polycysteine, calcium, calcium phosphate, calcium sulfate, manganese, manganese phosphate, manganese sulfate, magnesium, magnesium phosphate, magnesium sulfate, iron, iron phosphate, iron sulfate, lithium, lithium phosphate, and lithium sulfate. 
     
     
         45 . The multi-layered nanoparticle of  claim 43  or  claim 44 , comprising a surface coat surrounding the second sheddable layer. 
     
     
         46 . The multi-layered nanoparticle of  claim 45 , wherein the surface coat comprises a cationic component that interacts electrostatically with the second sheddable layer. 
     
     
         47 . The multi-layered nanoparticle of  claim 45  or  claim 46 , wherein the surface coat comprises one or more of: a polymer of a cationic amino acid, a poly(arginine), a cell penetrating peptide, a viral glycoprotein, a heparin sulfate proteoglycan, and a targeting ligand. 
     
     
         48 . The multi-layered nanoparticle of any one of  claims 43 - 47 , wherein the surface coat is zwitterionic and multivalent. 
     
     
         49 . The multi-layered nanoparticle of any one of  claims 43 - 48 , wherein the surface coat comprises one or more targeting ligands. 
     
     
         50 . A method of delivering a nucleic acid and/or protein payload to a target cell, the method comprising:
 contacting a eukaryotic target cell with the nanoparticle of any one of  claims 1 - 41 , the nanoparticle formulation of  claim 42 , and/or the multi-layered nanoparticle of any one of  claims 43 - 49 .   
     
     
         51 . The method of  claim 50 , wherein the payload includes a gene editing tool. 
     
     
         52 . The method of  claim 50  or  claim 51 , wherein the payload includes one or more of: a CRISPR/Cas guide RNA, a DNA molecule encoding a CRISPR/Cas guide RNA, a CRISPR/Cas RNA-guided polypeptide, a nucleic acid molecule encoding a CRISPR/Cas RNA-guided polypeptide, a zinc finger nuclease, a nucleic acid molecule encoding a zinc finger nuclease, a TALE or TALEN, a nucleic acid molecule encoding a TALE or TALEN, and DNA donor template. 
     
     
         53 . The method of any one of  claims 50 - 52 , wherein the target cell is a mammalian cell 
     
     
         54 . The method of any one of  claims 50 - 53 , wherein the target cell is a human cell 
     
     
         55 . The method of any one of  claims 50 - 54 , wherein the target cell is in culture in vitro. 
     
     
         56 . The method of any one of  claims 50 - 54 , wherein the target cell is in vivo. 
     
     
         57 . The method of  claim 56 , wherein said contacting includes a step of administering the nanoparticle to an individual 
     
     
         58 . The method of  claim 57 , wherein the individual has Huntington's disease, ALS, Parkinson's disease, pancreatic cancer, diabetes, or von Willebrand's disease. 
     
     
         59 . The method of any one of  claims 50 - 58 , wherein the nanoparticle includes a surface coat comprising a targeting ligand. 
     
     
         60 . The method of  claim 59 , wherein the targeting ligand provides for target binding to cells selected from: bone marrow cells, hematopoietic stem cells (HSCs), hematopoietic stem and progenitor cells (HSPCs), peripheral blood mononuclear cells (PBMCs), myeloid progenitor cells, lymphoid progenitor cells, T-cells, B-cells, NKT cells, NK cells, dendritic cells, monocytes, granulocytes, erythrocytes, megakaryocytes, mast cells, basophils, eosinophils, neutrophils, macrophages, erythroid progenitor cells, megakaryocyte-erythroid progenitor cells (MEPs), common myeloid progenitor cells (CMPs), multipotent progenitor cells (MPPs), hematopoietic stem cells (HSCs), short term HSCs (ST-HSCs), IT-HSCs, long term HSCs (LT-HSCs), endothelial cells, neurons, astrocytes, pancreatic cells, pancreatic β-islet cells, liver cells, muscle cells, skeletal muscle cells, cardiac muscle cells, hepatic cells, fat cells, intestinal cells, cells of the colon, and cells of the stomach. 
     
     
         61 . The method of any one of  claims 50 - 60 , wherein the target cell is selected from: a bone marrow cell, a hematopoietic stem cell (HSC), a hematopoietic stem and progenitor cell (HSPC), a peripheral blood mononuclear cell (PBMC), a myeloid progenitor cell, a lymphoid progenitor cell, a T-cell, a B-cell, a NKT cell, a NK cell, a dendritic cell, a monocyte, a granulocyte, an erythrocyte, a megakaryocyte, a mast cell, a basophil, an eosinophil, a neutrophil, a macrophage, an erythroid progenitor cell, a megakaryocyte-erythroid progenitor cell (MEP), a common myeloid progenitor cell (CMP), a multipotent progenitor cell (MPP), a hematopoietic stem cell (HSC), a short term HSC (ST-HSC), an IT-HSC, a long term HSC (LT-HSC), an endothelial cell, a neuron, an astrocyte, a pancreatic cell, a pancreatic β-islet cell, a liver cell, a muscle cell, a skeletal muscle cell, a cardiac muscle cell, a hepatic cell, a fat cell, an intestinal cell, a cell of the colon, and a cell of the stomach. 
     
     
         62 . A branched histone molecule, comprising: one or more histone tail peptides (HTPs) conjugated to side chains of a cationic polymer. 
     
     
         63 . The branched histone molecule of  claim 62 , wherein the cationic polymer comprises poly(arginine) or poly(lysine). 
     
     
         64 . The branched histone molecule of  claim 63 , wherein up to 40% of the side chains of the cationic polymer are conjugated to said one or more HTPs.