US20180179553A1 - Compositions and methods for nucleic acid and/or protein payload delivery - Google Patents

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

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Publication number
US20180179553A1
US20180179553A1 US15/842,829 US201715842829A US2018179553A1 US 20180179553 A1 US20180179553 A1 US 20180179553A1 US 201715842829 A US201715842829 A US 201715842829A US 2018179553 A1 US2018179553 A1 US 2018179553A1
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Prior art keywords
nanoparticle
cationic
cases
amino acid
poly
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US15/842,829
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English (en)
Inventor
Andre Ronald WATSON
Christian Foster
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Ligandal Inc
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Ligandal Inc
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Priority to US15/842,829 priority Critical patent/US20180179553A1/en
Publication of US20180179553A1 publication Critical patent/US20180179553A1/en
Assigned to Ligandal, Inc. reassignment Ligandal, Inc. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FOSTER, Christian, WATSON, Andre Ronald
Priority to US16/701,014 priority patent/US20200095605A1/en
Abandoned legal-status Critical Current

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    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3517—Marker; Tag
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • nucleic acid and/or protein payloads are an important objective for therapeutic strategies and for research methodologies.
  • To achieve effective introduction of a payload it is important to appropriately package the payload to protect it from degradation prior to cellular entry, to permit entry into cells, to direct the payload away from the lysosomal degradation pathway, and to direct delivery to the appropriate subcellular compartment.
  • the timing of release of a payload from the packaging following cellular entry can influence the effectiveness of the payload.
  • compositions and methods for delivery of payloads e.g., nucleic acid and/or protein payloads
  • cells e.g., nanoparticle, viral, and non-viral delivery of payloads to cells.
  • payloads e.g., nucleic acid and/or protein payloads
  • Nanoparticles designed for serum stability, targeted delivery to specific cell types, biomimicry of endogenous nucleic acid packaging via histones and nucleosome-like branched polymer, compartment-specific unpackaging within the nucleus, variable timed release kinetics, and methods of use thereof, are provided.
  • a subject nanoparticle includes a core and a sheddable layer encapsulating the core (e.g., providing for temporary stabilization of the core during cell delivery), 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 includes polymers of D-isomers of a cationic amino acid and polymers of L-isomers of a cationic amino acid.
  • the polymers of D-isomers of an anionic amino acid are present at a ratio, relative to the polymers of L-isomers of an anionic amino acid, in a range of from 10:1 to 1:10. In some cases, the 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.
  • a nanoparticle of the disclosure includes a surface coat, which surrounds the sheddable layer.
  • the surface coat can include a targeting ligand that provides for targeted binding to a surface molecule of a target cell.
  • the targeting ligand is conjugated (with or without a linker) to an anchoring domain, e.g., for anchoring the targeting ligand to the sheddable layer of the nanoparticle.
  • multi-layered nanoparticles that include a first payload (e.g., a DNA donor template) as part of the core, where the core is surrounded by a first sheddable layer, the first sheddable layer is surrounded by an intermediate layer that includes a second payload (e.g., a gene editing tool), and the intermediate layer is surrounded by a second sheddable layer.
  • a first payload e.g., a DNA donor template
  • the first sheddable layer is surrounded by an intermediate layer that includes a second payload (e.g., a gene editing tool)
  • the intermediate layer is surrounded by a second sheddable layer.
  • the second sheddable layer is coated with a surface coat (e.g., a surface coat that includes a targeting ligand.
  • nanoparticle formulations including two or more nanoparticles in which the payload of a first nanoparticle includes a donor DNA template and the payload of a second nanoparticle includes a gene editing tool (e.g., (i) a CRISPR/Cas guide RNA; (ii) a DNA encoding a CRISPR/Cas guide RNA; (iii) a DNA and/or RNA encoding a programmable gene editing protein; and/or (iv) a programmable gene editing protein).
  • a gene editing tool e.g., (i) a CRISPR/Cas guide RNA; (ii) a DNA encoding a CRISPR/Cas guide RNA; (iii) a DNA and/or RNA encoding a programmable gene editing protein; and/or (iv) a programmable gene editing protein).
  • a nucleic acid and/or protein payload to a target cell, where the method includes contacting a eukaryotic target cell with a viral or non-viral delivery vehicle that includes (a) a gene editing tool; and (b) a nucleic acid or protein agent that induces proliferation of and/or biases differentiation of the target cell.
  • FIG. 1 depicts results from a fluorimetric assay testing various parameters (e.g., cation:anion charge ratio) for condensation of nucleic acid payloads. The result showed, e.g., that a charge ratio of 2 works well for the condensation of plasmids encoding Cas9 and guide RNA molecules.
  • parameters e.g., cation:anion charge ratio
  • FIG. 2 depicts particle size and zeta potential distributions for nanoparticle cores that were generated. The data were obtained using a Particle Metrix ZetaView NTA instrument.
  • Nanoparticle Size (peak) was 128.8 nm, and Zeta potential (peak) was +10.5 mV (100%).
  • FIG. 3 depicts particle size and zeta potential distributions for stabilized nanoparticle cores (cores encapsulated by a sheddable layer).
  • the data were obtained using a Particle Metrix ZetaView NTA instrument.
  • the stabilized cores had a size of 110.6 nm and zeta potential of ⁇ 42.1 mV (95%).
  • FIG. 4 depicts data showing that nanoparticles with an outer shell (outer coat) that included RVG9R, which is Rabies Virus Glycoprotein (RVG) fused to a 9-Arg peptide sequence (as a cationic anchoring domain), had a characteristic particle size of 115.8 nm and a zeta potential of ⁇ 3.1 mV (100%).
  • Optimal outer coating yields a transition of zeta potential from ⁇ 50 mV (for the silica coated core) to between 0 and ⁇ 10 mV (after adding the outer shell).
  • FIG. 5 depicts results from cell culture experiments in which different nanoparticles were used to deliver nucleic acid payloads.
  • the figure compares nanoparticles that include poly(D-glutamic Acid) as part of the core (in addition to poly(L-arginine)) to those that do not.
  • the three rows represent replicates.
  • FIG. 6 depicts microscopy images of neural stem cells that were contacted with nanoparticles that included CRISPR/Cas9 expression vectors as the nucleic acid payload.
  • the core of the nanoparticles included poly(L-arginine) (a cationic polymer) tagged with a fluorophore (FITC).
  • FITC fluorophore
  • the endosome and nucleus were stained using Lysotracker (Red) and Hoescht 3342 (blue) respectively.
  • Nanoparticles (and Lipofectamine 3000 as a control) were introduced to cells 16 hours after seeding. Cells were incubated with Hoescht 3342 and Lysotracker Red prior to imaging.
  • Panels C-D present bar graphs that quantify colocalization of the nanoparticle core with the nucleus and with endosomes.
  • FIG. 7 depicts microscopy images of peripheral blood mononuclear cells (PBMCs) that were transfected with nanoparticles that included mRNA encoding GFP as a nucleic acid payload.
  • PBMCs peripheral blood mononuclear cells
  • the images demonstrate that mRNA expression can be extended to 16 days with nanoparticles that include a core with, at a defined ratio, a polymer of D-isomers of an anionic amino acid and a polymer of L-isomers of an anionic amino acid.
  • FIG. 8 depicts a schematic representation of an example embodiment of a subject nanoparticle.
  • FIG. 9 depicts a schematic representation of an example embodiment of a subject nanoparticle.
  • the nanoparticle is multi-layered, having a core (which includes a first payload) surrounded by a first sheddable layer, which is surrounded by an intermediate layer (which includes an additional payload), which is surrounded by a second sheddable layer, which is surface coated (i.e., includes an outer shell).
  • FIG. 10 depicts schematic representations of example configurations of a delivery molecule of a surface coat of a subject nanoparticle.
  • the delivery molecules depicted include a targeting ligand conjugated to an anchoring domain that is interacting electrostatically with a sheddable layer of a nanoparticle. Note that the targeting ligand can be conjugated at the N- or C-terminus (left of each panel), but can also be conjugated at an internal position (right of each panel).
  • the molecules in panel A include a linker while those in panel B do not.
  • FIG. 11 provides a schematic diagram of a family B GPCR, highlighting separate domains to be considered when evaluating a targeting ligand, e.g., for binding to allosteric/affinity N-terminal domains and orthosteric endosomal-sorting/signaling domains.
  • Figure is adapted from Siu, Fai Yiu, et al., Nature 499.7459 (2013): 444-449).
  • FIG. 12 provides an example of identifying an internal amino acid position for insertion and/or substitution (e.g., with a cysteine residue) for a targeting ligand such that affinity is maintained and the targeting ligand engages long endosomal recycling pathways that promote nucleic acid release and limit nucleic acid degradation.
  • the targeting ligand is exendin-4 and amino acid positions 10, 11, and 12 were identified as sites for possible insertion and/or substitution (e.g., with a cysteine residue, e.g., an S11C mutation).
  • the figure shows an alignment of simulated Exendin-4 (SEQ ID NO: 1) to known crystal structures of glucagon-GCGR (4ERS) and GLP1-GLP1R-ECD complex (PDB: 3IOL), and PDB renderings that were rotated in 3-dimensional space.
  • FIG. 13 shows a tbFGF fragment as part of a ternary FGF2-FGFR1-HEPARIN complex (1fq9 on PDB).
  • CKNGGFFLRIHPDGRVDGVREKS (highlighted) (SEQ ID NO: 43) was determined to be important for affinity to FGFR1.
  • FIG. 14 provides an alignment and PDB 3D rendering used to determine that HFKDPK (SEQ ID NO: 5) is a peptide that can be used for ligand-receptor orthosteric activity and affinity.
  • FIG. 15 provides an alignment and PDB 3D rendering used to determine that LESNNYNT (SEQ ID NO: 6) is a peptide that can be used for ligand-receptor orthosteric activity and affinity.
  • FIG. 16 provides non-limiting examples nuclear localization signals (NLSs) that can be used as part of a subject nanoparticle (e.g., as an NLS-containing peptide; as part of/conjugated to an NLS-containing peptide, an anionic polymer, a cationic polymer, and/or a cationic polypeptide; and the like).
  • NLSs nuclear localization signals
  • the figure is adapted from Kosugi et al., J Biol Chem. 2009 Jan. 2; 284(1):478-85.
  • FIG. 17 depicts schematic representations of the mouse (panel A) and human (panel B) hematopoietic cell lineage, and markers that have been identified for various cells within the lineage.
  • FIG. 18 depicts schematic representations of miRNA (panel A) and protein (panel B) factors that can be used to influence cell differentiation and/or proliferation
  • FIG. 19 provides condensation curves on nanoparticles with payload: VWF-EGFP pDNA with peptide nucleic acid (PNA) Binding Site.
  • FIG. 20 provides condensation curves on nanoparticles with payload: NLS-CAS9-NLS RNP complexed to HBB gRNA.
  • FIG. 21 provides condensation curves on nanoparticles with payload: HBB gRNA.
  • FIG. 22 provides condensation curves on nanoparticles with payload: HBB gRNA.
  • FIG. 23 provides condensation curves on nanoparticles with payload: NLS-CAS9-NLS RNP complexed to HBB gRNA.
  • FIG. 24 provides condensation curves on nanoparticles with payload: VWF-EGFP pDNA with peptide nucleic acid (PNA) Binding Site.
  • FIG. 25 provides condensation curves on nanoparticles with payload: VWF-EGFP pDNA with peptide nucleic acid (PNA) Binding Site.
  • FIG. 26 provides condensation curves on nanoparticles with payload: RNP of NLS-CAS9-NLS with HBB gRNA.
  • FIG. 27 provides condensation curves on nanoparticles with payload: VWF-EGFP pDNA with peptide nucleic acid (PNA) Binding Site.
  • FIG. 28 provides condensation curves on nanoparticles with payload: Cy5_EGFP mRNA.
  • FIG. 29 provides condensation curves on nanoparticles with payload: BLOCK-iT Alexa Fluor 555 siRNA.
  • FIG. 30 provides condensation curves on nanoparticles with payload: NLS-Cas9-EGFP RNP complexed to HBB gRNA.
  • FIG. 31 provides data collected when using nanoparticles with Alexa 555 Block-IT siRNA as payload.
  • FIG. 32 provides data collected when using nanoparticles with ribonuclear protein (RNP) formed by NLS-Cas9-GFP and HBB guide RNA as payload.
  • RNP ribonuclear protein
  • FIG. 33 provides data collected when using nanoparticles with Cy5 EGFP mRNA as payload.
  • FIG. 34 provides data collected when using nanoparticles with payload: VWF-EGFP pDNA with Cy5 tagged peptide nucleic acid (PNA) Binding Site.
  • FIG. 35 provides data from a SYBR Gold exclusion assay showing fluorescence intensity decrease by addition of cationic polypeptide CD45_mSiglec_(4GS)2_9R_C followed by PLE100 and by further addition of the cationic polypeptide to RNP.
  • FIG. 36 provides data from a SYBR Gold exclusion assay showing fluorescence intensity variations by addition of cationic polypeptide CD45_mSiglec_(4GS)2_9R_C followed by PLE100 and by further addition of the cationic polypeptide to siRNA and SYBR Gold.
  • FIG. 37 provides data from a SYBR Gold exclusion assay showing fluorescence intensity variations by addition of cationic polypeptide histone peptide H2A followed by CD45_mSiglec_(4GS)2_9R_C and by further addition of PLE100 to RNP of NLS-Cas9-EGFP with HBB gRNA and SYBR Gold.
  • FIG. 38 provides data from a SYBR Gold exclusion assay showing fluorescence intensity variations by addition of cationic polypeptide histone peptide H4 together with CD45_mSiglec_(4GS)2_9R_C and by further addition of PLE100 to RNP of NLS-Cas9-EGFP with HBB gRNA and SYBR Gold.
  • FIG. 39 provides data from a SYBR Gold exclusion assay showing fluorescence intensity variations by addition of cationic polypeptide CD45_mSiglec_(4GS)2_9R_C fand by further addition of PLE100 to mRNA.
  • FIG. 40 provides data from a SYBR Gold exclusion assay showing fluorescence intensity variations by addition histone H4 and by further addition of CD45-mSiglec-(4GS)2_9R_c and PLE100 to mRNA.
  • FIG. 41 provides data from a SYBR Gold exclusion assay showing fluorescence intensity variations by addition histone H2A and by further addition of CD45-mSiglec-(4GS)2_9R_c and PLE100 to mRNA.
  • FIG. 42 provides data from a SYBR Gold exclusion assay from intercalation with VWF_EGFP pDNA showing fluorescence intensity variations by addition of cationic polypeptide CD45_mSiglec_(4GS)2_9R_C followed by PLE100.
  • FIG. 43 provides data from a SYBR Gold exclusion assay from intercalation with VWF_EGFP pDNA showing fluorescence intensity variations by addition of histone H4, followed by cationic polypeptide CD45_mSiglec_(4GS)2_9R_C followed by PLE100.
  • FIG. 44 provides data from a SYBR Gold exclusion assay from intercalation with VWF_EGFP pDNA showing fluorescence intensity variations by addition of histone H4, followed by cationic polypeptide CD45_mSiglec_(4GS)2_9R_C followed by PLE100.
  • FIG. 45 panels A-C provide data related to polyplex size distribution, silica coated size and zeta potential distribution, and ligand coated/functionalized particle size and zeta potential distribution.
  • FIG. 46 provides data related to branched histone peptide conjugate pilot particles.
  • FIG. 47 provides data related to project HSC.001.001 (see Table 5).
  • FIG. 48 provides data related to project HSC.001.002 (see Table 5).
  • FIG. 49 provides data related to project HSC.002.01 (Targeting Ligand—ESELLg_mESEL_(4GS)2_9R_N) (see Table 5).
  • FIG. 50 provides data related to project HSC.002.02 (Targeting Ligand—ESELLg_mESEL_(4GS)2_9R_C) (see Table 5).
  • FIG. 51 provides data related to project HSC.002.03 (Targeting Ligand—CD45_mSiglec_(4GS)2_9R_C) (see Table 5).
  • FIG. 52 provides data related to project HSC.002.04 (Targeting Ligand—Cy5mRNA-SiO2-PEG) (see Table 5).
  • FIG. 53 provides data related to project BLOOD.002.88 (Targeting Ligand—CD45_mSiglec_(4GS)2_9R_C) (see Table 5).
  • FIG. 54 provides data related to project BLOOD.002.89 (Targeting Ligand—CD45_mSiglec_(4GS)2_9R_C) (see Table 5).
  • FIG. 55 provides data related to project BLOOD.002.90 (see Table 5).
  • FIG. 56 provides data related to project BLOOD.002.91 (PLR50) (see Table 5).
  • FIG. 57 provides data related to project BLOOD.002.92 (Targeting Ligand—CD45_mSiglec_(4GS)2_9R_C) (see Table 5).
  • FIG. 58 provides data related to project TCELL.001.1 (see Table 5).
  • FIG. 59 provides data related to project TCELL.001.3 (see Table 5).
  • FIG. 60 provides data related to project TCELL.001.13 (see Table 5).
  • FIG. 61 provides data related to project TCELL.001.14 (see Table 5).
  • FIG. 62 provides data related to project TCELL.001.16 (see Table 5).
  • FIG. 63 provides data related to project TCELL.001.18 (see Table 5).
  • FIG. 64 provides data related to project TCELL.001.28 (see Table 5).
  • FIG. 65 provides data related to project TCELL.001.29 (see Table 5).
  • FIG. 66 provides data related to project TCELL.001.31 (see Table 5).
  • FIG. 67 provides data related to project TCELL.001.33 (see Table 5).
  • FIG. 68 provides data related to project TCELL.001.43 (see Table 5).
  • FIG. 69 provides data related to project TCELL.001.44 (see Table 5).
  • FIG. 70 provides data related to project TCELL.001.46 (see Table 5).
  • FIG. 71 provides data related to project TCELL.001.48 (see Table 5).
  • FIG. 72 provides data related to project TCELL.001.58 (see Table 5).
  • FIG. 73 provides data related to project TCELL.001.59 (see Table 5).
  • FIG. 74 provides data related to project CYNOBM.002.82 (see Table 5).
  • FIG. 75 provides data related to project CYNOBM.002.83 (see Table 5).
  • FIG. 76 provides data related to project CYNOBM.002.84 (see Table 5).
  • FIG. 77 provides data related to project CYNOBM.002.85 (see Table 5).
  • FIG. 78 provides data related to project CYNOBM.002.86 (see Table 5).
  • FIG. 79 provides data related to project CYNOBM.002.76 (see Table 5).
  • FIG. 80 provides data related to project CYNOBM.002.77 (see Table 5).
  • FIG. 81 provides data related to project CYNOBM.002.78 (see Table 5).
  • FIG. 82 provides data related to project CYNOBM.002.79 (see Table 5).
  • FIG. 83 provides data related to project CYNOBM.002.80 (see Table 5).
  • FIG. 84 provides data related to untransfected controls for CynoBM.002 samples.
  • FIG. 85 provides data related to lipofectamine CRISPRMAX delivery of NLS-Cas9-EGFP BCL11a gRNA RNPs.
  • FIG. 86 provides data related to project CynoBM.002 RNP-Only controls (see Table 5).
  • FIG. 87 provides data related to project CynoBM.002.82 (see Table 5).
  • FIG. 88 provides data related to project CynoBM.002.83 (see Table 5).
  • FIG. 89 provides data related to project CYNOBM.002.84 (see Table 5).
  • FIG. 90 provides data related to project CynoBM.002.85 (see Table 5).
  • FIG. 91 provides data related to project CynoBM.002.86 (see Table 5).
  • FIG. 92 provides data related to project CynoBM.002.75 (see Table 5).
  • FIG. 93 provides data related to project CynoBM.002.76 (see Table 5).
  • FIG. 94 provides data related to project CynoBM.002.77 (see Table 5).
  • FIG. 95 provides data related to project CynoBM.002.78 (see Table 5).
  • FIG. 96 provides data related to project CynoBM.002.79 (see Table 5).
  • FIG. 97 provides data related to project CynoBM.002.80 (see Table 5).
  • FIG. 98 provides data related to project CynoBM.002.81 (see Table 5).
  • FIG. 99 provides qualitative images of CynoBM.002 RNP-Only controls.
  • FIG. 100 provides data related to project HSC.004 (see Table 5) high-content screening.
  • FIG. 101 provides data related to project TCELL.001 (see Table 5) high-content screening.
  • FIG. 102 provides data related to project TCELL.001 (see Table 5) lipofectamine CRISPRMAX controls.
  • FIG. 103 provides data related to project TCell.001.1 (see Table 5).
  • FIG. 104 provides data related to project TCell.001.2 (see Table 5).
  • FIG. 105 provides data related to project TCell.001.3 (see Table 5).
  • FIG. 106 provides data related to project TCell.001.4 (see Table 5).
  • FIG. 107 provides data related to project TCell.001.5 (see Table 5).
  • FIG. 108 provides data related to project TCell.001.6 (see Table 5).
  • FIG. 109 provides data related to project TCell.001.7 (see Table 5).
  • FIG. 110 provides data related to project TCell.001.8 (see Table 5).
  • FIG. 111 provides data related to project TCell.001.9 (see Table 5).
  • FIG. 112 provides data related to project TCell.001.10 (see Table 5).
  • FIG. 113 provides data related to project TCell.001.11 (see Table 5).
  • FIG. 114 provides data related to project TCell.001.12 (see Table 5).
  • FIG. 115 provides data related to project TCell.001.13 (see Table 5).
  • FIG. 116 provides data related to project TCell.001.14 (see Table 5).
  • FIG. 117 provides data related to project TCell.001.15 (see Table 5).
  • FIG. 118 provides data related to negative controls for project TCell.001 (see Table 5).
  • FIG. 119 provides data related to project Blood.002 (see Table 5).
  • FIG. 120 provides data related to project TCell.001.27 (see Table 5).
  • FIG. 121 depicts charge density plots of CRISPR RNP (a possible payload), which allows for determining whether an anionic or cationic peptide/material should be added to form a stable charged layer on the protein surface.
  • FIG. 122 depicts charge density plots of Sleeping Beauty Transposons (a possible payload), which allows for determining whether an anionic or cationic peptide/material should be added to form a stable charged layer on the protein surface.
  • FIG. 123 depicts (1) Exemplary anionic peptides (9-10 amino acids long, approximately to scale to 10 nm diameter CRISPR RNP) anchoring to cationic sites on the CRISPR RNP surface prior to (2) addition of cationic anchors as (2a) anchor-linker-ligands or standalone cationic anchors, with or without addition of (2b) subsequent multilayering chemistries, co-delivery of multiple nucleic acid or charged therapeutic agents, or layer stabilization through cross-linking.
  • FIG. 124 depicts examples of orders of addition and electrostatic matrix compositions based on core templates, which may include Cas9 RNP or any homogenously or zwitterionically charged surface.
  • FIG. 125 provides a modeled structure of IL2 bound to IL2R.
  • FIG. 126 provides a modeled structure of single chain CD3 antibody fragments.
  • FIG. 127 provides a modeled structure of sialoadhesin N-terminal in complex with N-Acetylneuraminic acid (Neu5Ac).
  • FIG. 128 provides a modeled structure of Stem Cell Factor (SCF).
  • SCF Stem Cell Factor
  • FIG. 129 provides example images generated during rational design of a cKit Receptor Fragment.
  • FIG. 130 provides example images generated during rational design of a cKit Receptor Fragment.
  • FIG. 131 provides example images generated during rational design of a cKit Receptor Fragment.
  • FIG. 132 provides circular dichroism data from analyzing the rationally designed cKit Receptor Fragment.
  • FIG. 133 depicts modeling of the stabilized conformation of the rationally designed cKit Receptor Fragment.
  • FIG. 134 depicts an example of a branched histone structure in which HTPs are conjugated to the side chains of a cationic polymer backbone.
  • the polymer on the right represents the precursor backbone molecule and the molecule on the left is an example of a segment of a branched structure.
  • a subject nanoparticle includes a core and a sheddable layer encapsulating the core (e.g., providing for temporary stabilization of the core during cell delivery), 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.
  • the anionic polymer composition includes polymers of D-isomers of an anionic amino acid and polymers of L-isomers of an anionic amino acid
  • the cationic polymer composition comprises polymers of D-isomers of a cationic
  • the polymers of D-isomers of an anionic amino acid are present at a ratio, relative to the polymers of L-isomers of an anionic amino acid, in a range of from 10:1 to 1:10. In some cases, the 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.
  • a nanoparticle of the disclosure includes a surface coat, which surrounds the sheddable layer.
  • the surface coat can include a targeting ligand that provides for targeted binding to a surface molecule of a target cell.
  • the targeting ligand is conjugated (with or without a linker) to an anchoring domain, e.g., for anchoring the targeting ligand to the sheddable layer of the nanoparticle.
  • multi-layered nanoparticles the include a first payload (e.g., a DNA donor template) as part of the core, where the core is surrounded by a first sheddable layer, the first sheddable layer is surrounded by an intermediate layer that includes a second payload (e.g., a gene editing tool), and the intermediate layer is surround by a second sheddable layer.
  • a first payload e.g., a DNA donor template
  • the first sheddable layer is surrounded by an intermediate layer that includes a second payload (e.g., a gene editing tool)
  • the intermediate layer is surround by a second sheddable layer.
  • the second sheddable layer is coated with a surface coat (e.g., a surface coat that includes a targeting ligand.
  • nanoparticle formulations including two or more nanoparticles in which the payload of a first nanoparticle includes a donor DNA template and the payload of a second nanoparticle includes a gene editing tool (e.g., (i) a CRISPR/Cas guide RNA; (ii) a DNA encoding a CRISPR/Cas guide RNA; (iii) a DNA and/or RNA encoding a programmable gene editing protein; and/or (iv) a programmable gene editing protein).
  • a gene editing tool e.g., (i) a CRISPR/Cas guide RNA; (ii) a DNA encoding a CRISPR/Cas guide RNA; (iii) a DNA and/or RNA encoding a programmable gene editing protein; and/or (iv) a programmable gene editing protein).
  • a subject nanoparticle includes (i) a core that is encapsulated by (ii) a sheddable layer, and the sheddable layer is in some cases surrounded by (iii) a surface coat, which can include a targeting ligand.
  • WO2015042585 is hereby incorporated by reference in its entirety.
  • the core of a subject nanoparticle includes an anionic polymer composition (e.g., poly(glutamic acid)), a cationic polymer composition (e.g., poly(arginine), a cationic polypeptide composition (e.g., a histone tail peptide), and a payload (e.g., nucleic acid and/or protein payload).
  • an anionic polymer composition e.g., poly(glutamic acid)
  • a cationic polymer composition e.g., poly(arginine
  • a cationic polypeptide composition e.g., a histone tail peptide
  • a payload e.g., nucleic acid and/or protein payload.
  • the core is generated by condensation of a cationic amino acid polymer and payload in the presence of an anionic amino acid polymer (and in some cases in the presence of a cationic polypeptide of a cationic polypeptide composition).
  • condensation of the components that make up the core can mediate increased transfection efficiency compared to conjugates of cationic polymers with a payload.
  • Inclusion of an anionic polymer in a nanoparticle core may prolong the duration of intracellular residence of the nanoparticle and release of payload.
  • ratios of D-isomer polymers to L-isomer polymers can be controlled in order to control the timed release of payload, where increased ration of D-isomer polymers to L-isomer polymers leads to increased stability (reduced payload release rate), which for example can enable longer lasting gene expression from a payload delivered by a subject nanoparticle.
  • modifying the ratio of D-to-L isomer polypeptides within the nanoparticle core can cause gene expression profiles (e.g., expression of a protein encoded by a payload molecule) to be on the order of from 1-90 days (e.g.
  • the control of payload release (e.g., when delivering a gene editing tool), can be particularly effective for performing genomic edits e.g., in some cases where homology-directed repair is desired.
  • a nanoparticle includes a core and a sheddable layer encapsulating the core, where the core includes: (a) an anionic polymer composition; (b) a cationic polymer composition; (c) a cationic polypeptide composition; and (d) a nucleic acid and/or protein payload, where one of (a) and (b) includes a D-isomer polymer of an amino acid, and the other of (a) and (b) includes an L-isomer polymer of an amino acid, and where the ratio of the D-isomer polymer to the L-isomer polymer is in a range of from 10:1 to 1.5:1 (e.g., from 8:1 to 1.5:1, 6:1 to 1.5:1, 5:1 to 1.5:1, 4:1 to 1.5:1, 3:1 to 1.5:1, 2:1 to 1.5:1, 10:1 to 2:1; 8:1 to 2:1, 6:1 to 2:1, 5:1 to 2:1, 10:1 to 3:1; 8:1 to 3:1, 6:1 to 3:1, 6:1
  • the ratio of the D-isomer polymer to the L-isomer polymer not 1:1.
  • the anionic polymer composition includes an anionic polymer selected from poly(D-glutamic acid) (PDEA) and poly(D-aspartic acid) (PDDA), where (optionally) the cationic polymer composition can include a cationic polymer selected from poly(L-arginine), poly(L-lysine), poly(L-histidine), poly(L-ornithine), and poly(L-citrulline).
  • the cationic polymer composition comprises a cationic polymer selected from poly(D-arginine), poly(D-lysine), poly(D-histidine), poly(D-ornithine), and poly(D-citrulline), where (optionally) the anionic polymer composition can include an anionic polymer selected from poly(L-glutamic acid) (PLEA) and poly(L-aspartic acid) (PLDA).
  • a cationic polymer selected from poly(D-arginine), poly(D-lysine), poly(D-histidine), poly(D-ornithine), and poly(D-citrulline)
  • the anionic polymer composition can include an anionic polymer selected from poly(L-glutamic acid) (PLEA) and poly(L-aspartic acid) (PLDA).
  • a 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, wherein (a) said 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) said cationic polymer composition includes polymers of D-isomers of a cationic amino acid and polymers of L-isomers of a cationic amino acid.
  • the 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).
  • PDEA poly(D-glutamic acid)
  • PDDA poly(D-aspartic acid)
  • PDA poly(L-glutamic acid)
  • PLDA poly(L-aspartic acid)
  • the 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).
  • the 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.
  • the 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.
  • timing of payload release can be controlled by selecting particular types of proteins, e.g., as part of the core (e.g., part of a cationic polypeptide composition, part of a cationic polymer composition, and/or part of an anionic polymer composition). For example, it may be desirable to delay payload release for a particular range of time, or until the payload is present at a particular cellular location (e.g., cytosol, nucleus, lysosome, endosome) or under a particular condition (e.g., low pH, high pH, etc.).
  • a particular cellular location e.g., cytosol, nucleus, lysosome, endosome
  • a particular condition e.g., low pH, high pH, etc.
  • a protein is used (e.g., as part of the core) that is susceptible to a specific protein activity (e.g., enzymatic activity), e.g., is a substrate for a specific protein activity (e.g., enzymatic activity), and this is in contrast to being susceptible to general ubiquitous cellular machinery, e.g., general degradation machinery.
  • ESP enzymatically susceptible protein
  • ESPs include but are not limited to: (i) proteins that are substrates for matrix metalloproteinase (MMP) activity (an example of an extracellular activity), e.g., a protein that includes a motif recognized by an MMP; (ii) proteins that are substrates for cathepsin activity (an example of an intracellular endosomal activity), e.g., a protein that includes a motif recognized by a cathepsin; and (iii) proteins such as histone tails peptides (HTPs) that are substrates for methyltransferase and/or acetyltransferase activity (an example of an intracellular nuclear activity), e.g., a protein that includes a motif that can be enzymatically methylated/de-methylated and/or a motif that can be enzymatically acetylated/de-acetylated.
  • MMP matrix metalloproteinase
  • cathepsin activity an example of
  • a nucleic acid payload is condensed with a protein (such as a histone tails peptide) that is a substrate for acetyltransferase activity, and acetylation of the protein causes the protein to release the payload—as such, one can exercise control over payload release by choosing to use a protein that is more or less susceptible to acetylation.
  • a protein such as a histone tails peptide
  • a core of a subject nanoparticle includes an enzymatically neutral polypeptide (ENP), which is a polypeptide homopolymer (i.e., a protein having a repeat sequence) where the polypeptide does not have a particular activity and is neutral.
  • ENP enzymatically neutral polypeptide
  • a core of a subject nanoparticle includes a enzymatically protected polypeptide (EPP), which is a protein that is resistant to enzymatic activity.
  • EPP enzymatically protected polypeptide
  • examples of PPs include but are not limited to: (i) polypeptides that include D-isomer amino acids (e.g., D-isomer polymers), which can resist proteolytic degradation; and (ii) self-sheltering domains such as a polyglutamine repeat domains (e.g., QQQQQQQQQ) (SEQ ID NO: 170).
  • ESPs susceptible proteins
  • EPPs protected proteins
  • use of more ESPs can in general lead to quicker release of payload than use of more EPPs.
  • use of more ESPs can in general lead to release of payload that depends upon a particular set of conditions/circumstances, e.g., conditions/circumstances that lead to activity of proteins (e.g., enzymes) to which the ESP is susceptible.
  • An anionic polymer composition can include one or more anionic amino acid polymers.
  • a subject anionic polymer composition includes a polymer selected from: poly(glutamic acid)(PEA), poly(aspartic acid)(PDA), and a combination thereof.
  • a given anionic amino acid polymer can include a mix of aspartic and glutamic acid residues.
  • Each polymer can be present in the composition as a polymer of L-isomers or D-isomers, where D-isomers are more stable in a target cell because they take longer to degrade.
  • inclusion of D-isomer poly(amino acids) in the nanoparticle core delays degradation of the core and subsequent payload release.
  • the payload release rate can therefore be controlled and is proportional to the ratio of polymers of D-isomers to polymers of L-isomers, where a higher ratio of D-isomer to L-isomer increases duration of payload release (i.e., decreases release rate).
  • the relative amounts of D- and L- isomers can modulate the nanoparticle core's timed release kinetics and enzymatic susceptibility to degradation and payload release.
  • an anionic polymer composition of a subject nanoparticle includes polymers of D-isomers and polymers of L-isomers of an anionic amino acid polymer (e.g., poly(glutamic acid)(PEA) and poly(aspartic acid)(PDA)).
  • an anionic amino acid polymer e.g., poly(glutamic acid)(PEA) and poly(aspartic acid)(PDA)
  • the D- to L- isomer ratio is in a range of from 10:1-1:10 (e.g., from 8:1-1:10, 6:1-1:10, 4:1-1:10, 3:1-1:10, 2:1-1:10, 1:1-1:10, 10:1-1:8, 8:1-1:8, 6:1-1:8, 4:1-1:8, 3:1-1:8, 2:1-1:8, 1:1-1:8, 10:1-1:6, 8:1-1:6, 6:1-1:6, 4:1-1:6, 3:1-1:6, 2:1-1:6, 1:1-1:6, 10:1-1:4, 8:1-1:4, 6:1-1:4, 4:1-1:4, 3:1-1:4, 2:1-1:4, 1:1-1:4, 10:1-1:3, 8:1-1:3, 6:1-1:3, 4:1-1:3, 3:1-1:3, 2:1-1:3, 1:1-1:3, 10:1-1:2, 8:1-1:2, 6:1-1:2, 4:1-1:2, 3:1-1:2, 2:1-1:2, 1:1-1:2, 10:1-1:1, 8:1-1:1, 6:1-1:1, 3:1:
  • an anionic polymer composition includes a first anionic polymer (e.g., amino acid polymer) that is a polymer of D-isomers (e.g., selected from poly(D-glutamic acid) (PDEA) and poly(D-aspartic acid) (PDDA)); and includes a second anionic polymer (e.g., amino acid polymer) that is a polymer of L-isomers (e.g., selected from poly(L-glutamic acid) (PLEA) and poly(L-aspartic acid) (PLDA)).
  • a first anionic polymer e.g., amino acid polymer
  • D-isomers e.g., selected from poly(D-glutamic acid) (PDEA) and poly(D-aspartic acid) (PDDA)
  • PDDA poly(D-aspartic acid)
  • second anionic polymer e.g., amino acid polymer
  • L-isomers e.g., selected from poly(
  • the ratio of the first anionic polymer (D-isomers) to the second anionic polymer (L-isomers) is in a range of from 10:1-1:10 (e.g., from 8:1-1:10, 6:1-1:10, 4:1-1:10, 3:1-1:10, 2:1-1:10, 1:1-1:10, 10:1-1:8, 8:1-1:8, 6:1-1:8, 4:1-1:8, 3:1-1:8, 2:1-1:8, 1:1-1:8, 10:1-1:6, 8:1-1:6, 6:1-1:6, 4:1-1:6, 3:1-1:6, 2:1-1:6, 1:1-1:6, 10:1-1:4, 8:1-1:4, 6:1-1:4, 4:1-1:4, 3:1-1:4, 2:1-1:4, 1:1-1:4, 10:1-1:3, 8:1-1:3, 6:1-1:3, 4:1-1:3, 3:1-1:3, 2:1-1:3, 1:1-1:3, 10:1-1:2, 8:1-1:2, 6:1-1:2, 4:1-1:2, 3:1-1:2, 2:1-1:2, 1:1-1:2,
  • an anionic polymer composition of a core of a subject nanoparticle includes (e.g., in addition to or in place of any of the foregoing examples of anionic polymers) a glycosaminoglycan, a glycoprotein, a polysaccharide, poly(mannuronic acid), poly(guluronic acid), heparin, heparin sulfate, chondroitin, chondroitin sulfate, keratan, keratan sulfate, aggrecan, poly(glucosamine), or an anionic polymer that comprises any combination thereof.
  • an anionic polymer within the core can have a molecular weight in a range of from 1-200 kDa (e.g., from 1-150, 1-100, 1-50, 5-200, 5-150, 5-100, 5-50, 10-200, 10-150, 10-100, 10-50, 15-200, 15-150, 15-100, or 15-50 kDa).
  • an anionic polymer includes poly(glutamic acid) with a molecular weight of approximately 15 kDa.
  • an anionic amino acid polymer includes a cysteine residue, which can facilitate conjugation, e.g., to a linker, an NLS, and/or a cationic polypeptide (e.g., a histone or HTP).
  • a cysteine residue can be used for crosslinking (conjugation) via sulfhydryl chemistry (e.g., a disulfide bond) and/or amine-reactive chemistry.
  • an anionic amino acid polymer e.g., poly(glutamic acid) (PEA), poly(aspartic acid) (PDA), poly(D-glutamic acid) (PDEA), poly(D-aspartic acid) (PDDA), poly(L-glutamic acid) (PLEA), poly(L-aspartic acid) (PLDA)
  • PEA poly(glutamic acid)
  • PDA poly(D-glutamic acid)
  • PDA poly(L-glutamic acid)
  • PDA poly(L-aspartic acid)
  • PLDA poly(L-aspartic acid)
  • an anionic amino acid polymer composition includes a cysteine residue.
  • the anionic amino acid polymer includes cysteine residue on the N- and/or C-terminus.
  • the anionic amino acid polymer includes an internal cysteine residue.
  • an anionic amino acid polymer includes (and/or is conjugated to) a nuclear localization signal (NLS) (described in more detail below).
  • NLS nuclear localization signal
  • an anionic amino acid polymer e.g., poly(glutamic acid) (PEA), poly(aspartic acid) (PDA), poly(D-glutamic acid) (PDEA), poly(D-aspartic acid) (PDDA), poly(L-glutamic acid) (PLEA), poly(L-aspartic acid) (PLDA)
  • PDA nuclear localization signal
  • an anionic amino acid polymer e.g., poly(glutamic acid) (PEA), poly(aspartic acid) (PDA), poly(D-glutamic acid) (PDEA), poly(D-aspartic acid) (PDDA), poly(L-glutamic acid) (PLEA), poly(L-aspartic acid) (PLDA)
  • an anionic amino acid polymer composition includes (and/or is conjugated to
  • an anionic polymer is added prior to a cationic polymer when generating a subject nanoparticle core.
  • a cationic polymer composition can include one or more cationic amino acid polymers.
  • a subject cationic polymer composition includes a polymer selected from: poly(arginine)(PR), poly(lysine)(PK), poly(histidine)(PH), poly(ornithine), poly(citrulline), and a combination thereof.
  • a given cationic amino acid polymer can include a mix of arginine, lysine, histidine, ornithine, and citrulline residues (in any convenient combination).
  • Each polymer can be present in the composition as a polymer of L-isomers or D-isomers, where D-isomers are more stable in a target cell because they take longer to degrade.
  • D-isomer poly(amino acids) delays degradation of the core and subsequent payload release.
  • the payload release rate can therefore be controlled and is proportional to the ratio of polymers of D-isomers to polymers of L-isomers, where a higher ratio of D-isomer to L-isomer increases duration of payload release (i.e., decreases release rate).
  • the relative amounts of D- and L- isomers can modulate the nanoparticle core's timed release kinetics and enzymatic susceptibility to degradation and payload release.
  • a cationic polymer composition of a subject nanoparticle includes polymers of D-isomers and polymers of L-isomers of an cationic amino acid polymer (e.g., poly(arginine)(PR), poly(lysine)(PK), poly(histidine)(PH), poly(ornithine), poly(citrulline)).
  • an cationic amino acid polymer e.g., poly(arginine)(PR), poly(lysine)(PK), poly(histidine)(PH), poly(ornithine), poly(citrulline)
  • the D- to L- isomer ratio is in a range of from 10:1-1:10 (e.g., from 8:1-1:10, 6:1-1:10, 4:1-1:10, 3:1-1:10, 2:1-1:10, 1:1-1:10, 10:1-1:8, 8:1-1:8, 6:1-1:8, 4:1-1:8, 3:1-1:8, 2:1-1:8, 1:1-1:8, 10:1-1:6, 8:1-1:6, 6:1-1:6, 4:1-1:6, 3:1-1:6, 2:1-1:6, 1:1-1:6, 10:1-1:4, 8:1-1:4, 6:1-1:4, 4:1-1:4, 3:1-1:4, 2:1-1:4, 1:1-1:4, 10:1-1:3, 8:1-1:3, 6:1-1:3, 4:1-1:3, 3:1-1:3, 2:1-1:3, 1:1-1:3, 10:1-1:2, 8:1-1:2, 6:1-1:2, 4:1-1:2, 3:1-1:2, 2:1-1:2, 1:1-1:2, 10:1-1:1, 8:1-1:1, 6:1-1:1, 3:1:
  • a cationic polymer composition includes a first cationic polymer (e.g., amino acid polymer) that is a polymer of D-isomers (e.g., selected from poly(D-arginine), poly(D-lysine), poly(D-histidine), poly(D-ornithine), and poly(D-citrulline)); and includes a second cationic polymer (e.g., amino acid polymer) that is a polymer of L-isomers (e.g., selected from poly(L-arginine), poly(L-lysine), poly(L-histidine), poly(L-ornithine), and poly(L-citrulline)).
  • a first cationic polymer e.g., amino acid polymer
  • D-isomers e.g., selected from poly(D-arginine), poly(D-lysine), poly(D-histidine), poly(D-ornithine), and poly(D-c
  • the ratio of the first cationic polymer (D-isomers) to the second cationic polymer (L-isomers) is in a range of from 10:1-1:10 (e.g., from 8:1-1:10, 6:1-1:10, 4:1-1:10, 3:1-1:10, 2:1-1:10, 1:1-1:10, 10:1-1:8, 8:1-1:8, 6:1-1:8, 4:1-1:8, 3:1-1:8, 2:1-1:8, 1:1-1:8, 10:1-1:6, 8:1-1:6, 6:1-1:6, 4:1-1:6, 3:1-1:6, 2:1-1:6, 1:1-1:6, 10:1-1:4, 8:1-1:4, 6:1-1:4, 4:1-1:4, 3:1-1:4, 2:1-1:4, 1:1-1:4, 10:1-1:3, 8:1-1:3, 6:1-1:3, 4:1-1:3, 3:1-1:3, 2:1-1:3, 1:1-1:3, 10:1-1:2, 8:1-1:2, 6:1-1:2, 4:1-1:2, 3:1-1:2, 2:1-1:2, 2:1-1
  • an cationic polymer composition of a core of a subject nanoparticle includes (e.g., in addition to or in place of any of the foregoing examples of cationic polymers) poly(ethylenimine), poly(amidoamine) (PAMAM), poly(aspartamide), polypeptoids (e.g., for forming “spiderweb”-like branches for core condensation), a charge-functionalized polyester, a cationic polysaccharide, an acetylated amino sugar, chitosan, or a cationic polymer that comprises any combination thereof (e.g., in linear or branched forms).
  • an cationic polymer within the core can have a molecular weight in a range of from 1-200 kDa (e.g., from 1-150, 1-100, 1-50, 5-200, 5-150, 5-100, 5-50, 10-200, 10-150, 10-100, 10-50, 15-200, 15-150, 15-100, or 15-50 kDa).
  • an cationic polymer includes poly(L-arginine), e.g., with a molecular weight of approximately 29 kDa.
  • a cationic polymer includes linear poly(ethylenimine) with a molecular weight of approximately 25 kDa (PEI).
  • a cationic polymer includes branched poly(ethylenimine) with a molecular weight of approximately 10 kDa.
  • a cationic polymer includes branched poly(ethylenimine) with a molecular weight of approximately 70 kDa.
  • a cationic polymer includes PAMAM.
  • a cationic amino acid polymer includes a cysteine residue, which can facilitate conjugation, e.g., to a linker, an NLS, and/or a cationic polypeptide (e.g., a histone or HTP).
  • a cysteine residue can be used for crosslinking (conjugation) via sulfhydryl chemistry (e.g., a disulfide bond) and/or amine-reactive chemistry.
  • the cationic amino acid polymer includes cysteine residue on the N- and/or C-terminus.
  • the cationic amino acid polymer includes an internal cysteine residue.
  • a cationic amino acid polymer includes (and/or is conjugated to) a nuclear localization signal (NLS) (described in more detail below).
  • NLS nuclear localization signal
  • the cationic polypeptide composition of a nanoparticle can mediate stability, subcellular compartmentalization, and/or payload release.
  • fragments of the N-terminus of histone proteins, referred to generally as histone tail peptides, within a subject nanoparticle core are in some case not only capable of being deprotonated by various histone modifications, such as in the case of histone acetyltransferase-mediated acetylation, but may also mediate effective nuclear-specific unpackaging of components (e.g., a payload) of a nanoparticle core.
  • a cationic polypeptide composition includes a histone and/or histone tail peptide (e.g., a cationic polypeptide can be a histone and/or histone tail peptide).
  • a cationic polypeptide composition includes an NLS-containing peptide (e.g., a cationic polypeptide can be an NLS-containing peptide).
  • a cationic polypeptide composition includes a peptide that includes a mitochondrial localization signal (e.g., a cationic polypeptide can be a peptide that includes a mitochondrial localization signal).
  • HTPs Histone Tail Peptide
  • a cationic polypeptide composition of a subject nanoparticle includes a histone peptide or a fragment of a histone peptide, such as an N-terminal histone tail (e.g., a histone tail of an H1, H2 (e.g., H2A, H2AX, H2B), H3, or H4 histone protein).
  • a histone tail peptide H1, H2 (e.g., H2A, H2AX, H2B), H3, or H4 histone protein).
  • H1, H2 e.g., H2A, H2AX, H2B
  • H3 histone tail peptide
  • a core that includes one or more histones or HTPs is sometimes referred to herein as a nucleosome-mimetic core.
  • Histones and/or HTPs can be included as monomers, and in some cases form dimers, trimers, tetramers and/or octamers when condensing a nucleic acid payload into a nanoparticle core.
  • HTPs are not only capable of being deprotonated by various histone modifications, such as in the case of histone acetyltransferase-mediated acetylation, but may also mediate effective nuclear-specific unpackaging of components of the core (e.g., release of a payload). Trafficking of a core that includes a histone and/or HTP may be reliant on alternative endocytotic pathways utilizing retrograde transport through the Golgi and endoplasmic reticulum. Furthermore, some histones include an innate nuclear localization sequence and inclusion of an NLS in the core can direct the core (including the payload) to the nucleus of a target cell.
  • a subject cationic polypeptide composition includes a protein having an amino acid sequence of an H2A, H2AX, H2B, H3, or H4 protein.
  • a subject cationic polypeptide composition includes a protein having an amino acid sequence that corresponds to the N-terminal region of a histone protein.
  • the fragment can include the first 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 N-terminal amino acids of a histone protein.
  • a subject HTP includes from 5-50 amino acids (e.g., from 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 8-50, 8-45, 8-40, 8-35, 8-30, 10-50, 10-45, 10-40, 10-35, or 10-30 amino acids) from the N-terminal region of a histone protein.
  • a subject a cationic polypeptide includes from 5-150 amino acids (e.g., from 5-100, 5-50, 5-35, 5-30, 5-25, 5-20, 8-150, 8-100, 8-50, 8-40, 8-35, 8-30, 10-150, 10-100, 10-50, 10-40, 10-35, or 10-30 amino acids).
  • a cationic polypeptide e.g., a histone or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4
  • a post-translational modification e.g., in some cases on one or more histidine, lysine, arginine, or other complementary residues.
  • the cationic polypeptide is methylated (and/or susceptible to methylation/demethylation), acetylated (and/or susceptible to acetylation/deacetylation), crotonylated (and/or susceptible to crotonylation/decrotonylation), ubiquitinylated (and/or susceptible to ubiquitinylation/deubiquitinylation), phosphorylated (and/or susceptible to phosphorylation/dephosphorylation), SUMOylated (and/or susceptible to SUMOylation/deSUMOylation), farnesylated (and/or susceptible to farnesylation/defarnesylation), sulfated (and/or susceptible to sulfation/desulfation) or otherwise post-translationally modified.
  • a cationic polypeptide e.g., a histone or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4
  • HTP histone or HTP
  • H1H2, H2A, H2AX, H2B, H3, or H4 a cationic polypeptide composition
  • p300/CBP substrate e.g., see example HTPs below, e.g., SEQ ID NOs: 129-130.
  • a cationic polypeptide e.g., a histone or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4
  • a cationic polypeptide composition includes one or more thiol residues (e.g., can include a cysteine and/or methionine residue) that is sulfated or susceptible to sulfation (e.g., as a thiosulfate sulfurtransferase substrate).
  • a cationic polypeptide e.g., a histone or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4
  • H1, H2, H2A, H2AX, H2B, H3, or H4 e.g., H1, H2, H2A, H2AX, H2B, H3, or H4
  • Histones H2A, H2B, H3, and H4 (and/or HTPs) may be monomethylated, dimethylated, or trimethylated at any of their lysines to promote or suppress transcriptional activity and alter nuclear-specific release kinetics.
  • a cationic polypeptide can be synthesized with a desired modification or can be modified in an in vitro reaction.
  • a cationic polypeptide e.g., a histone or HTP
  • the desired modified protein can be isolated/purified.
  • the cationic polypeptide composition of a subject nanoparticle includes a methylated HTP, e.g., includes the HTP sequence of H3K4(Me3)—includes the amino acid sequence set forth as SEQ ID NO: 75 or 88).
  • a cationic polypeptide e.g., a histone or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4
  • H1, H2, H2A, H2AX, H2B, H3, or H4 e.g., H1, H2, H2A, H2AX, H2B, H3, or H4
  • H2A (SEQ ID NO: 62) SGRGKQGGKARAKAKTRSSR [1-20] (SEQ ID NO: 63) SGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGGG [1-39] (SEQ ID NO: 64) MSGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYAERVGAGAP VYLAAVLEYLTAEILELAGNAARDNKKTRIIPRHLQLAIRNDEELNKLL GKVTIAQGGVLPNIQAVLLPKKTESHHKAKGK [1-130] H2AX (SEQ ID NO: 65) CKATQASQEY [134-143] (SEQ ID NO: 66) KKTSATVGPKAPSGGKKATQASQEY [KK 120-129] (SEQ ID NO: 67) MSGRGKTGGKARAKAKSRSSRAGLQFPVGRVHRLLRKGHYAERVGAGAP VYLAAVLEYLTAEILELAGN
  • a cationic polypeptide of a subject cationic polypeptide composition can include an amino acid sequence having the amino acid sequence set forth in any of SEQ ID NOs: 62-139.
  • a cationic polypeptide of subject a cationic polypeptide composition includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% sequence identity) with the amino acid sequence set forth in any of SEQ ID NOs: 62-139.
  • a cationic polypeptide of subject a cationic polypeptide composition includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 98% or more, 99% or more, or 100% sequence identity) with the amino acid sequence set forth in any of SEQ ID NOs: 62-139.
  • the cationic polypeptide can include any convenient modification, and a number of such contemplated modifications are discussed above, e.g., methylated, acetylated, crotonylated, ubiquitinylated, phosphorylated, SUMOylated, farnesylated, sulfated, and the like.
  • a cationic polypeptide of a cationic polypeptide composition includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% sequence identity) with the amino acid sequence set forth in SEQ ID NO: 94. In some cases a cationic polypeptide of a cationic polypeptide composition includes an amino acid sequence having 95% or more sequence identity (e.g., 98% or more, 99% or more, or 100% sequence identity) with the amino acid sequence set forth in SEQ ID NO: 94. In some cases a cationic polypeptide of a cationic polypeptide composition includes the amino acid sequence set forth in SEQ ID NO: 94.
  • a cationic polypeptide of a cationic polypeptide composition includes the sequence represented by H3K4(Me3) (SEQ ID NO: 95), which comprises the first 25 amino acids of the human histone 3 protein, and tri-methylated on the lysine 4 (e.g., in some cases amidated on the C-terminus).
  • a cationic polypeptide e.g., a histone or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4
  • a cationic polypeptide composition includes a cysteine residue, which can facilitate conjugation to: a cationic (or in some cases anionic) amino acid polymer, a linker, an NLS, and/or other cationic polypeptides (e.g., in some cases to form a branched histone structure).
  • a cysteine residue can be used for crosslinking (conjugation) via sulfhydryl chemistry (e.g., a disulfide bond) and/or amine-reactive chemistry.
  • the cysteine residue is internal.
  • the cysteine residue is positioned at the N-terminus and/or C-terminus.
  • a cationic polypeptide e.g., a histone or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4
  • HTPs that include a cysteine include but are not limited to:
  • a cationic polypeptide e.g., a histone or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4
  • a cationic polypeptide composition is conjugated to a cationic (and/or anionic) amino acid polymer of the core of a subject nanoparticle.
  • a histone or HTP can be conjugated to a cationic amino acid polymer (e.g., one that includes poly(lysine)), via a cysteine residue, e.g., where the pyridyl disulfide group(s) of lysine(s) of the polymer are substituted with a disulfide bond to the cysteine of a histone or HTP.
  • a cationic amino acid polymer e.g., one that includes poly(lysine)
  • cysteine residue e.g., where the pyridyl disulfide group(s) of lysine(s) of the polymer are substituted with a disulfide bond to the cysteine of a histone or HTP.
  • a cationic polypeptide of a subject a cationic polypeptide composition has a linear structure. In some embodiments a cationic polypeptide of a subject a cationic polypeptide composition has a branched structure.
  • a cationic polypeptide e.g., HTPs, e.g., HTPs with a cysteine residue
  • a cationic polymer e.g., poly(L-arginine), poly(D-lysine), poly(L-lysine), poly(D-lysine)
  • a cationic polymer e.g., poly(L-arginine), poly(D-lysine), poly(L-lysine), poly(D-lysine)
  • one or more (two or more, three or more, etc.) cationic polypeptides are conjugated (e.g., at their C-termini) to the end(s) of a cationic polymer (e.g., poly(L-arginine), poly(D-lysine), poly(L-lysine), poly(D-lysine)), thus forming an extended linear polypeptide.
  • a cationic polymer e.g., poly(L-arginine), poly(D-lysine), poly(L-lysine), poly(D-lysine)
  • the cationic polymer has a molecular weight in a range of from 4,500-150,000 Da).
  • one or more (two or more, three or more, etc.) cationic polypeptides are conjugated (e.g., at their C-termini) to the side-chains of a cationic polymer (e.g., poly(L-arginine), poly(D-lysine), poly(L-lysine), poly(D-lysine)), thus forming a branched structure (branched polypeptide).
  • a cationic polymer e.g., poly(L-arginine), poly(D-lysine), poly(L-lysine), poly(D-lysine)
  • Formation of a branched structure by components of the nanoparticle core can in some cases increase the amount of core condensation (e.g., of a nucleic acid payload) that can be achieved. Thus, in some cases it is desirable to used components that form a branched structure.
  • branches structures are of interest, and examples of branches structures that can be generated (e.g., using subject cationic polypeptides such as HTPs, e.g., HTPs with a cysteine residue; peptoids, polyamides, and the like) include but are not limited to: brush polymers, webs (e.g., spider webs), graft polymers, star-shaped polymers, comb polymers, polymer networks, dendrimers, and the like.
  • subject cationic polypeptides such as HTPs, e.g., HTPs with a cysteine residue; peptoids, polyamides, and the like
  • brush polymers e.g., webs
  • graft polymers graft polymers
  • star-shaped polymers e.g., comb polymers
  • polymer networks e.g., dendrimers, and the like.
  • FIG. 134 depicts a brush type of branched structure.
  • a branched structure includes from 2-30 cationic polypeptides (e.g., HTPs) (e.g., from 2-25, 2-20, 2-15, 2-10, 2-5, 4-30, 4-25, 4-20, 4-15, or 4-10 cationic polypeptides), where each can be the same or different than the other cationic polypeptides of the branched structure (see, e.g., FIG. 134 ).
  • the cationic polymer has a molecular weight in a range of from 4,500-150,000 Da).
  • 5% or more (e.g., 10% or more, 20% or more, 25% or more, 30% or more, 40% or more, or 50% or more) of the side-chains of a cationic polymer e.g., poly(L-arginine), poly(D-lysine), poly(L-lysine), poly(D-lysine)
  • a subject cationic polypeptide e.g., HTP, e.g., HTP with a cysteine residue
  • a cationic polymer e.g., poly(L-arginine), poly(D-lysine), poly(L-lysine), poly(D-lysine)
  • a subject cationic polypeptide e.g., HTP, e.g., HTP with a cysteine residue
  • HTP e.g., HTP with a cysteine residue
  • branched structures can be facilitated using components such as peptoids (polypeptoids), polyamides, dendrimers, and the like.
  • peptoids e.g., polypeptoids
  • a nanoparticle core e.g., in order to generate a web (e.g., spider web) structure, which can in some cases facilitate condensation of the nanoparticle core.
  • each polypeptide is included in equal amine molarities within a nanoparticle core.
  • each polypeptide's C-terminus can be modified with 5R (5 arginines).
  • each polypeptide's C-terminus can be modified with 9R (9 arginines).
  • each polypeptide's N-terminus can be modified with 5R (5 arginines).
  • each polypeptide's N-terminus can be modified with 9R (9 arginines).
  • an H2A, H2B, H3 and/or H4 histone fragment are each bridged in series with a FKFL Cathepsin B proteolytic cleavage domain or RGFFP Cathepsin D proteolytic cleavage domain.
  • an H2A, H2B, H3 and/or H4 histone fragment can be bridged in series by a 5R (5 arginines), 9R (9 arginines), 5K (5 lysines), 9K (9 lysines), 5H (5 histidines), or 9H (9 histidines) cationic spacer domain.
  • one or more H2A, H2B, H3 and/or H4 histone fragments are disulfide-bonded at their N-terminus to protamine.
  • a 29 ⁇ L aqueous solution of 700 ⁇ M Cys-modified histone/NLS (20 nmol) can be added to 57 ⁇ L of 0.2 M phosphate buffer (pH 8.0).
  • 14 ⁇ L of 100 ⁇ M pyridyl disulfide protected poly(lysine) solution can then be added to the histone solution bringing the final volume to 100 ⁇ L with a 1:2 ratio of pyridyl disulfide groups to Cysteine residues.
  • This reaction can be carried out at room temperature for 3 h.
  • the reaction can be repeated four times and degree of conjugation can be determined via absorbance of pyridine-2-thione at 343 nm.
  • a 29 ⁇ L aqueous solution of 700 ⁇ M Cys-modified histone (20 nmol) can be added to 57 ⁇ L of 0.2 M phosphate buffer (pH 8.0).
  • 14 ⁇ L of 100 ⁇ M pyridyl disulfide protected poly(lysine) solution can then be added to the histone solution bringing the final volume to 100 ⁇ L with a 1:2 ratio of pyridyl disulfide groups to Cysteine residues.
  • This reaction can be carried out at room temperature for 3 h. The reaction can be repeated four times and degree of conjugation can be determined via absorbance of pyridine-2-thione at 343 nm.
  • an anionic polymer is conjugated to a targeting ligand.
  • a cationic polypeptide e.g., a histone or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4
  • a cationic polypeptide composition of a subject nanoparticle includes (and/or is conjugated to) one or more (e.g., two or more, three or more, or four or more) nuclear localization sequences (NLSs).
  • NLSs nuclear localization sequences
  • the cationic polypeptide composition of a subject nanoparticle includes a peptide that includes an NLS.
  • a histone protein (or an HTP) of a subject nanoparticle includes one or more (e.g., two or more, three or more) natural nuclear localization signals (NLSs).
  • a histone protein (or an HTP) of a subject nanoparticle includes one or more (e.g., two or more, three or more) NLSs that are heterologous to the histone protein (i.e., NLSs that do not naturally occur as part of the histone/HTP, e.g., an NLS can be added by humans).
  • the HTP includes an NLS on the N- and/or C-terminus.
  • the cationic amino acid polymer includes an NLS on the N- and/or C-terminus.
  • the cationic amino acid polymer includes an N
  • an anionic amino acid polymer e.g., poly(glutamic acid) (PEA), poly(aspartic acid) (PDA), poly(D-glutamic acid) (PDEA), poly(D-aspartic acid) (PDDA), poly(L-glutamic acid) (PLEA), or poly(L-aspartic acid) (PLDA)
  • an anionic polymer composition includes (and/or is conjugated to) one or more (e.g., two or more, three or more, or four or more) NLSs.
  • the anionic amino acid polymer includes an NLS on the N- and/or C-terminus.
  • the anionic amino acid polymer includes an internal NLS.
  • NLS any convenient NLS can be used (e.g., conjugated to a histone, an HTP, a cationic amino acid polymer, an anionic amino acid polymer, and the like). Examples include, but are not limited to Class 1 and Class 2 ‘monopartite NLSs’, as well as NLSs of Classes 3-5 (see, e.g., FIG. 16 , which is adapted from Kosugi et al., J Biol Chem. 2009 Jan. 2; 284(1):478-85). In some cases, an NLS has the formula: (K/R) (K/R) X10-12 (K/R) 3-5 . In some cases, an NLS has the formula: K(K/R)X(K/R).
  • a cationic polypeptide of a cationic polypeptide composition includes one more (e.g., two or more, three or more, or four or more) NLSs.
  • the cationic polypeptide is not a histone protein or histone fragment (e.g., is not an HTP).
  • the cationic polypeptide of a cationic polypeptide composition is an NLS-containing peptide.
  • the NLS-containing peptide includes a cysteine residue, which can facilitate conjugation to: a cationic (or in some cases anionic) amino acid polymer, a linker, histone protein for HTP, and/or other cationic polypeptides (e.g., in some cases as part of a branched histone structure).
  • a cysteine residue can be used for crosslinking (conjugation) via sulfhydryl chemistry (e.g., a disulfide bond) and/or amine-reactive chemistry.
  • the cysteine residue is internal.
  • the cysteine residue is positioned at the N-terminus and/or C-terminus.
  • an NLS-containing peptide of a cationic polypeptide composition includes a mutation (e.g., insertion or substitution) (e.g., relative to a wild type amino acid sequence) that adds a cysteine residue.
  • NLSs that can be used as an NLS-containing peptide (or conjugated to any convenient cationic polypeptide such as an HTP or cationic polymer or cationic amino acid polymer or anionic amino acid polymer) include but are not limited to (some of which include a cysteine residue):
  • a cationic polypeptide e.g., a histone or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4
  • an anionic polymer e.g., H1, H2, H2A, H2AX, H2B, H3, or H4
  • a cationic polymer of a subject nanoparticle includes (and/or is conjugated to) one or more (e.g., two or more, three or more, or four or more) mitochondrial localization sequences. Any convenient mitochondrial localization sequence can be used.
  • mitochondrial localization sequences include but are not limited to: PEDEIWLPEPESVDVPAKPISTSSMMMP (SEQ ID NO: 149), a mitochondrial localization sequence of SDHB, mono/di/triphenylphosphonium or other phosphoniums, VAMP 1A, VAMP 1B, the 67 N-terminal amino acids of DGAT2, and the 20 N-terminal amino acids of Bax.
  • Nanoparticles of the disclosure include a payload, which can be made of nucleic acid and/or protein.
  • a subject nanoparticle is used to deliver a nucleic acid payload (e.g., a DNA and/or RNA).
  • the nucleic acid payload can be any nucleic acid of interest, e.g., the nucleic acid payload can be linear or circular, and can be a plasmid, a viral genome, an RNA (e.g., a coding RNA such as an mRNA or a non-coding RNA such as a guide RNA, a short interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), and the like), a DNA, etc.
  • the nucleic payload is an RNAi agent (e.g., an shRNA, an siRNA, a miRNA, etc.) or a DNA template encoding an RNAi agent.
  • the nucleic acid payload is an siRNA molecule (e.g., one that targets an mRNA, one that targets a miRNA). In some cases, the nucleic acid payload is an LNA molecule (e.g., one that targets a miRNA). In some cases, the nucleic acid payload is a miRNA.
  • the nucleic acid payload includes an mRNA that encodes a protein of interest (e.g., one or more reprograming and/or transdifferentiation factors such as Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28, e.g., alone or in any desired combination such as (i) Oct4, Sox2, Klf4, and c-Myc; (ii) Oct4, Sox2, Nanog, and Lin28; and the like; a gene editing endonuclease; a therapeutic protein; and the like).
  • a protein of interest e.g., one or more reprograming and/or transdifferentiation factors such as Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28, e.g., alone or in any desired combination such as (i) Oct4, Sox2, Klf4, and c-Myc; (ii) Oct4, Sox2, Nanog, and Lin28; and the like; a gene editing endonuclease;
  • the nucleic acid payload includes a non-coding RNA (e.g., an RNAi agent, a CRISPR/Cas guide RNA, etc.) and/or a DNA molecule encoding the non-coding RNA.
  • a nucleic acid payload includes a nucleic acid (DNA and/or mRNA) that encodes IL2R ⁇ and IL12R ⁇ (e.g., to modulate the behavior or survival of a target cell), and in some cases the payload is released intracellularly from a subject nanoparticle over the course of from 7-90 days (e.g., from 7-80, 7-60, 7-50, 7-40, 7-35, or 7-30 days).
  • a nucleic acid payload includes a nucleic acid (DNA and/or mRNA) that encodes BCL-XL (e.g., to prevent apoptosis of a target cell due to engagement of Fas or TNF ⁇ receptors).
  • a nucleic acid payload includes a nucleic acid (DNA and/or mRNA) that encodes
  • a nucleic acid payload includes a nucleic acid (DNA and/or mRNA) that encodes SCF.
  • a nucleic acid payload includes a nucleic acid (DNA and/or mRNA) that encodes HoxB4.
  • a nucleic acid payload includes a nucleic acid (DNA and/or mRNA) that encodes SIRT6.
  • a nucleic acid payload includes a nucleic acid molecule (e.g., an siRNA, an LNA, etc.) that targets (reduces expression of) a microRNA such as miR-155 (see, e.g., MiR Base accession: MI0000681 and MI0000177).
  • a nucleic acid payload includes an siRNA that targets ku70 and/or an siRNA that targets ku80.
  • nucleic acid payload encompasses modified nucleic acids.
  • RNAi agent and “siRNA” encompass modified nucleic acids.
  • the nucleic acid molecule can be a mimetic, can include a modified sugar backbone, one or more modified internucleoside linkages (e.g., one or more phosphorothioate and/or heteroatom internucleoside linkages), one or more modified bases, and the like.
  • a subject payload includes triplex-forming peptide nucleic acids (PNAs) (see, e.g., McNeer et al., Gene Ther. 2013 June; 20(6):658-69).
  • PNAs triplex-forming peptide nucleic acids
  • a subject nucleic acid payload can have a morpholino backbone structure.
  • a subject nucleic acid payload e.g., an siRNA
  • Suitable sugar substituent groups include methoxy (—O—CH 3 ), aminopropoxy (—OCH 2 CH 2 CH 2 NH 2 ), allyl (—CH 2 —CH ⁇ CH 2 ), —O-allyl (—O—CH 2 —CH ⁇ CH 2 ) and fluoro (F). 2′-sugar substituent groups may be in the arabino (up) position or ribo (down) position.
  • Suitable base modifications include synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C ⁇ C—CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-
  • nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g.
  • a nucleic acid payload can include a conjugate moiety (e.g., one that enhances the activity, stability, cellular distribution or cellular uptake of the nucleic acid payload).
  • conjugate moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups.
  • Conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
  • Suitable conjugate groups include, but are not limited to, cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid.
  • Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of a subject nucleic acid.
  • Any convenient polynucleotide can be used as a subject nucleic acid payload.
  • examples include but are not limited to: species of RNA and DNA including mRNA, m1A modified mRNA (monomethylation at position 1 of Adenosine), siRNA, miRNA, aptamers, shRNA, AAV-derived nucleic acids and scaffolds, morpholino RNA, peptoid and peptide nucleic acids, cDNA, DNA origami, DNA and RNA with synthetic nucleotides, DNA and RNA with predefined secondary structures, multimers and oligomers of the aforementioned, and payloads whose sequence may encode other products such as any protein or polypeptide whose expression is desired.
  • a payload of a subject nanoparticle includes a protein.
  • protein payloads include, but are not limited to: programmable gene editing proteins (e.g., transcription activator-like (TAL) effectors (TALEs), TALE nucleases (TALENs), zinc-finger proteins (ZFPs), zinc-finger nucleases (ZFNs), DNA-guided polypeptides such as Natronobacterium gregoryi Argonaute (NgAgo), CRISPR/Cas RNA-guided polypeptides such as Cas9, CasX, CasY, Cpf1, and the like); transposons (e.g., a Class I or Class II transposon—e.g., piggybac, sleeping beauty, Tc1/mariner, Tol2, PIF/harbinger, hAT, mutator, merlin, transib, helitron, maverick, frog prince, minos, Himar1 and the like); meganucleases
  • a payload of a subject nanoparticle can include a nucleic acid (DNA and/or mRNA) encoding the protein, and/or can include the actual protein.
  • a nucleic acid payload includes or encodes a gene editing tool (i.e., a component of a gene editing system, e.g., a site specific gene editing system such as a programmable gene editing system).
  • a nucleic acid payload can include one or more of: (i) a CRISPR/Cas guide RNA, (ii) a DNA encoding a CRISPR/Cas guide RNA, (iii) a DNA and/or RNA encoding a programmable gene editing protein such as a zinc finger protein (ZFP) (e.g., a zinc finger nuclease—ZFN), a transcription activator-like effector (TALE) protein (e.g., fused to a nuclease—TALEN), a DNA-guided polypeptide such as Natronobacterium gregoryi Argonaute (NgAgo), and/or a CRISPR/Cas RNA-guided poly
  • ZFP
  • a subject nanoparticle is used to deliver a protein payload, e.g., a gene editing protein such as a ZFP (e.g., ZFN), a TALE (e.g., TALEN), a DNA-guided polypeptide such as Natronobacterium gregoryi Argonaute (NgAgo), a CRISPR/Cas RNA-guided polypeptide (e.g., Cas9, CasX, CasY, Cpf1, and the like), a site-specific recombinase (e.g., Cre recombinase, Dre recombinase, Flp recombinase, KD recombinase, B2 recombinase, B3 recombinase, R recombinase, Hin recombinase, Tre recombinase, PhiC31 integrase, Bxb1 integras
  • a gene editing system e.g. a site specific gene editing system such as a programmable gene editing system
  • a gene editing system can include a single component (e.g., a ZFP, a ZFN, a TALE, a TALEN, a site-specific recombinase, a resolvase/integrase, a transpose, a transposon, and the like) or can include multiple components.
  • a gene editing system includes at least two components.
  • a gene editing system e.g.
  • a programmable gene editing system includes (i) a donor template nucleic acid; and (ii) a gene editing protein (e.g., a programmable gene editing protein such as a ZFP, a ZFN, a TALE, a TALEN, a DNA-guided polypeptide such as Natronobacterium gregoryi Argonaute (NgAgo), a CRISPR/Cas RNA-guided polypeptide such as Cas9, CasX, CasY, or Cpf1, and the like), or a nucleic acid molecule encoding the gene editing protein (e.g., DNA or RNA such as a plasmid or mRNA).
  • a gene editing protein e.g., a programmable gene editing protein such as a ZFP, a ZFN, a TALE, a TALEN, a DNA-guided polypeptide such as Natronobacterium gregoryi Argonaute (NgAgo),
  • a gene editing system (e.g. a programmable gene editing system) includes (i) a CRISPR/Cas guide RNA, or a DNA encoding the CRISPR/Cas guide RNA; and (ii) a CRISPR/CAS RNA-guided polypeptide (e.g., Cas9, CasX, CasY, Cpf1, and the like), or a nucleic acid molecule encoding the RNA-guided polypeptide (e.g., DNA or RNA such as a plasmid or mRNA).
  • a gene editing system e.g.
  • a programmable gene editing system includes (i) an NgAgo-like guide DNA; and (ii) a DNA-guided polypeptide (e.g., NgAgo), or a nucleic acid molecule encoding the DNA-guided polypeptide (e.g., DNA or RNA such as a plasmid or mRNA).
  • a gene editing system e.g.
  • a programmable gene editing system includes at least three components: (i) a donor DNA template; (ii) a CRISPR/Cas guide RNA, or a DNA encoding the CRISPR/Cas guide RNA; and (iii) a CRISPR/Cas RNA-guided polypeptide (e.g., Cas9, CasX, CasY, or Cpf1), or a nucleic acid molecule encoding the RNA-guided polypeptide (e.g., DNA or RNA such as a plasmid or mRNA).
  • a gene editing system e.g.
  • a programmable gene editing system includes at least three components: (i) a donor DNA template; (ii) an NgAgo-like guide DNA, or a DNA encoding the NgAgo-like guide DNA; and (iii) a DNA-guided polypeptide (e.g., NgAgo), or a nucleic acid molecule encoding the DNA-guided polypeptide (e.g., DNA or RNA such as a plasmid or mRNA).
  • a donor DNA template includes at least three components: (i) a donor DNA template; (ii) an NgAgo-like guide DNA, or a DNA encoding the NgAgo-like guide DNA; and (iii) a DNA-guided polypeptide (e.g., NgAgo), or a nucleic acid molecule encoding the DNA-guided polypeptide (e.g., DNA or RNA such as a plasmid or mRNA).
  • a subject nanoparticle is used to deliver a gene editing tool.
  • the payload includes one or more gene editing tools.
  • the term “gene editing tool” is used herein to refer to one or more components of a gene editing system.
  • the payload includes a gene editing system and in some cases the payload includes one or more components of a gene editing system (i.e., one or more gene editing tools).
  • a target cell might already include one of the components of a gene editing system and the user need only add the remaining components.
  • the payload of a subject nanoparticle does not necessarily include all of the components of a given gene editing system.
  • a payload includes one or more gene editing tools.
  • a target cell might already include a gene editing protein (e.g., a ZFP, a TALE, a DNA-guided polypeptide (e.g., NgAgo), a CRISPR/Cas RNA-guided polypeptide such Cas9, CasX, CasY, Cpf1, and the like, a site-specific recombinase such as Cre recombinase, Dre recombinase, Flp recombinase, KD recombinase, B2 recombinase, B3 recombinase, R recombinase, Hin recombinase, Tre recombinase, PhiC31 integrase, Bxb1 integrase, R4 integrase, lambda integrase, HK022 integrase, HP1 integrase, and the like,
  • a gene editing protein e
  • the target cell may already include a CRISPR/Cas guide RNA and/or a DNA encoding the guide RNA or an NgAgo-like guide DNA
  • the payload can include one or more of: (i) a donor template; and (ii) a CRISPR/Cas RNA-guided polypeptide (e.g., Cas9, CasX, CasY, Cpf1, and the like), or a nucleic acid molecule encoding the RNA-guided polypeptide (e.g., DNA or RNA such as a plasmid or mRNA); or a DNA-guided polypeptide (e.g., NgAgo), or a nucleic acid molecule encoding the DNA-guided polypeptide.
  • a CRISPR/Cas RNA-guided polypeptide e.g., Cas9, CasX, CasY, Cpf1, and the like
  • a nucleic acid molecule encoding the
  • a gene editing system need not be a system that ‘edits’ a nucleic acid.
  • a gene editing system can be used to modify target nucleic acids (e.g., DNA and/or RNA) in a variety of ways without creating a double strand break (DSB) in the target DNA.
  • target nucleic acids e.g., DNA and/or RNA
  • a double stranded target DNA is nicked (one strand is cleaved), and in some cases (e.g., in some cases where the gene editing protein is devoid of nuclease activity, e.g., a CRISPR/Cas RNA-guided polypeptide may harbor mutations in the catalytic nuclease domains), the target nucleic acid is not cleaved at all.
  • a CRISPR/Cas protein e.g., Cas9, CasX, CasY, Cpf1 with or without nuclease activity, is fused to a heterologous protein domain.
  • the heterologous protein domain can provide an activity to the fusion protein such as (i) a DNA-modifying activity (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity), (ii) a transcription modulation activity (e.g., fusion to a transcriptional repressor or activator), or (iii) an activity that modifies a protein (e.g., a histone) that is associated with target DNA (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase
  • programmable gene editing tools e.g., CRISPR/Cas RNa-guided proteins such as Cas9, CasX, CasY, and Cpf1
  • Zinc finger proteins such as Zinc finger nucleases
  • TALE proteins such as TALENs, CRISPR/Cas guide RNAs, and the like
  • Dreier et al., (2001) J Biol Chem 276:29466-78; Dreier, et al., (2000) J Mol Biol 303:489-502; Liu, et al., (2002) J Biol Chem 277:3850-6); Dreier, et al., (2005) J Biol Chem 280:35588-97; Jamieson, et al., (2003) Nature Rev Drug Discov 2:361-8; Durai, et al., (2005) Nucleic Acids Res 33:5978-90; Segal, (2002) Methods 26:76-83; Porteus and
  • more than one payload is delivered as part of the same package (e.g., nanoparticle), e.g., in some cases different payloads are part of different cores.
  • One advantage of delivering multiple payloads as part of the same package (e.g., nanoparticle) is that the efficiency of each payload is not diluted. As an illustrative example, if payload A and payload B are delivered in two separate packages (package A and package B, respectively), then the efficiencies are multiplicative, e.g., if package A and package B each have a 1% transfection efficiency, the chance of delivering payload A and payload B to the same cell is 0.01% (1% ⁇ 1%).
  • payload A and payload B are both delivered as part of the same package (e.g., part of the same nanoparticle—package A), then the chance of delivering payload A and payload B to the same cell is 1%, a 100-fold improvement over 0.01%.
  • the chance of delivering payload A and payload B to the same cell is 0.0001% (0.1% ⁇ 0.1%).
  • payload A and payload B are both delivered as part of the same package (e.g., part of the same nanoparticle—package A) in this scenario, then the chance of delivering payload A and payload B to the same cell is 0.1%, a 1000-fold improvement over 0.0001%.
  • one or more gene editing tools is delivered in combination with (e.g., as part of the same nanoparticle) a protein (and/or a DNA or mRNA encoding same) and/or a non-coding RNA that increases genomic editing efficiency.
  • one or more gene editing tools is delivered in combination with (e.g., as part of the same nanoparticle) a protein (and/or a DNA or mRNA encoding same) and/or a non-coding RNA that controls cell division and/or differentiation.
  • one or more gene editing tools (e.g., as described above) is delivered in combination with (e.g., as part of the same nanoparticle) a protein (and/or a DNA or mRNA encoding same) and/or a non-coding RNA that biases the cell DNA repair machinery toward non-homologous end joining (NHEJ) or homology directed repair (HDR).
  • NHEJ non-homologous end joining
  • HDR homology directed repair
  • one or more gene editing tools can be delivered in combination with one or more of: SCF (and/or a DNA or mRNA encoding SCF), HoxB4 (and/or a DNA or mRNA encoding HoxB4), BCL-XL (and/or a DNA or mRNA encoding BCL-XL), SIRT6 (and/or a DNA or mRNA encoding SIRT6), a nucleic acid molecule (e.g., an siRNA and/or an LNA) that suppresses miR-155, a nucleic acid molecule (e.g., an siRNA, an shRNA, a microRNA) that reduces ku70 expression, and a nucleic acid molecule (e.g., an siRNA, an shRNA, a microRNA) that reduces ku80 expression.
  • SCF and/or a DNA or mRNA encoding SCF
  • HoxB4 and/or a DNA or mRNA encoding HoxB4
  • microRNAs that can be delivered in combination with a gene editing tool, see FIG. 18A .
  • the following microRNAs can be used for the following purposes: for blocking differentiation of a pluripotent stem cell toward ectoderm lineage: miR-430/427/302 (see, e.g., MiR Base accession: MI0000738, MI0000772, MI0000773, MI0000774, MI0006417, MI0006418, MI0000402, MI0003716, MI0003717, and MI0003718); for blocking differentiation of a pluripotent stem cell toward endoderm lineage: miR-109 and/or miR-24 (see, e.g., MiR Base accession: MI0000080, MI0000081, MI0000231, and MI0000572); for driving differentiation of a pluripotent stem cell toward endoderm lineage: miR-122 (see, e.g., MiR Base accession: MI0000442 and MI0000256) and/or miR-192 (see, e
  • signaling proteins e.g., extracellular signaling proteins
  • a gene editing tool see FIG. 18B .
  • the same proteins can be used as part of the outer shell of a subject nanoparticle in a similar manner as a targeting ligand, e.g., for the purpose of biasing differentiation in target cells that receive the nanoparticle.
  • the following signaling proteins can be used for the following purposes: for driving differentiation of a hematopoietic stem cell toward a common lymphoid progenitor cell lineage: IL-7 (see, e.g., NCBI Gene ID 3574); for driving differentiation of a hematopoietic stem cell toward a common myeloid progenitor cell lineage: IL-3 (see, e.g., NCBI Gene ID 3562), GM-CSF (see, e.g., NCBI Gene ID 1437), and/or M-CSF (see, e.g., NCBI Gene ID 1435); for driving differentiation of a common lymphoid progenitor cell toward a B-cell fate: IL-3, IL-4 (see, e.g., NCBI Gene ID: 3565), and/or IL-7; for driving differentiation of a common lymphoid progenitor cell toward a Natural Killer Cell fate: IL-15 (
  • proteins that can be delivered include but are not limited to: SOX17, HEX, OSKM (Oct4/Sox2/Klf4/c-myc), and/or bFGF (e.g., to drive differentiation toward hepatic stem cell lineage); HNF4a (e.g., to drive differentiation toward hepatocyte fate); Poly (1:0), BMP-4, bFGF, and/or 8-Br-cAMP (e.g., to drive differentiation toward endothelial stem cell/progenitor lineage); VEGF (e.g., to drive differentiation toward arterial endothelium fate); Sox-2, Brn4, Mytl1, Neurod2, Ascl1 (e.g., to drive differentiation toward neural stem cell/progenitor lineage); and BDNF, FCS, Forskolin, and/or SHH (e
  • signaling proteins e.g., extracellular signaling proteins
  • cytokines e.g., IL-2 and/or IL-15, e.g., for activating CD8+ T-cells
  • ligands and or signaling proteins that modulate one or more of the Notch, Wnt, and/or Smad signaling pathways
  • SCF stem cell differentiating factors
  • a fibroblast may be converted into a neural stem cell via delivery of Sox2, while it will turn into a cardiomyocyte in the presence of Oct3/4 and small molecule “epigenetic resetting factors.”
  • these fibroblasts may respectively encode diseased phenotypic traits associated with neurons and cardiac cells.
  • the packaging of multiple payloads in the same package does not preclude one from achieving different release times and/or locations for different payloads.
  • the release of the above proteins (and/or a DNAs or mRNAs encoding same) and/or non-coding RNAs can be controlled separately from the release of the one or more gene editing tools that are part of the same package.
  • proteins and/or nucleic acids e.g., DNAs, mRNAs, non-coding RNAs, miRNAs
  • proteins and/or nucleic acids can be released earlier than the one or more gene editing tools or can be released later than the one or more gene editing tools.
  • This can be achieved, e.g., by using more than one sheddable layer and/or by using more than one core (e.g., where one core has a different release profile than the other, e.g., uses a different D- to L-isomer ratio, uses a different ESP:ENP:EPP profile, and the like).
  • a subject nanoparticle includes a sheddable layer (also referred to herein as a “transient stabilizing layer”) that surrounds (encapsulates) the core.
  • a subject sheddable layer can protect the payload before and during initial cellular uptake. For example, without a sheddable layer, much of the payload can be lost during cellular internalization.
  • a sheddable layer ‘sheds’ (e.g., the layer can be pH- and/or or glutathione-sensitive), exposing the components of the core.
  • a subject sheddable layer includes silica.
  • a subject nanoparticle includes a sheddable layer (e.g., of silica)
  • greater intracellular delivery efficiency can be observed despite decreased probability of cellular uptake.
  • coating a nanoparticle core with a sheddable layer e.g., silica coating
  • nanoparticle cores encapsulated by a sheddable layer can be stable in serum and can be suitable for administration in vivo.
  • Any desired sheddable layer can be used, and one of ordinary skill in the art can take into account where in the target cell (e.g., under what conditions, such as low pH) they desire the payload to be released (e.g., endosome, cytosol, nucleus, lysosome, and the like).
  • Different sheddable layers may be more desirable depending on when, where, and/or under what conditions it would be desirable for the sheddable coat to shed (and therefore release the payload).
  • a sheddable layer can be acid labile.
  • the sheddable layer is an anionic sheddable layer (an anionic coat).
  • the sheddable layer comprises silica, a peptoid, a polycysteine, and/or a ceramic (e.g., a bioceramic).
  • the sheddable includes one or more of: calcium, manganese, magnesium, iron (e.g., the sheddable layer can be magnetic, e.g., Fe 3 MnO 2 ), and lithium. Each of these can include phosphate or sulfate.
  • the sheddable includes one or more of: calcium phosphate, calcium sulfate, manganese phosphate, manganese sulfate, magnesium phosphate, magnesium sulfate, iron phosphate, iron sulfate, lithium phosphate, and lithium sulfate; each of which can have a particular effect on how and/or under which conditions the sheddable layer will ‘shed.’
  • the sheddable layer includes one or more of: silica, a peptoid, a polycysteine, a ceramic (e.g., a bioceramic), 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 (in any combination thereof) (e.g., the sheddable layer can be
  • different release times for different payloads are desirable. For example, in some cases it is desirable to release a payload early (e.g., within 0.5-7 days of contacting a target cell) and in some cases it is desirable to release a payload late (e.g., within 6 days-30 days of contacting a target cell).
  • a payload e.g., a gene editing tool such as a CRISPR/Cas guide RNA, a DNA molecule encoding said CRISPR/Cas guide RNA, a CRISPR/Cas RNA-guided polypeptide, and/or a nucleic acid molecule encoding said CRISPR/Cas RNA-guided polypeptide
  • a payload e.g., a gene editing tool such as a CRISPR/Cas guide RNA, a DNA molecule encoding said CRISPR/Cas guide RNA, a CRISPR/Cas RNA-guided polypeptide, and/or a nucleic acid molecule encoding said CRISPR/Cas RNA-guided polypeptide
  • a payload e.g., a DNA donor template, e.g., for homology directed repair—HDR
  • a target cell e.g., within 6-30, 6-20, 6-15, 7-40, 7-30, 7-20, 7-15, 9-40, 9-30, 9-20, or 9-15 days of contacting a target cell.
  • release times can be controlled by delivering nanoparticles having different payloads at different times.
  • release times can be controlled by delivering nanoparticles at the same time (as part of different formulations or as part of the same formulation), where the components of the nanoparticle are designed to achieve the desired release times.
  • a sheddable layer that degrades faster or slower, core components that are more or less resistant to degradation, core components that are more or less susceptible to de-condensation, etc.—and any or all of the components can be selected in any convenient combination to achieve the desired timing.
  • a first nanoparticle includes a donor DNA template as a payload is designed such that the payload is released within 6-40 days of contacting a target cell (e.g., within 6-30, 6-20, 6-15, 7-40, 7-30, 7-20, 7-15, 9-40, 9-30, 9-20, or 9-15 days of contacting a target cell), while a second nanoparticle that includes one or more gene editing tools (e.g., a ZFP or nucleic acid encoding the ZFP, a TALE or a nucleic acid encoding the TALE, a ZFN or nucleic acid encoding the ZFN, a TALEN or a nucleic acid encoding the TALEN, a CRISPR/Cas guide RNA or DNA molecule encoding the CRISPR/Ca
  • a nanoparticle includes more than one payload, where it is desirable for the payloads to be released at different times.
  • a nanoparticle can have more than one core, where one core is made with components that can release the payload early (e.g., within 0.5-7 days of contacting a target cell, e.g., within 0.5-5 days, 0.5-3 days, 1-7 days, 1-5 days, or 1-3 days of contacting a target cell) (e.g., an siRNA, an mRNA, and/or a genome editing tool such as a ZFP or nucleic acid encoding the ZFP, a TALE or a nucleic acid encoding the TALE, a ZFN or nucleic acid encoding the ZFN, a TALEN or a nucleic acid encoding the TALEN, a CRISPR/Cas guide RNA or DNA molecule encoding the CRISPR/Cas guide RNA,
  • a ZFP or nucleic acid encoding the ZFP
  • a nanoparticle can include more than one sheddable layer, where the outer sheddable layer is shed (releasing a payload) prior to an inner sheddable layer being shed (releasing another payload).
  • the inner payload is a DNA donor template (e.g., for homology directed repair—HDR) and the outer payload is one or more gene editing tools (e.g., a ZFP or nucleic acid encoding the ZFP, a TALE or a nucleic acid encoding the TALE, a ZFN or nucleic acid encoding the ZFN, a TALEN or a nucleic acid encoding the TALEN, a CRISPR/Cas guide RNA or DNA molecule encoding the CRISPR/Cas guide RNA, a CRISPR/Cas RNA-guided polypeptide or a nucleic acid molecule encoding the CRISPR/Cas RNA-guided polypeptide, and the like).
  • the inner and outer payloads can be any desired payload and either or both can include, for example, one or more siRNAs and/or one or more mRNAs.
  • a nanoparticle can have more than one sheddable layer and can be designed to release one payload early (e.g., within 0.5-7 days of contacting a target cell, e.g., within 0.5-5 days, 0.5-3 days, 1-7 days, 1-5 days, or 1-3 days of contacting a target cell) (e.g., an siRNA, an mRNA, a genome editing tool such as a ZFP or nucleic acid encoding the ZFP, a TALE or a nucleic acid encoding the TALE, a ZFN or nucleic acid encoding the ZFN, a TALEN or a nucleic acid encoding the TALEN, a CRISPR/Cas guide RNA or DNA molecule encoding the CRISPR/Cas guide RNA, a CRISPR/C
  • time of altered gene expression can be used as a proxy for the time of payload release.
  • time of altered gene expression can be used as a proxy for the time of payload release.
  • one can assay for the desired result of nanoparticle delivery on day 12. For example, if the desired result was to reduce the expression of a target gene of the target cell, e.g., by delivering an siRNA, then the expression of the target gene can be assayed/monitored to determine if the siRNA has been released.
  • the desired result was to express a protein of interest, e.g., by delivering a DNA or mRNA encoding the protein of interest, then the expression of the protein of interest can be assayed/monitored to determine if the payload has been released.
  • the desired result was to alter the genome of the target cell, e.g., via cleaving genomic DNA and/or inserting a sequence of a donor DNA template, the expression from the targeted locus and/or the presence of genomic alterations can be assayed/monitored to determine if the payload has been released.
  • a sheddable layer provides for a staged release of nanoparticle components.
  • a nanoparticle has more than one (e.g., two, three, or four) sheddable layers.
  • a nanoparticle with two sheddable layers can have, from inner-most to outer-most: a core, e.g., with a first payload; a first sheddable layer, an intermediate layer e.g., with a second payload; and a second sheddable layer surrounding the intermediate layer (see, e.g., FIG. 9 ).
  • Such a configuration facilitates staged release of various desired payloads.
  • a nanoparticle with two sheddable layers can include one or more desired gene editing tools in the core (e.g., one or more of: a DNA donor template, a CRISPR/Cas guide RNA, a DNA encoding a CRISPR/Cas guide RNA, and the like), and another desired gene editing tool in the intermediate layer (e.g., one or more of: a programmable gene editing protein such as a CRISPR/Cas protein, a ZFP, a ZFN, a TALE, a TALEN, etc.; a DNA or RNA encoding a programmable gene editing protein; a CRISPR/Cas guide RNA; a DNA encoding a CRISPR/Cas guide RNA; and the like)—in any desired combination.
  • desired gene editing tools in the core e.g., one or more of: a DNA donor template, a CRISPR/Cas guide RNA, a DNA encoding a CRISPR/C
  • a subject core (e.g., including any combination of components and/or configurations described above) is part of a lipid-based delivery system, e.g., a cationic lipid delivery system (see, e.g., Chesnoy and Huang, Annu Rev Biophys Biomol Struct. 2000, 29:27-47; Hirko et al., Curr Med Chem. 2003 Jul. 10(14):1185-93; and Liu et al., Curr Med Chem. 2003 Jul. 10(14):1307-15).
  • a subject core (e.g., including any combination of components and/or configurations described above) is not surrounded by a sheddable layer.
  • a core can include an anionic polymer composition (e.g., poly(glutamic acid)), a cationic polymer composition (e.g., poly(arginine), a cationic polypeptide composition (e.g., a histone tail peptide), and a payload (e.g., nucleic acid and/or protein payload).
  • anionic polymer composition e.g., poly(glutamic acid)
  • a cationic polymer composition e.g., poly(arginine
  • a cationic polypeptide composition e.g., a histone tail peptide
  • a payload e.g., nucleic acid and/or protein payload
  • the core was designed with timed and/or positional release in mind.
  • the core includes ESPs, ENPs, and/or EPPs, and in some such cases these components are present at ratios such that payload release is delayed until a desired condition (e.g., cellular location, cellular condition such as pH, presence of a particular enzyme, and the like) is encountered by the core (e.g., described above).
  • a desired condition e.g., cellular location, cellular condition such as pH, presence of a particular enzyme, and the like
  • the core includes polymers of D-isomers of an anionic amino acid and polymers of L-isomers of an anionic amino acid, and in some cases the polymers of D- and L- isomers are present, relative to one another, within a particular range of ratios (e.g., described above).
  • the core includes polymers of D-isomers of a cationic amino acid and polymers of L-isomers of a cationic amino acid, and in some cases the polymers of D- and L- isomers are present, relative to one another, within a particular range of ratios (e.g., described above).
  • the core includes polymers of D-isomers of an anionic amino acid and polymers of L-isomers of a cationic amino acid, and in some cases the polymers of D- and L-isomers are present, relative to one another, within a particular range of ratios (e.g., described above).
  • the core includes polymers of L-isomers of an anionic amino acid and polymers of D-isomers of a cationic amino acid, and in some cases the polymers of D- and L-isomers are present, relative to one another, within a particular range of ratios (e.g., described above).
  • the core includes a protein that includes an NLS (e.g., described above).
  • the core includes an HTP (e.g., described above).
  • Cationic lipids are nonviral vectors that can be used for gene delivery and have the ability to condense plasmid DNA.
  • N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride for lipofection improving molecular structures of cationic lipids has been an active area, including head group, linker, and hydrophobic domain modifications. Modifications have included the use of multivalent polyamines, which can improve DNA binding and delivery via enhanced surface charge density, and the use of sterol-based hydrophobic groups such as 3B—[N—(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol, which can limit toxicity.
  • helper lipids such as dioleoyl phosphatidylethanolamine (DOPE) can be used to improve transgene expression via enhanced liposomal hydrophobicity and hexagonal inverted-phase transition to facilitate endosomal escape.
  • DOPE dioleoyl phosphatidylethanolamine
  • a lipid formulation includes one or more of: DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, C12-200, a cholesterol a PEG-lipid, a lipiopolyamine, dexamethasone-spermine (DS), and disubstituted spermine (D 2 S) (e.g., resulting from the conjugation of dexamethasone to polyamine spermine).
  • D 2 S disubstituted spermine
  • DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200 and DLin-MC3-DMA can be synthesized by methods outlined in the art (see, e.g., Heyes et. al, J. Control Release, 2005, 107, 276-287; Semple et. al, Nature Biotechnology, 2010, 28, 172-176; Akinc et. al, Nature Biotechnology, 2008, 26, 561-569; Love et. al, PNAS, 2010, 107, 1864-1869; international patent application publication WO2010054401; all of which are hereby incorporated by reference in their entirety.
  • lipid-based delivery systems include, but are not limited to those described in the following publications: international patent publication No. WO2016081029; U.S. patent application publication Nos. US20160263047 and US20160237455; and U.S. Pat. Nos. 9,533,047; 9,504,747; 9,504,651; 9,486,538; 9,393,200; 9,326,940; 9,315,828; and 9,308,267; all of which are hereby incorporated by reference in their entirety.
  • a subject core is surrounded by a lipid (e.g., a cationic lipid such as a LIPOFECTAMINE transfection reagent).
  • a subject core is present in a lipid formulation (e.g., a lipid nanoparticle formulation).
  • a lipid formulation can include a liposome and/or a lipoplex.
  • a lipid formulation can include a Spontaneous Vesicle Formation by Ethanol Dilution (SNALP) liposome (e.g., one that includes cationic lipids together with neutral helper lipids which can be coated with polyethylene glycol (PEG) and/or protamine).
  • SNALP Spontaneous Vesicle Formation by Ethanol Dilution
  • a lipid formulation can be a lipidoid-based formulation.
  • the synthesis of lipidoids has been extensively described and formulations containing these compounds can be included in a subject lipid formulation (see, e.g., Mahon et al., Bioconjug Chem. 2010 21:1448-1454; Schroeder et al., J Intern Med. 2010 267:9-21; Akinc et al., Nat Biotechnol. 2008 26:561-569; Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869; and Siegwart et al., Proc Natl Acad Sci USA. 2011 108:12996-3001; all of which are incorporated herein by reference in their entirety).
  • a subject lipid formulation can include one or more of (in any desired combination): 1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC); 1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE); N-[1-(2,3-Dioleyloxy)prophyl]N,N,N-trimethylammonium chloride (DOTMA); 1,2-Dioleoyloxy-3-trimethylammonium-propane (DOTAP); Dioctadecylamidoglycylspermine (DOGS); N-(3-Aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1 (GAP-DLRIE); propanaminium bromide; cetyltrimethylammonium bromide (CTAB); 6-Lauroxyhexyl ornithinate (LHON); Dioleoyl
  • the sheddable layer (the coat), is itself coated by an additional layer, referred to herein as an “outer shell,” “outer coat,” or “surface coat.”
  • a surface coat can serve multiple different functions. For example, a surface coat can increase delivery efficiency and/or can target a subject nanoparticle to a particular cell type.
  • the surface coat can include a peptide, a polymer, or a ligand-polymer conjugate.
  • the surface coat can include a targeting ligand.
  • an aqueous solution of one or more targeting ligands can be added to a coated nanoparticle suspension (suspension of nanoparticles coated with a sheddable layer).
  • the final concentration of protonated anchoring residues is between 25 and 300 ⁇ M.
  • the process of adding the surface coat yields a monodispersed suspension of particles with a mean particle size between 50 and 150 nm and a zeta potential between 0 and ⁇ 10 mV.
  • the surface coat interacts electrostatically with the outermost sheddable layer.
  • a nanoparticle has two sheddable layers (e.g., from inner-most to outer-most: a core, e.g., with a first payload; a first sheddable layer, an intermediate layer e.g., with a second payload; and a second sheddable layer surrounding the intermediate layer), and the outer shell (surface coat) can interact with (e.g., electrostatically) the second sheddable layer.
  • a nanoparticle has only one sheddable layer (e.g., an anionic silica layer), and the outer shell can in some cases electrostatically interact with the sheddable layer.
  • the surface coat can interact electrostatically with the sheddable layer if the surface coat includes a cationic component.
  • the surface coat includes a delivery molecule in which a targeting ligand is conjugated to a cationic anchoring domain.
  • the cationic anchoring domain interacts electrostatically with the sheddable layer and anchors the delivery molecule to the nanoparticle.
  • the surface coat can interact electrostatically with the sheddable layer if the surface coat includes an anionic component.
  • the surface coat includes a cell penetrating peptide (CPP).
  • CPP cell penetrating peptide
  • a polymer of a cationic amino acid can function as a CPP (also referred to as a ‘protein transduction domain’—PTD), which is a term used to refer to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane.
  • PTD protein transduction domain
  • a PTD attached to another molecule e.g., embedded in and/or interacting with a sheddable layer of a subject nanoparticle
  • a sheddable layer of a subject nanoparticle which can range from a small polar molecule to a large macromolecule and/or a nanoparticle, facilitates the molecule traversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle (e.g., the nucleus).
  • CPPs include but are not limited to a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR (SEQ ID NO: 160); a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); an Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm.
  • a minimal undecapeptide protein transduction domain corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR (SEQ ID NO: 160
  • a polyarginine sequence comprising a number of arginines sufficient to
  • Example CPPs include but are not limited to: YGRKKRRQRRR (SEQ ID NO: 160), RKKRRQRRR (SEQ ID NO: 165), an arginine homopolymer of from 3 arginine residues to 50 arginine residues, RKKRRQRR (SEQ ID NO: 166), YARAAARQARA (SEQ ID NO: 167), THRLPRRRRRR (SEQ ID NO: 168), and GGRRARRRRRR (SEQ ID NO: 169).
  • the CPP is an activatable CPP (ACPP) (Aguilera et al. (2009) lntegr Biol (Camb) June; 1(5-6): 371-381).
  • ACPPs comprise a polycationic CPP (e.g., Arg9 or “R9”) connected via a cleavable linker to a matching polyanion (e.g., Glu9 or “E9”), which reduces the net charge to nearly zero and thereby inhibits adhesion and uptake into cells.
  • a polycationic CPP e.g., Arg9 or “R9”
  • a matching polyanion e.g., Glu9 or “E9”
  • a CPP can be added to the nanoparticle by contacting a coated core (a core that is surrounded by a sheddable layer) with a composition (e.g., solution) that includes the CPP.
  • the CPP can then interact with the sheddable layer (e.g., electrostatically).
  • the surface coat includes a polymer of a cationic amino acid (e.g., a poly(arginine) such as poly(L-arginine) and/or poly(D-arginine), a poly(lysine) such as poly(L-lysine) and/or poly(D-lysine), a poly(histidine) such as poly(L- histidine) and/or poly(D-histidine), a poly(ornithine) such as poly(L-ornithine) and/or poly(D-ornithine), poly(citrulline) such as poly(L-citrulline) and/or poly(D-citrulline), and the like).
  • the surface coat includes poly(arginine), e.g., poly(L-arginine).
  • the surface coat includes a heptapeptide such as selank (TKPRPGP—SEQ ID NO: 147) (e.g., N-acetyl selank) and/or semax (MEHFPGP—SEQ ID NO: 148) (e.g., N-acetyl semax).
  • TKPRPGP—SEQ ID NO: 147) e.g., N-acetyl selank
  • MEHFPGP—SEQ ID NO: 1408 e.g., N-acetyl semax
  • the surface coat includes selank (e.g., N-acetyl selank).
  • semax e.g., N-acetyl semax.
  • the surface coat includes a delivery molecule.
  • a delivery molecule includes a targeting ligand and in some cases the targeting ligand is conjugated to an anchoring domain (e.g. a cationic anchoring domain). In some case a targeting ligand is conjugated to an anchoring domain (e.g. a cationic anchoring domain) via an intervening linker.
  • targeting ligands e.g., as part of a subject delivery molecule
  • a targeting ligand can be used as part of a surface coat, and numerous different targeting ligands are envisioned.
  • the targeting ligand is a fragment (e.g., a binding domain) of a wild type protein.
  • the peptide targeting ligand of a subject delivery molecule can have a length of from 4-50 amino acids (e.g., from 4-40, 4-35, 4-30, 4-25, 4-20, 4-15, 5-50, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 7-50, 7-40, 7-35, 7-30, 7-25, 7-20, 7-15, 8-50, 8-40, 8-35, 8-30, 8-25, 8-20, or 8-15 amino acids).
  • the targeting ligand can be a fragment of a wild type protein, but in some cases has a mutation (e.g., insertion, deletion, substitution) relative to the wild type amino acid sequence (i.e., a mutation relative to a corresponding wild type protein sequence).
  • a targeting ligand can include a mutation that increases or decreases binding affinity with a target cell surface protein.
  • the targeting ligand is an antigen-binding region of an antibody (e.g., an ScFv).
  • Fv is the minimum antibody fragment which contains a complete antigen-recognition and -binding site.
  • this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association.
  • scFv single-chain Fv species
  • one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species.
  • a targeting ligand includes a viral glycoprotein, which in some cases binds to ubiqituous surface markers such as heparin sulfate proteoglycans, and may induce micropinocytosis in some cell populations through membrane ruffling associated processes.
  • Poly(L-arginine) is another example targeting ligand that can also be used for binding to surface markers such as heparin sulfate proteoglycans.
  • a targeting ligand can include a mutation that adds a cysteine residue, which can facilitate conjugation to a linker and/or an anchoring domain (e.g., cationic anchoring domain).
  • cysteine can be used for crosslinking (conjugation) via sulfhydryl chemistry (e.g., a disulfide bond) and/or amine-reactive chemistry.
  • a targeting ligand includes an internal cysteine residue. In some cases, a targeting ligand includes a cysteine residue at the N- and/or C-terminus. In some cases, in order to include a cysteine residue, a targeting ligand is mutated (e.g., insertion or substitution), e.g., relative to a corresponding wild type sequence. As such, any of the targeting ligands described herein can be modified by inserting and/or substituting in a cysteine residue (e.g., internal, N-terminal, C-terminal insertion of or substitution with a cysteine residue).
  • a cysteine residue e.g., internal, N-terminal, C-terminal insertion of or substitution with a cysteine residue.
  • corresponding wild type sequence is meant a wild type sequence from which the subject sequence was or could have been derived (e.g., a wild type protein sequence having high sequence identity to the sequence of interest).
  • a targeting ligand that has one or more mutations (e.g., substitution, insertion) but is otherwise highly similar to a wild type sequence
  • the amino acid sequence to which it is most similar may be considered to be a corresponding wild type amino acid sequence.
  • a corresponding wild type protein/sequence does not have to be 100% identical (e.g., can be 85% or more identical, 90% or more identical, 95% or more identical, 98% or more identical, 99% or more identical, etc.) (outside of the position(s) that is modified), but the targeting ligand and corresponding wild type protein (e.g., fragment of a wild protein) can bind to the intended cell surface protein, and retain enough sequence identity (outside of the region that is modified) that they can be considered homologous.
  • the amino acid sequence of a “corresponding” wild type protein sequence can be identified/evaluated using any convenient method (e.g., using any convenient sequence comparison/alignment software such as BLAST, MUSCLE, T-COFFEE, etc.).
  • targeting ligands that can be used as part of a surface coat (e.g., as part of a delivery molecule of a surface coat) include, but are not limited to, those listed in Table 1.
  • Examples of targeting ligands that can be used as part of a subject delivery molecule include, but are not limited to, those listed in Table 3 (many of the sequences listed in Table 3 include the targeting ligand (e.g., SNRWLDVK for row 2) conjugated to a cationic polypeptide domain, e.g., 9R, 6R, etc., via a linker (e.g., GGGGSGGGGS).
  • amino acid sequences that can be included in a targeting ligand include, but are not limited to: NPKLTRMLTFKFY (SEQ ID NO: xx) (IL2), TSVGKYPNTGYYGD (SEQ ID NO: xx) (CD3), SNRWLDVK (Siglec), EKFILKVRPAFKAV (SEQ ID NO: xx) (SCF); EKFILKVRPAFKAV (SEQ ID NO: xx) (SCF), EKFILKVRPAFKAV (SEQ ID NO: xx) (SCF), SNYSIIDKLVNIVDDLVECVKENS (SEQ ID NO: xx) (cKit), and Ac-SNYSAibADKAibANAibADDAibAEAibAKENS (SEQ ID NO: xx) (cKit).
  • a targeting ligand includes an amino acid sequence that has 85% or more (e.g., 90% or more, 95% or more, 98% or more, 99% or more, or 100%) sequence identity with NPKLTRMLTFKFY (SEQ ID NO: xx) (IL2), TSVGKYPNTGYYGD (SEQ ID NO: xx) (CD3), SNRWLDVK (Siglec), EKFILKVRPAFKAV (SEQ ID NO: xx) (SCF); EKFILKVRPAFKAV (SEQ ID NO: xx) (SCF), EKFILKVRPAFKAV (SEQ ID NO: xx) (SCF), or SNYSIIDKLVNIVDDLVECVKENS (SEQ ID NO: xx) (cKit).
  • a targeting ligand (e.g., of a delivery molecule) can include the amino acid sequence RGD and/or an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 1-12.
  • a targeting ligand includes the amino acid sequence RGD and/or the amino acid sequence set forth in any one of SEQ ID NOs: 1-12.
  • a targeting ligand can include a cysteine (internal, C-terminal, or N-terminal), and can also include the amino acid sequence RGD and/or an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 1-12.
  • a targeting ligand (e.g., of a delivery molecule) can include the amino acid sequence RGD and/or an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 1-12 and 181-187.
  • a targeting ligand includes the amino acid sequence RGD and/or the amino acid sequence set forth in any one of SEQ ID NOs: 1-12 and 181-187.
  • a targeting ligand can include a cysteine (internal, C-terminal, or N-terminal), and can also include the amino acid sequence RGD and/or an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 1-12 and 181-187.
  • a targeting ligand (e.g., of a delivery molecule) can include the amino acid sequence RGD and/or an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 1-12, 181-187, and 271-277.
  • a targeting ligand includes the amino acid sequence RGD and/or the amino acid sequence set forth in any one of SEQ ID NOs: 1-12, 181-187, and 271-277.
  • a targeting ligand can include a cysteine (internal, C-terminal, or N-terminal), and can also include the amino acid sequence RGD and/or an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 1-12, 181-187, and 271-277.
  • a targeting ligand (e.g., of a delivery molecule) can include an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 181-187, and 271-277.
  • a targeting ligand includes the amino acid sequence set forth in any one of SEQ ID NOs: 181-187, and 271-277.
  • a targeting ligand can include a cysteine (internal, C-terminal, or N-terminal), and can also include an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 181-187, and 271-277.
  • a targeting ligand (e.g., of a delivery molecule) can include an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 181-187.
  • a targeting ligand includes the amino acid sequence set forth in any one of SEQ ID NOs: 181-187.
  • a targeting ligand can include a cysteine (internal, C-terminal, or N-terminal), and can also include an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 181-187.
  • cysteine internal, C-terminal, or N-terminal
  • amino acid sequence having 85% or more sequence identity e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity
  • a targeting ligand (e.g., of a delivery molecule) can include an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 271-277.
  • a targeting ligand includes the amino acid sequence set forth in any one of SEQ ID NOs: 271-277.
  • a targeting ligand can include a cysteine (internal, C-terminal, or N-terminal), and can also include an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth in any one of SEQ ID NOs: 271-277.
  • cysteine internal, C-terminal, or N-terminal
  • amino acid sequence having 85% or more sequence identity e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity
  • targets and “targeted binding” are used herein to refer to specific binding.
  • the terms “specific binding,” “specifically binds,” and the like, refer to non-covalent or covalent preferential binding to a molecule relative to other molecules or moieties in a solution or reaction mixture (e.g., an antibody specifically binds to a particular polypeptide or epitope relative to other available polypeptides, a ligand specifically binds to a particular receptor relative to other available receptors).
  • the affinity of one molecule for another molecule to which it specifically binds is characterized by a K d (dissociation constant) of 10 ⁇ 5 M or less (e.g., 10 ⁇ 6 M or less, 10 ⁇ 7 M or less, 10 ⁇ 8 M or less, 10 ⁇ 9 M or less, 10 ⁇ 10 M or less, 10 11 M or less, 10 ⁇ 12 M or less, 10 ⁇ 13 M or less, 10 ⁇ 14 M or less, 10 ⁇ 15 M or less, or 10 ⁇ 16 M or less).
  • K d dissociation constant
  • the targeting ligand provides for targeted binding to a cell surface protein selected from a family B G-protein coupled receptor (GPCR), a receptor tyrosine kinase (RTK), a cell surface glycoprotein, and a cell-cell adhesion molecule.
  • GPCR family B G-protein coupled receptor
  • RTK receptor tyrosine kinase
  • a cell surface glycoprotein e.g., a cell surface glycoprotein
  • a cell-cell adhesion molecule e.g., cell surface protein selected from a family B G-protein coupled receptor (GPCR), a receptor tyrosine kinase (RTK), a cell surface glycoprotein, and a cell-cell adhesion molecule.
  • RTK receptor tyrosine kinase
  • a cell surface glycoprotein e.g., cell surface glycoprotein
  • cell-cell adhesion molecule e.g., a cell surface protein selected from a family B G-protein coupled receptor (GPCR),
  • a crystal structure of a desired target (cell surface protein) bound to its ligand is available (or where such a structure is available for a related protein)
  • 3D structure modeling and sequence threading can visualize sites of interaction between the ligand and the target. This can facilitate, e.g., selection of internal sites for placement of substitutions and/or insertions (e.g., of a cysteine residue).
  • the targeting ligand provides for binding to a family B G protein coupled receptor (GPCR) (also known as the ‘secretin-family’).
  • GPCR family B G protein coupled receptor
  • the targeting ligand provides for binding to both an allosteric-affinity domain and an orthosteric domain of the family B GPCR to provide for the targeted binding and the engagement of long endosomal recycling pathways, respectively (see e.g., the examples section below as well as FIG. 11 and FIG. 12 ).
  • G-protein-coupled receptors share a common molecular architecture (with seven putative transmembrane segments) and a common signaling mechanism, in that they interact with G proteins (heterotrimeric GTPases) to regulate the synthesis of intracellular second messengers such as cyclic AMP, inositol phosphates, diacylglycerol and calcium ions.
  • Family B the secretin-receptor family or ‘family 2’ of the GPCRs is a small but structurally and functionally diverse group of proteins that includes receptors for polypeptide hormones and molecules thought to mediate intercellular interactions at the plasma membrane (see e.g., Harmar et al., Genome Biol.
  • a targeting ligand that provides for targeting binding to GLP1R can be used to target the brain and pancreas.
  • targeting GLP1R facilitates methods (e.g., treatment methods) focused on treating diseases (e.g., via delivery of one or more gene editing tools) such as Huntington's disease (CAG repeat expansion mutations), Parkinson's disease (LRRK2 mutations), ALS (SOD1 mutations), and other CNS diseases.
  • Targeting GLP1R also facilitates methods (e.g., treatment methods) focused on delivering a payload to pancreatic ⁇ -islets for the treatment of diseases such as diabetes mellitus type I, diabetes mellitus type II, and pancreatic cancer (e.g., via delivery of one or more gene editing tools).
  • an amino acid for cysteine substitution and/or insertion (e.g., for conjugation to a nucleic acid payload) can be identified by aligning the Exendin-4 amino acid sequence, which is HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO.
  • a desirable cross-linking site e.g., site for substitution/insertion of a cysteine residue
  • a targeting ligand that targets a family B GPCR
  • high-affinity binding may occur as well as concomitant long endosomal recycling pathway sequestration (e.g., for optimal payload release).
  • the cysteine substitution at amino acid positions 10, 11, and/or 12 of SEQ ID NO: 1 confers bimodal binding and specific initiation of a Gs-biased signaling cascade, engagement of beta arrestin, and receptor dissociation from the actin cytoskeleton.
  • this targeting ligand triggers internalization of the nanoparticle via receptor-mediated endocytosis, a mechanism that is not engaged via mere binding to the GPCR's N-terminal domain without concomitant orthosteric site engagement (as is the case with mere binding of the affinity strand, Exendin-4 [31-39]).
  • a subject targeting ligand includes an amino acid sequence having 85% or more (e.g., 90% or more, 95% or more, 98% or more, 99% or more, or 100%) identity to the exendin-4 amino acid sequence (SEQ ID NO: 1).
  • the targeting ligand includes a cysteine substitution or insertion at one or more of positions corresponding to L10, S11, and K12 of the amino acid sequence set forth in SEQ ID NO: 1.
  • the targeting ligand includes a cysteine substitution or insertion at a position corresponding to S11 of the amino acid sequence set forth in SEQ ID NO: 1.
  • a subject targeting ligand includes an amino acid sequence having the exendin-4 amino acid sequence (SEQ ID NO: 1).
  • the targeting ligand is conjugated (with or without a linker) to an anchoring domain (e.g., a cationic anchoring domain).
  • a targeting ligand provides for binding to a receptor tyrosine kinase (RTK) such as fibroblast growth factor (FGF) receptor (FGFR).
  • RTK receptor tyrosine kinase
  • FGF fibroblast growth factor receptor
  • the targeting ligand is a fragment of an FGF (i.e., comprises an amino acid sequence of an FGF).
  • the targeting ligand binds to a segment of the RTK that is occupied during orthosteric binding (e.g., see the examples section below).
  • the targeting ligand binds to a heparin-affinity domain of the RTK.
  • the targeting ligand provides for targeted binding to an FGF receptor and comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence KNGGFFLRIHPDGRVDGVREKS (SEQ ID NO: 4).
  • the targeting ligand provides for targeted binding to an FGF receptor and comprises the amino acid sequence set forth as SEQ ID NO: 4.
  • small domains that occupy the orthosteric site of the RTK may be used to engage endocytotic pathways relating to nuclear sorting of the RTK (e.g., FGFR) without engagement of cell-proliferative and proto-oncogenic signaling cascades, which can be endemic to the natural growth factor ligands.
  • the truncated bFGF (tbFGF) peptide (a.a.30-115), contains a bFGF receptor binding site and a part of a heparin-binding site, and this peptide can effectively bind to FGFRs on a cell surface, without stimulating cell proliferation.
  • tbFGF The sequences of tbFGF are KRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLAS KCVTDECFFFERLESNNYNTY (SEQ ID NO: 13) (see, e.g., Cai et al., Int J Pharm. 2011 Apr. 15; 408(1-2):173-82).
  • the targeting ligand provides for targeted binding to an FGF receptor and comprises the amino acid sequence HFKDPK (SEQ ID NO: 5) (see, e.g., the examples section below). In some cases, the targeting ligand provides for targeted binding to an FGF receptor, and comprises the amino acid sequence LESNNYNT (SEQ ID NO: 6) (see, e.g., the examples section below).
  • a targeting ligand according to the present disclosure provides for targeted binding to a cell surface glycoprotein.
  • the targeting ligand provides for targeted binding to a cell-cell adhesion molecule.
  • the targeting ligand provides for targeted binding to CD34, which is a cell surface glycoprotein that functions as a cell-cell adhesion factor, and which is protein found on hematopoietic stem cells (e.g., of the bone marrow).
  • the targeting ligand is a fragment of a selectin such as E-selectin, L-selectin, or P-selectin (e.g., a signal peptide found in the first 40 amino acids of a selectin).
  • a subject targeting ligand includes sushi domains of a selectin (e.g., E-selectin, L-selectin, P-selectin).
  • the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence MIASQFLSALTLVLLIKESGA (SEQ ID NO: 7). In some cases, the targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 7.
  • the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence MVFPWRCEGTYWGSRNILKLWVWTLLCCDFLIHHGTHC (SEQ ID NO: 8).
  • the targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 8.
  • targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence MIFPWKCQSTQRDLWNIFKLWGWTMLCCDFLAHHGTDC (SEQ ID NO: 9).
  • targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 9.
  • targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence MIFPWKCQSTQRDLWNIFKLWGWTMLCC (SEQ ID NO: 10). In some cases, targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 10.
  • Fragments of selectins that can be used as a subject targeting ligand can in some cases attain strong binding to specifically-modified sialomucins, e.g., various Sialyl Lewis x modifications/O-sialylation of extracellular CD34 can lead to differential affinity for P-selectin, L-selectin and E-selectin to bone marrow, lymph, spleen and tonsillar compartments.
  • a targeting ligand can be an extracellular portion of CD34.
  • modifications of sialylation of the ligand can be utilized to differentially target the targeting ligand to various selectins.
  • a targeting ligand provides for targeted binding to E-selectin.
  • E-selectin can mediate the adhesion of tumor cells to endothelial cells and ligands for E-selectin can play a role in cancer metastasis.
  • P-selectin glycoprotein-1 e.g., derived from human neutrophils
  • a subject targeting ligand can therefore in some cases include the PSGL-1 amino acid sequence (or a fragment thereof the binds to E-selectin).
  • E-selectin ligand-1 can bind E-selectin and a subject targeting ligand can therefore in some cases include the ESL-1 amino acid sequence (or a fragment thereof the binds to E-selectin).
  • a targeting ligand with the PSGL-1 and/or ESL-1 amino acid sequence (or a fragment thereof the binds to E-selectin) bears one or more sialyl Lewis modifications in order to bind E-selectin.
  • CD44, death receptor-3 (DR3), LAMP1, LAMP2, and Mac2-BP can bind E-selectin and a subject targeting ligand can therefore in some cases include the amino acid sequence (or a fragment thereof the binds to E-selectin) of any one of: CD44, death receptor-3 (DR3), LAMP1, LAMP2, and Mac2-BP.
  • a targeting ligand according to the present disclosure provides for targeted binding to P-selectin.
  • PSGL-1 can provide for such targeted binding.
  • a subject targeting ligand can therefore in some cases include the PSGL-1 amino acid sequence (or a fragment thereof the binds to P-selectin).
  • a targeting ligand with the PSGL-1 amino acid sequence (or a fragment thereof the binds to P-selectin) bears one or more sialyl Lewis modifications in order to bind P-selectin.
  • a targeting ligand according to the present disclosure provides for targeted binding to a transferrin receptor.
  • the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence THRPPMWSPVWP (SEQ ID NO: 11).
  • targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 11.
  • a targeting ligand according to the present disclosure provides for targeted binding to an integrin (e.g., ⁇ 5 ⁇ 1 integrin).
  • the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence RRETAWA (SEQ ID NO: 12).
  • targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 12.
  • the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence RGDGW (SEQ ID NO: 181).
  • targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 181.
  • the targeting ligand comprises the amino acid sequence RGD.
  • a targeting ligand according to the present disclosure provides for targeted binding to an integrin.
  • the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence GCGYGRGDSPG (SEQ ID NO: 182).
  • the targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 182.
  • such a targeting ligand is acetylated on the N-terminus and/or amidated (NH2) on the C-terminus.
  • a targeting ligand according to the present disclosure provides for targeted binding to an integrin (e.g., ⁇ 5 ⁇ 3 integrin).
  • the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence DGARYCRGDCFDG (SEQ ID NO: 187).
  • the targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 187.
  • a targeting ligand used to target the brain includes an amino acid sequence from rabies virus glycoprotein (RVG) (e.g., YTIWMPENPRPGTPCDIFTNSRGKRASNGGGG (SEQ ID NO: 183)).
  • the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 183.
  • RVG can be conjugated and/or fused to an anchoring domain (e.g., 9R peptide sequence).
  • a subject delivery molecule used as part of a surface coat of a subject nanoparticle can include the sequence YTIWMPENPRPGTPCDIFTNSRGKRASNGGGGRRRRRRRRR (SEQ ID NO: 180).
  • a targeting ligand according to the present disclosure provides for targeted binding to c-Kit receptor.
  • the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 184.
  • the targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 184.
  • a targeting ligand according to the present disclosure provides for targeted binding to CD27.
  • the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 185.
  • the targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 185.
  • a targeting ligand according to the present disclosure provides for targeted binding to CD150.
  • the targeting ligand comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 186.
  • the targeting ligand comprises the amino acid sequence set forth as SEQ ID NO: 186.
  • a targeting ligand provides for targeted binding to KLS CD27+/IL-7Ra ⁇ /CD150+/CD34 ⁇ hematopoietic stem and progenitor cells (HSPCs).
  • HSPCs hematopoietic stem and progenitor cells
  • a gene editing tool(s) can be introduced in order to disrupt expression of a BCL11a transcription factor and consequently generate fetal hemoglobin.
  • the beta-globin (HBB) gene may be targeted directly to correct the altered E7V substitution with a corresponding homology-directed repair donor template.
  • a CRISPR/Cas RNA-guided polypeptide e.g., Cas9, CasX, CasY, Cpf1
  • an appropriate guide RNA such that it will bind to loci in the HBB gene and create double-stranded or single-stranded breaks in the genome, initiating genomic repair.
  • a DNA donor template single stranded or double stranded
  • a guide RNA/CRISPR/Cas protein complex a ribonucleoprotein complex
  • a payload can include an siRNA for ku70 or ku80, e.g., which can be used to promote homologous directed repair (HDR) and limit indel formation.
  • an mRNA for SIRT6 is released over 14-30 d to promote HDR-driven insertion of a donor strand following nuclease-mediated site-specific cleavage.
  • a targeting ligand provides for targeted binding to CD4+ or CD8+ T-cells, hematopoietic stem and progenitor cells (HSPCs), or peripheral blood mononuclear cells (PBMCs), in order to modify the T-cell receptor.
  • HSPCs hematopoietic stem and progenitor cells
  • PBMCs peripheral blood mononuclear cells
  • a gene editing tool(s) can be introduced in order to modify the T-cell receptor.
  • the T-cell receptor may be targeted directly and substituted with a corresponding homology-directed repair donor template for a novel T-cell receptor.
  • a CRISPR/Cas RNA-guided polypeptide e.g., Cas9, CasX, CasY, Cpf1
  • an appropriate guide RNA such that it will bind to loci in the TCR gene and create double-stranded or single-stranded breaks in the genome, initiating genomic repair.
  • a DNA donor template single stranded or double stranded
  • HDR high-density lipothelial growth factor
  • other CRISPR guide RNA and HDR donor sequences, targeting beta-globin, CCR5, the T-cell receptor, or any other gene of interest, and/or other expression vectors may be employed in accordance with the present disclosure.
  • a targeting ligand is bivalent (e.g., heterobivalent).
  • cell-penetrating peptides and/or heparin sulfate proteoglycan binding ligands are used as heterobivalent endocytotic triggers along with any of the targeting ligands of this disclosure.
  • a heterobivalent targeting ligand can include an affinity sequence from one of targeting ligand and an orthosteric binding sequence (e.g., one known to engage a desired endocytic trafficking pathway) from a different targeting ligand.
  • the surface coat includes a delivery molecule that includes a targeting ligand conjugated to an anchoring domain (e.g., cationic anchoring domain) (see e.g., FIG. 10 , panels A-B).
  • a targeting ligand is conjugate to an anchoring domain (or to a linker) distal to the active region of the targeting ligand, e.g., in order to preserve activity.
  • Anchoring domains e.g., cationic anchoring domains
  • a cationic anchoring domain has a length in a range of from 3 to 30 amino acids (e.g., from 3-28, 3-25, 3-24, 3-20, 4-30, 4-28, 4-25, 4-24, or 4-20 amino acids). In some cases, a cationic anchoring domain has a length in a range of from 4 to 24 amino acids.
  • Suitable examples of an anchoring domain include, but are not limited to: RRRRRRRRR (9R)(SEQ ID NO: 15) and HHHHHH (6H)(SEQ ID NO: 16).
  • an anchoring domain e.g., cationic anchoring domain
  • a subject delivery molecule is used as an anchor to coat the surface of a nanoparticle with the delivery molecule, e.g., so that the targeting ligand is used to target the nanoparticle to a desired cell/cell surface protein (see e.g., FIG. 8 , FIG. 9 , and FIG. 10 ).
  • the anchoring domain e.g., cationic anchoring domain
  • the stabilization layer has a negative charge and a positively anchoring domain (e.g., cationic anchoring domain) can therefore interact with the stabilization layer, effectively anchoring the delivery molecule to the nanoparticle and coating the nanoparticle surface with a subject targeting ligand (e.g., see FIG. 8 , FIG. 9 , and FIG. 10 ).
  • a positively anchoring domain e.g., cationic anchoring domain
  • Conjugation of a targeting ligand to an anchoring domain can be accomplished by any convenient technique and many different conjugation chemistries will be known to one of ordinary skill in the art.
  • the conjugation is via sulfhydryl chemistry (e.g., a disulfide bond).
  • the conjugation is accomplished using amine-reactive chemistry (e.g., an amine present on a side chain from an amino acid residue in the anchoring domain).
  • the targeting ligand can include a cysteine residue, which can facilitate conjugation.
  • an anchoring domain e.g., a cationic anchoring domain
  • an anchoring domain can include a cysteine residue, which can facilitate conjugation.
  • the targeting ligand and the anchoring domain are conjugated by virtue of being part of the same polypeptide.
  • a targeting ligand according to the present disclosure is conjugated to an anchoring domain (e.g., a cationic anchoring domain) via an intervening linker (e.g., see FIG. 10 ).
  • the linker can be a protein linker or non-protein linker.
  • a linker can in some cases aid in stability, prevent complement activation, and/or provide flexibility to the ligand relative to the anchoring domain.
  • Conjugation of a targeting ligand to a linker or a linker to an anchoring domain can be accomplished in a number of different ways.
  • the conjugation is via sulfhydryl chemistry (e.g., a disulfide bond, e.g., between two cysteine residues, e.g., see FIG. 10 ).
  • the conjugation is accomplished using amine-reactive chemistry.
  • a targeting ligand includes a cysteine residue and is conjugated to the linker via the cysteine residue; and/or an anchoring domain includes a cysteine residue and is conjugated to the linker via the cysteine residue.
  • the linker is a peptide linker and includes a cysteine residue.
  • the targeting ligand and a peptide linker are conjugated by virtue of being part of the same polypeptide; and/or the anchoring domain and a peptide linker are conjugated by virtue of being part of the same polypeptide.
  • a subject linker is a polypeptide and can be referred to as a polypeptide linker. It is to be understood that while polypeptide linkers are contemplated, non-polypeptide linkers (chemical linkers) are used in some cases.
  • the linker is a polyethylene glycol (PEG) linker.
  • Suitable protein linkers include polypeptides of between 4 amino acids and 60 amino acids in length (e.g., 4-50, 4-40, 4-30, 4-25, 4-20, 4-15, 4-10, 6-60, 6-50, 6-40, 6-30, 6-25, 6-20, 6-15, 6-10, 8-60, 8-50, 8-40, 8-30, 8-25, 8-20, or 8-15 amino acids in length).
  • a subject linker is rigid (e.g., a linker that include one or more proline residues).
  • a rigid linker is GAPGAPGAP (SEQ ID NO: 17).
  • a polypeptide linker includes a C residue at the N- or C-terminal end.
  • a rigid linker is selected from: GAPGAPGAPC (SEQ ID NO: 18) and CGAPGAPGAP (SEQ ID NO: 19).
  • Peptide linkers with a degree of flexibility can be used.
  • a subject linker is flexible.
  • the linking peptides may have virtually any amino acid sequence, bearing in mind that flexible linkers will have a sequence that results in a generally flexible peptide.
  • small amino acids such as glycine and alanine, are of use in creating a flexible peptide.
  • the creation of such sequences is routine to those of skill in the art.
  • a variety of different linkers are commercially available and are considered suitable for use.
  • Example linker polypeptides include glycine polymers (G) n , glycine-serine polymers (including, for example, (GS) n , GSGGS n (SEQ ID NO: 20), GGSGGS n (SEQ ID NO: 21), and GGGS n (SEQ ID NO: 22), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers.
  • Example linkers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 23), GGSGG (SEQ ID NO: 24), GSGSG (SEQ ID NO: 25), GSGGG (SEQ ID NO: 26), GGGSG (SEQ ID NO: 27), GSSSG (SEQ ID NO: 28), and the like.
  • GGSG SEQ ID NO: 23
  • GGSGG SEQ ID NO: 24
  • GSGSG SEQ ID NO: 25
  • GSGGG SEQ ID NO: 26
  • GGGSG SEQ ID NO: 27
  • GSSSG SEQ ID NO: 28
  • the ordinarily skilled artisan will recognize that design of a peptide conjugated to any elements described above can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure. Additional examples of flexible linkers include, but are not limited to: GGGGGSGGGGG (SEQ ID NO: 29) and GGGGGSGGGGS (SEQ ID NO
  • a polypeptide linker includes a C residue at the N- or C-terminal end.
  • a flexible linker includes an amino acid sequence selected from: GGGGGSGGGGGC (SEQ ID NO: 31), CGGGGGSGGGGG (SEQ ID NO: 32), GGGGGSGGGGSC (SEQ ID NO: 33), and CGGGGGSGGGGS (SEQ ID NO: 34).
  • a subject polypeptide linker is endosomolytic.
  • Endosomolytic polypeptide linkers include but are not limited to: KALA (SEQ ID NO: 35) and GALA (SEQ ID NO: 36).
  • a polypeptide linker includes a C residue at the N- or C-terminal end.
  • a subject linker includes an amino acid sequence selected from: CKALA (SEQ ID NO: 37), KALAC (SEQ ID NO: 38), CGALA (SEQ ID NO: 39), and GALAC (SEQ ID NO: 40).
  • Cysteine residues in the reduced state containing free sulfhydryl groups, readily form disulfide bonds with protected thiols in a typical disulfide exchange reaction.
  • Sulfhydryl groups of cysteine react with maleimide and acyl halide groups, forming stable thioether and thioester bonds respectively.
  • This conjugation is facilitated by chemical modification of the cysteine residue to contain an alkyne bond, or by the use of L-propargyl cysteine (pictured below) in synthetic peptide preparation. Coupling is then achieved by means of Cu promoted click chemistry.
  • targeting ligands include, but are not limited, to those that include to the following amino acid sequences:
  • SCF targets/binds to c-Kit receptor
  • SCF EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVV QLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKK SFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPEKDS RVSVTKPFMLPPVA; CD70 (targets/binds to CD27) (SEQ ID NO: 185) PEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPLE SLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRD GIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGC TIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQVVVRP; and (SEQ ID NO: 186)
  • RRRRRRR M EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLP SHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECV KENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVV SSTLSPEKDSRVSVTKPFMLPPVA 9R-CD70 (SEQ ID NO: 190)
  • This can be conjugated to CCA1 (see above) either via sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive chemistry.
  • This can be conjugated to CCA2 (see above) either via sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive chemistry.
  • This can be conjugated to CCA1 (see above) either via sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive chemistry.
  • This can be conjugated to CCA2 (see above) either via sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive chemistry.
  • CTHRPPMWSPVWP (SEQ ID NO: 53)
  • This can be conjugated to CCA1 (see above) either via sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive chemistry.
  • This can be conjugated to CCA2 (see above) either via sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive chemistry.
  • This can be conjugated to CCA1 (see above) either via sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive chemistry.
  • MIASQFLSALTLVLLIKESGAC (SEQ ID NO: 59)
  • This can be conjugated to CCA2 (see above) either via sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive chemistry.
  • GPGAPGAP Targeting ligand—linker
  • anchoring domain e.g., cationic anchoring domain
  • KNGGFFLRIHPDGRVDGVREKS GAPGAPGAP RRRRRRRRR (SEQ ID NO: 61)
  • This can be conjugated to CCA1 (see above) either via sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive chemistry.
  • This can be conjugated to CCA1 (see above) either via sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive chemistry.
  • This can be conjugated to CCA2 (see above) either via sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive chemistry.
  • HGEGTFTSDL C KQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 2)
  • This can be conjugated to CCA1 (see above) either via sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive chemistry.
  • the surface coat includes any one or more of (in any desired combination): (i) one or more of the above described polymers, (ii) one or more targeting ligands, one or more CPPs, and one or more heptapeptides.
  • a surface coat can include one or more (e.g., two or more, three or more) targeting ligands, but can also include one or more of the above described cationic polymers.
  • a surface coat can include one or more (e.g., two or more, three or more) targeting ligands, but can also include one or more CPPs.
  • a surface coat includes a combination of targeting ligands that provides for targeted binding to CD34 and heparin sulfate proteoglycans.
  • poly(L-arginine) can be used as part of a surface coat to provide for targeted binding to heparin sulfate proteoglycans.
  • a nanoparticle with a cationic polymer e.g., poly(L-arginine)
  • the coated nanoparticle is incubated with hyaluronic acid, thereby forming a zwitterionic and multivalent surface.
  • the surface coat is multivalent.
  • a multivalent surface coat is one that includes two or more targeting ligands (e.g., two or more delivery molecules that include different ligands).
  • An example of a multimeric (in this case trimeric) surface coat (outer shell) is one that includes the targeting ligands stem cell factor (SCF) (which targets c-Kit receptor, also known as CD117), CD70 (which targets CD27), and SH2 domain-containing protein 1A (SH2D1A) (which targets CD150).
  • SCF stem cell factor
  • CD70 which targets CD27
  • SH2D1A SH2 domain-containing protein 1A
  • a subject nanoparticle includes a surface coat that includes a combination of the targeting ligands SCF, CD70, and SH2 domain-containing protein 1A (SH2D1A), which target c-Kit, CD27, and CD150, respectively (see, e.g., Table 1).
  • HSCs hematopoietic stem cells
  • SH2D1A SH2 domain-containing protein 1A
  • such a surface coat can selectively target HSPCs and long-term HSCs (c-Kit+/Lin ⁇ /Sca-1+/CD27+/IL-7Ra ⁇ /CD150+/CD34 ⁇ ) over other lymphoid and myeloid progenitors.
  • all three targeting ligands are anchored to the nanoparticle via fusion to a cationic anchoring domain (e.g., a poly-histidine such as 6H, a poly-arginine such as 9R, and the like).
  • a cationic anchoring domain e.g., a poly-histidine such as 6H, a poly-arginine such as 9R, and the like.
  • the targeting polypeptide SCF (which targets c-Kit receptor) can include XMEGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFS NISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVAS ETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAX (SEQ ID NO: 194), where the X is a cationic anchoring domain (e.g., a poly-histidine such as 6H, a poly-arginine such as 9R, and the like), e.g., which can in some cases be present at the N- and/or C-terminal end, or can be embedded within the polypeptide sequence;
  • the targeting polypeptide CD70 (which targets CD27) can include XPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLP
  • nanoparticles of the disclosure can include multiple targeting ligands (as part of a surface coat) in order to target a desired cell type, or in order to target a desired combination of cell types.
  • targeting ligands as part of a surface coat
  • FIG. 17 panels A-B
  • various combinations of cell surface markers of interest include, but are not limited to: [Mouse] (i) CD150; (ii) Sca1, cKit, CD150; (iii) CD150 and CD49b; (iv) Sca1, cKit, CD150, and CD49b; (v) CD150 and Flt3; (vi) Sca1, cKit, CD150, and Flt3; (vii) Flt3 and CD34; (viii) Flt3, CD34, Sca1, and cKit; (ix) Flt3 and CD127; (x) Sca1, cKit, Flt3, and CD127; (xi) CD34; (xii) cKit and CD34; (xiii) CD16/32 and CD34; (xiv) cKit, CD16/32, and CD34; and (xv) cKit; and [Human] (i) CD90 and CD49f; (ii) CD34, CD90, and CD49f
  • a surface coat includes one or more targeting ligands that provide targeted binding to a surface protein or combination of surface proteins selected from: [Mouse] (i) CD150; (ii) Sca1, cKit, CD150; (iii) CD150 and CD49b; (iv) Sca1, cKit, CD150, and CD49b; (v) CD150 and Flt3; (vi) Sca1, cKit, CD150, and Flt3; (vii) Flt3 and CD34; (viii) Flt3, CD34, Sca1, and cKit; (ix) Flt3 and CD127; (x) Sca1, cKit, Flt3, and CD127; (xi) CD34; (xii) cKit and CD34; (xiii) CD16/32 and CD34; (xiv) cKit, CD16/32, and CD34; and (xv) cKit; and [Human] (i) CD90 and CD49
  • a subject nanoparticle can include more than one targeting ligand, and because some cells include overlapping markers, multiple different cell types can be targeted using combinations of surface coats, e.g., in some cases a surface coat may target one specific cell type while in other cases a surface coat may target more than one specific cell type (e.g., 2 or more, 3 or more, 4 or more cell types). For example, any combination of cells within the hematopoietic lineage can be targeted.
  • targeting CD34 using a targeting ligand that provides for targeted binding to CD34
  • a nucleic acid, protein, or ribonucleoprotein payload to a cell.
  • the payload includes a gene editing tool.
  • a subject method is used to perform site-specific genome editing, which in some cases, e.g., when performed in the presence of a donor DNA template, leads to and homology-directed repair.
  • Such methods include a step of contacting a cell with a subject nanoparticle (or subject viral or non-viral delivery vehicle).
  • a subject nanoparticle (or subject viral or non-viral delivery vehicle) can be used to deliver a payload to any desired eukaryotic target cell.
  • the target cell is a mammalian cell (e.g., a cell of a rodent, a mouse, a rat, an ungulate, a cow, a sheep, a pig, a horse, a camel, a rabbit, a canine (dog), a feline (cat), a primate, a non-human primate, or a human).
  • any cell type can be targeted, and in some cases specific targeting of particular cells depends on the presence of targeting ligands, e.g., as part of the surface coat, where the targeting ligands provide for targeting binding to a particular cell type.
  • cells that can be targeted include but are not limited to bone marrow cells, hematopoietic stem cells (HSCs), long-term HSCs, short-term 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 (e.g., HUDEP cells), megakaryocyte-eryth
  • Hematopoietic stem cells and multipotent progenitors can be targeted for gene editing (e.g., insertion) in vivo. Even editing 1% of bone marrow cells in vivo (approximately 15 billion cells) would target more cells than an ex vivo therapy (approximately 10 billion cells).
  • pancreatic cells e.g., ⁇ islet cells
  • pancreatic cancer can be targeted, e.g., to treat pancreatic cancer, to treat diabetes, etc.
  • somatic cells in the brain such as neurons can be targeted (e.g., to treat indications such as Huntington's disease, Parkinson's (e.g., LRRK2 mutations), and ALS (e.g., SOD1 mutations)). In some cases this can be achieved through direct intracranial injections.
  • endothelial cells and cells of the hematopoietic system can be targeted with a subject nanoparticle (or subject viral or non-viral delivery vehicle) to treat Von Willebrand's disease.
  • a megakaryocyte-erythroid progenitor cell MEP
  • CMP common myeloid progenitor cell
  • MPP multipotent progenitor cell
  • HSC hematopoietic stem cells
  • ST-HSC short term HSC
  • IT-HSC IT-HSC
  • LT-HSC long term HSC
  • a cell e.g., an endothelial cell, a megakaryocyte and/or any progenitor cell upstream of a megakaryocyte such as an MEP, a CMP, an MPP, an HSC such as an ST-HSC, an IT-HSC, and/or an LT-HSC
  • VWF von Willebrand factor
  • an active protein e.g., via delivery of a functional VWF protein and/or a nucleic acid encoding a functional VWF protein
  • a replacement sequence e.g., via delivery of a gene editing tool and delivery of a DNA donor template.
  • a subject targeting ligand provides for targeted binding to E-selectin.
  • a cell of a stem cell lineage e.g., a stem and/or progenitor cell of the hematopoietic lineage, e.g., a GMP, MEP, CMP, MLP, MPP, and/or an HSC
  • a subject nanoparticle or subject viral or non-viral delivery vehicle
  • SCF stem cell factor
  • a subject nanoparticle or subject viral or non-viral delivery vehicle
  • a subject nanoparticle can be used to deliver SCF and/or a nucleic acid (DNA or mRNA) encoding SCF to the targeted cell.
  • Methods and compositions of this disclosure can be used to treat any number of diseases, including any disease that is linked to a known causative mutation, e.g., a mutation in the genome.
  • methods and compositions of this disclosure can be used to treat sickle cell disease, ß thalassemia, HIV, myelodysplastic syndromes, JAK2-mediated polycythemia vera, JAK2-mediated primary myelofibrosis, JAK2-mediated leukemia, and various hematological disorders.
  • the methods and compositions of this disclosure can also be used for B-cell antibody generation, immunotherapies (e.g., delivery of a checkpoint blocking reagent), and stem cell differentiation applications.
  • a targeting ligand provides for targeted binding to KLS CD27+/IL-7Ra ⁇ /CD150+/CD34 ⁇ hematopoietic stem and progenitor cells (HSPCs).
  • HSPCs hematopoietic stem and progenitor cells
  • a gene editing tool(s) can be introduced in order to disrupt expression of a BCL11a transcription factor and consequently generate fetal hemoglobin.
  • the beta-globin (HBB) gene may be targeted directly to correct the altered E7V substitution with a corresponding homology-directed repair donor template.
  • a CRISPR/Cas RNA-guided polypeptide e.g., Cas9, CasX, CasY, Cpf1
  • an appropriate guide RNA such that it will bind to loci in the HBB gene and create double-stranded or single-stranded breaks in the genome, initiating genomic repair.
  • a DNA donor template single stranded or double stranded
  • a guide RNA/CRISPR/Cas protein complex a ribonucleoprotein complex
  • a payload can include an siRNA for ku70 or ku80, e.g., which can be used to promote homologous directed repair (HDR) and limit indel formation.
  • an mRNA for SIRT6 is released over 14-30 d to promote HDR-driven insertion of a donor strand following nuclease-mediated site-specific cleavage.
  • a targeting ligand provides for targeted binding to CD4+ or CD8+ T-cells, hematopoietic stem and progenitor cells (HSPCs), or peripheral blood mononuclear cells (PBMCs), in order to modify the T-cell receptor.
  • HSPCs hematopoietic stem and progenitor cells
  • PBMCs peripheral blood mononuclear cells
  • a gene editing tool(s) can be introduced in order to modify the T-cell receptor.
  • the T-cell receptor may be targeted directly and substituted with a corresponding homology-directed repair donor template for a novel T-cell receptor.
  • a CRISPR/Cas RNA-guided polypeptide e.g., Cas9, CasX, CasY, Cpf1
  • an appropriate guide RNA such that it will bind to loci in the TCR gene and create double-stranded or single-stranded breaks in the genome, initiating genomic repair.
  • a DNA donor template single stranded or double stranded
  • HDR high-density lipothelial growth factor
  • other CRISPR guide RNA and HDR donor sequences, targeting beta-globin, CCR5, the T-cell receptor, or any other gene of interest, and/or other expression vectors may be employed in accordance with the present disclosure.
  • the contacting when contacting a cell with a subject nanoparticle (or subject viral or non-viral delivery vehicle), the contacting is in vitro (e.g., the cell is in culture), e.g., the cell can be a cell of an established tissue culture cell line.
  • the contacting is ex vivo (e.g., the cell is a primary cell (or a recent descendant) isolated from an individual, e.g. a patient).
  • the cell is in vivo and is therefore inside of (part of) an organism.
  • the contacting step includes administration of a subject nanoparticle (or subject viral or non-viral delivery vehicle) to an individual.
  • a subject nanoparticle may be introduced to the subject (i.e., administered to an individual) via any of the following routes: systemic, local, parenteral, subcutaneous (s.c.), intravenous (i.v.), intracranial (i.c.), intraspinal, intraocular, intradermal (i.d.), intramuscular (i.m.), intralymphatic (LI.), or into spinal fluid.
  • a subject nanoparticle (or subject viral or non-viral delivery vehicle) may be introduced by injection (e.g., systemic injection, direct local injection, local injection into or near a tumor and/or a site of tumor resection, etc.), catheter, or the like.
  • Examples of methods for local delivery include, e.g., by bolus injection, e.g. by a syringe, e.g. into a joint, tumor, or organ, or near a joint, tumor, or organ; e.g., by continuous infusion, e.g. by cannulation, e.g. with convection (see e.g. US Application No. 20070254842, incorporated here by reference).
  • the number of administrations of treatment to a subject may vary. Introducing a subject nanoparticle (or subject viral or non-viral delivery vehicle) into an individual may be a one-time event; but in certain situations, such treatment may elicit improvement for a limited period of time and require an on-going series of repeated treatments. In other situations, multiple administrations of a subject nanoparticle (or subject viral or non-viral delivery vehicle) may be required before an effect is observed. As will be readily understood by one of ordinary skill in the art, the exact protocols depend upon the disease or condition, the stage of the disease and parameters of the individual being treated.
  • a “therapeutically effective dose” or “therapeutic dose” is an amount sufficient to effect desired clinical results (i.e., achieve therapeutic efficacy).
  • a therapeutically effective dose can be administered in one or more administrations.
  • a therapeutically effective dose of a subject nanoparticle or subject viral or non-viral delivery vehicle is an amount that is sufficient, when administered to the individual, to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of a disease state/ailment.
  • An example therapeutic intervention is one that creates resistance to HIV infection in addition to ablating any retroviral DNA that has been integrated into the host genome.
  • T-cells are directly affected by HIV and thus a hybrid blood targeting strategy for CD34+ and CD45+ cells may be explored for delivering dual guided nucleases.
  • a hybrid blood targeting strategy for CD34+ and CD45+ cells may be explored for delivering dual guided nucleases.
  • one advantage of delivering multiple payloads as part of the same package is that the efficiency of each payload is not diluted.
  • one or more gene editing tools e.g., as described above
  • is delivered in combination with e.g., as part of the same package/delivery vehicle, where the delivery vehicle does not need to be a nanoparticle of the disclosure
  • a protein and/or a DNA or mRNA encoding same
  • a non-coding RNA that increases genomic editing efficiency.
  • one or more gene editing tools is delivered in combination with (e.g., as part of the same package/delivery vehicle, where the delivery vehicle does not need to be a nanoparticle of the disclosure) a protein (and/or a DNA or mRNA encoding same) and/or a non-coding RNA that controls cell division and/or differentiation.
  • a protein and/or a DNA or mRNA encoding same
  • a non-coding RNA that controls cell division and/or differentiation.
  • one or more gene editing tools is delivered in combination with (e.g., as part of the same package/delivery vehicle, where the delivery vehicle does not need to be a nanoparticle of the disclosure) a protein (and/or a DNA or mRNA encoding same) and/or a non-coding RNA that controls differentiation.
  • one or more gene editing tools is delivered in combination with (e.g., as part of the same package/delivery vehicle, where the delivery vehicle does not need to be a nanoparticle of the disclosure) a protein (and/or a DNA or mRNA encoding same) and/or a non-coding RNA that biases the cell DNA repair machinery toward non-homologous end joining (NHEJ) or homology directed repair (HDR).
  • a protein and/or a DNA or mRNA encoding same
  • HDR homology directed repair
  • the delivery vehicle does not need to be a nanoparticle of the disclosure.
  • the delivery vehicle is viral and in some cases the delivery vehicle is non-viral.
  • non-viral delivery systems include materials that can be used to co-condense multiple nucleic acid payloads, or combinations of protein and nucleic acid payloads.
  • Examples include, but are not limited to: (1) lipid based particles such as zwitterionic or cationic lipids, and exosome or exosome-derived vesicles; (2) inorganic/hybrid composite particles such as those that include ionic complexes co-condensed with nucleic acids and/or protein payloads, and complexes that can be condensed from cationic ionic states of Ca, Mg, Si, Fe and physiological anions such as O 2 ⁇ , OH, PO 4 3 ⁇ , SO 4 2 ⁇ ; (3) carbohydrate Delivery vehicles such as cyclodextrin and/or alginate; (4) polymeric and/or co-polymeric complexes such as poly(amino-acid) based electrostatic complexes, poly(Amido-Amine), and cationic poly(B-Amino Ester); and (5) virus like particles (e.g., protein and nucleic acid based) such as Li2016 artificial viruses.
  • viral delivery systems include but are
  • one or more gene editing tools can be delivered in combination with (e.g., as part of the same package/delivery vehicle, where the delivery vehicle does not need to be a nanoparticle of the disclosure) one or more of: SCF (and/or a DNA or mRNA encoding SCF), HoxB4 (and/or a DNA or mRNA encoding HoxB4), BCL-XL (and/or a DNA or mRNA encoding BCL-XL), SIRT6 (and/or a DNA or mRNA encoding SIRT6), a nucleic acid molecule (e.g., an siRNA and/or an LNA) that suppresses miR-155, a nucleic acid molecule (e.g., an siRNA, an shRNA, a microRNA) that reduces ku70 expression, and a nucleic acid molecule (e.g., an siRNA, an shRNA, a microRNA) that reduces ku80 expression.
  • SCF and/or a DNA
  • microRNAs livered as RNAs or as DNA encoding the RNAs
  • the following microRNAs can be used for the following purposes: for blocking differentiation of a pluripotent stem cell toward ectoderm lineage: miR-430/427/302; for blocking differentiation of a pluripotent stem cell toward endoderm lineage: miR-109 and/or miR-24; for driving differentiation of a pluripotent stem cell toward endoderm lineage: miR-122 and/or miR-192; for driving differentiation of an ectoderm progenitor cell toward a keratinocyte fate: miR-203; for driving differentiation of a neural crest stem cell toward a smooth muscle fate: miR-145; for driving differentiation of a neural stem cell toward a glial cell fate and/or toward a neuron fate: miR-9 and/or miR-124a; for blocking differentiation of a mesoderm progenitor cell toward
  • signaling proteins e.g., extracellular signaling proteins
  • the following signaling proteins e.g., extracellular signaling proteins
  • the following signaling proteins can be used for the following purposes: for driving differentiation of a hematopoietic stem cell toward a common lymphoid progenitor cell lineage: IL-7; for driving differentiation of a hematopoietic stem cell toward a common myeloid progenitor cell lineage: IL-3, GM-CSF, and/or M-CSF; for driving differentiation of a common lymphoid progenitor cell toward a B-cell fate: IL-3, IL-4, and/or IL-7; for driving differentiation of a common lymphoid progenitor cell toward a Natural Killer Cell fate: IL
  • proteins that can be delivered include but are not limited to: SOX17, HEX, OSKM (Oct4/Sox2/Klf4/c-myc), and/or bFGF (e.g., to drive differentiation toward hepatic stem cell lineage); HNF4a (e.g., to drive differentiation toward hepatocyte fate); Poly (I:C), BMP-4, bFGF, and/or 8-Br-cAMP (e.g., to drive differentiation toward endothelial stem cell/progenitor lineage); VEGF (e.g., to drive differentiation toward arterial endothelium fate); Sox-2, Brn4, Myt11, Neurod2, Ascl1 (e.g., to drive differentiation toward neural stem cell/progenitor lineage); and BDNF, FCS
  • signaling proteins e.g., extracellular signaling proteins
  • cytokines e.g., IL-2 and/or IL-15, e.g., for activating CD8+ T-cells
  • ligands and or signaling proteins that modulate one or more of the Notch, Wnt, and/or Smad signaling pathways
  • SCF stem cell differentiating factors
  • a fibroblast may be converted into a neural stem cell via delivery of Sox2, while it will turn into a cardiomyocyte in the presence of Oct3/4 and small molecule “epigenetic resetting factors.”
  • these fibroblasts may respectively encode diseased phenotypic traits associated with neurons and cardiac cells.
  • a cell death cue may be conditional upon a gene edit not being successful, and cell differentiation/proliferation/activation is tied to a tissue/organ-specific promoter and/or exogenous factor.
  • a diseased cell receiving a gene edit may activate and proliferate, but due to the presence of another promoter-driven expression cassette (e.g. one tied to the absence of tumor suppressor such as p21 or p53), those cells will subsequently be eliminated.
  • the cells expressing desired characteristics may be triggered to further differentiate into the desired downstream lineages.
  • kits can include one or more of (in any combination): (i) a targeting ligand, (ii) a linker, (iii) a targeting ligand conjugated to a linker, (iv) a targeting ligand conjugated to an anchoring domain (e.g., with or without a linker), (v) an agent for use as a sheddable layer (e.g., silica), (vi) a payload, e.g., an siRNA or a transcription template for an siRNA or shRNA; a gene editing tool, and the like, (vii) a polymer that can be used as a cationic polymer, (viii) a polymer that can be used as an anionic polymer, (ix) a polypeptide that can be used as a cationic polypeptide, e.g., one or more HTPs, and (x) a subject viral or non-viral delivery vehicle.
  • a targeting ligand e.g., a linker
  • a nanoparticle comprising a core and a sheddable layer encapsulating the core, wherein the core comprises:
  • nucleic acid and/or protein payload (iv) a nucleic acid and/or protein payload
  • 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.
  • 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).
  • 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).
  • one of (a) and (b) comprises a D-isomer polymer of an amino acid
  • the other of (a) and (b) comprises an L-isomer polymer of an amino acid
  • the ratio of the D-isomer polymer to the L-isomer polymer is in a range of from 10:1 to 1.5:1, or from 1:1.5 to 1:10.
  • said anionic polymer composition comprises an anionic polymer selected from poly(D-glutamic acid) (PDEA) and poly(D-aspartic acid) (PDDA).
  • said cationic polymer composition comprises a cationic polymer selected from poly(L-arginine), poly(L-lysine), poly(L-histidine), poly(L-ornithine), and poly(L-citrulline).
  • said cationic polymer composition comprises a cationic polymer selected from poly(D-arginine), poly(D-lysine), poly(D-histidine), poly(D-ornithine), and poly(D-citrulline).
  • said anionic polymer composition comprises an anionic polymer selected from poly(L-glutamic acid) (PLEA) and poly(L-aspartic acid) (PLDA).
  • PDA poly(L-glutamic acid)
  • PLDA poly(L-aspartic acid)
  • the nanoparticle of any one of 1-11, wherein the sheddable layer is pH and/or glutathione sensitive.
  • the nanoparticle of 15, wherein the surface coat comprises a cationic component that interacts electrostatically with the sheddable layer.
  • 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.
  • the nanoparticle of any one of 15-17, wherein the surface coat is zwitterionic and multivalent.
  • the nanoparticle of any one of 15-18, wherein the surface coat comprises one or more targeting ligands. 20.
  • 26. The nanoparticle of any one of 22-25, 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.
  • 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 ⁇ 5 ⁇ 1, 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, cer
  • RVG rabies virus glycoprotein
  • SCF stem cell factor
  • SH2D1A SH2 domain-containing protein 1A
  • 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
  • any one of 19-34, wherein the surface coat comprises 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 (e.g., HUDEP cells), megakaryocyte-erythroid progenitor cells (MEPs), common myeloid progenitor cells (CMPs), multipotent progenitor cells (MPPs), hematopoi
  • HSCs
  • the cationic polypeptide composition comprises a polypeptide that comprises a nuclear localization signal (NLS). 38. The nanoparticle of 37, wherein the NLS comprises the amino acid sequence set forth in any one of SEQ ID NOs: 151-157 and 201-264. 39. The nanoparticle of any one of 1-38, wherein the cationic polypeptide composition comprises a histone tail peptide (HTP). 40. The nanoparticle of 39, wherein the HTP is conjugated to a cationic amino acid polymer. 41. The nanoparticle of 40, wherein the HTP is conjugated to a cationic amino acid polymer via a cysteine residue. 42.
  • NLS nuclear localization signal
  • HTP histone tail peptide
  • 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.
  • ZFP zinc finger protein
  • ZFP zinc finger protein
  • ZFP zinc finger protein
  • ZFP zinc finger protein
  • 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.
  • the payload further comprises a DNA donor template.
  • a nanoparticle formulation comprising:
  • a first nanoparticle according to any one of 1-47, 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
  • a second nanoparticle comprising a nucleic acid payload that comprises a DNA donor template.
  • a multi-layered nanoparticle comprising:
  • an intermediate core surrounding the first sheddable layer 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
  • 54. The multi-layered nanoparticle of any one of 49-53, wherein the surface coat is zwitterionic and multivalent.
  • a method of delivering a nucleic acid and/or protein payload to a target cell comprising: contacting a eukaryotic target cell with the nanoparticle of any one of 1-47, the nanoparticle formulation of 48, and/or the multi-layered nanoparticle of any one of 49-55.
  • the payload includes a gene editing tool.
  • 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, DNA donor template, a nucleic acid molecule encoding a site-specific recombinase (e.g., Cre recombinase, Dre recombinase, Flp recombinase, KD recombinase, B2 recombinase, B3 recombinase, R re
  • the method of any one of 56-58, wherein the target cell is a mammalian cell 60.
  • the method of any one of 56-59, wherein the target cell is a human cell 61.
  • the method of any one of 56-60, wherein the target cell is in culture in vitro.
  • the method of any one of 56-60, wherein the target cell is in vivo.
  • the method of 62, wherein said contacting includes a step of administering the nanoparticle to an individual 64.
  • the method of 63, wherein the individual has Huntington's disease, ALS, Parkinson's disease, pancreatic cancer, diabetes, or von Willebrand's disease. 65.
  • the nanoparticle includes a surface coat comprising a targeting ligand.
  • 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 (e.g., HUDEP cells), megakaryocyte-erythroid progenitor cells (MEPs), common myeloid progenitor cells (CMPs), multipotent progenitor
  • HSCs hematopoietic stem cells
  • 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 hematopo
  • the target cell is a stem and/or progenitor cell and the payload comprises stem cell factor (SCF) and/or a nucleic acid encoding SCF.
  • SCF stem cell factor
  • the target cell wherein (i) the target cell is from an individual with von Willebrand's disease and/or the target cell includes a genomic mutation in the gene encoding VWF such that the cell produces sub-normal levels of functional VWF; (ii) the target cell is any one of: a megakaryocyte, an endothelial cell, an MEP, a CMP, an MPP, an HSC, a ST-HSC, and a LT-HSC; and (iii) the payload includes a functional VWF protein and/or a nucleic acid encoding a functional VWF.
  • a branched histone molecule comprising: one or more histone tail peptides (HTPs) conjugated to side chains of a cationic polymer.
  • HTPs histone tail peptides
  • 71. The branched histone molecule of 70, wherein the cationic polymer comprises poly(arginine) or poly(lysine).
  • 72. The branched histone molecule of 70 or 71, wherein up to 40% of the side chains of the cationic polymer are conjugated to said one or more HTPs.
  • HTPs histone tail peptides
  • a branched histone molecule comprising: one or more histone tail peptides (HTPs) conjugated to one another such that the branched histone molecule forms a structure selected from: a brush polymer, a web (e.g., spider web structure), a graft polymer, a star-shaped polymer, a comb polymer, a polymer network, and a dendrimer.
  • HTPs histone tail peptides
  • a lipid formulation for delivering a protein and/or nucleic acid payload comprising: a lipid and a core, wherein the core comprises:
  • nucleic acid and/or protein payload (iv) a nucleic acid and/or protein payload
  • 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.
  • 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).
  • 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).
  • a lipid formulation for delivering a protein and/or nucleic acid payload comprising: a lipid and a core, wherein the core comprises:
  • one of (a) and (b) comprises a D-isomer polymer of an amino acid
  • the other of (a) and (b) comprises an L-isomer polymer of an amino acid
  • the lipid formulation of 6 wherein the ratio of the D-isomer polymer to the L-isomer polymer is in a range of from 10:1 to 1.5:1, or from 1:1.5 to 1:10.
  • said anionic polymer composition comprises an anionic polymer selected from poly(D-glutamic acid) (PDEA) and poly(D-aspartic acid) (PDDA).
  • said cationic polymer composition comprises a cationic polymer selected from poly(L-arginine), poly(L-lysine), poly(L-histidine), poly(L-ornithine), and poly(L-citrulline).
  • said cationic polymer composition comprises a cationic polymer selected from poly(D-arginine), poly(D-lysine), poly(D-histidine), poly(D-ornithine), and poly(D-citrulline).
  • said anionic polymer composition comprises an anionic polymer selected from poly(L-glutamic acid) (PLEA) and poly(L-aspartic acid) (PLDA).
  • PKA poly(L-glutamic acid)
  • PLDA poly(L-aspartic acid)
  • the lipid formulation of 12 wherein the NLS comprises the amino acid sequence set forth in any one of SEQ ID NOs: 151-157 and 201-264. 14.
  • the lipid formulation of 14 or 15, wherein the cationic amino acid polymer comprises poly(lysine). 18.
  • 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, (
  • 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.
  • 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 lipid formulation of any one of 1-21. 23.
  • the payload includes a gene editing tool.
  • 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, DNA donor template, a nucleic acid molecule encoding a site-specific recombinase (e.g., Cre recombinase, Dre recombinase, Flp recombinase, KD recombinase, B2 recombina
  • the method of any one of 22-24, wherein the target cell is a mammalian cell 26.
  • the method of any one of 22-25, wherein the target cell is a human cell 27.
  • the method of any one of 22-26, wherein the target cell is in culture in vitro.
  • 28. The method of any one of 22-26, wherein the target cell is in vivo.
  • 29. The method of 28, wherein said contacting includes a step of administering the lipid formulation to an individual 30.
  • the method of 29, wherein the individual has Huntington's disease, ALS, Parkinson's disease, pancreatic cancer, diabetes, or von Willebrand's disease. 31.
  • 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 hematopoi
  • the target cell is a stem and/or progenitor cell and the payload comprises stem cell factor (SCF) and/or a nucleic acid encoding SCF.
  • SCF stem cell factor
  • a method of delivering a nucleic acid and/or protein payload to a target cell comprising: contacting a eukaryotic target cell with a viral or non-viral delivery vehicle comprising:
  • nucleic acid or protein agent that induces proliferation of and/or biases differentiation of the target cell.
  • a CRISPR/Cas guide RNA 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, (x) a DNA donor template, (xi) a nucleic acid molecule encoding a site-specific recomb
  • (b) comprises one or more of: SCF, a nucleic acid encoding SCF, HoxB4, a nucleic acid encoding HoxB4, BCL-XL, a nucleic acid encoding BCL-XL, SIRT6, a nucleic acid encoding SIRT6, a nucleic acid molecule (e.g., an siRNA, an LNA) that suppresses miR-155, a nucleic acid molecule (e.g., an siRNA, an shRNA, a microRNA) that reduces ku70 expression, and a nucleic acid molecule (e.g., an siRNA, an shRNA, a microRNA) that reduces ku80 expression.
  • SCF a nucleic acid encoding SCF
  • HoxB4 a nucleic acid encoding HoxB4
  • BCL-XL a nucleic acid encoding BCL-XL
  • SIRT6 a nucleic acid encoding SIRT6, a nu
  • Ethidium bromide Ethidium bromide
  • FIG. 1 depicts results from a fluorimetric assay testing various parameters (e.g., cation: anion charge ratio) for condensation of nucleic acid payloads. The result showed, e.g., that a charge ratio of 2 works well for the condensation of plasmids encoding Cas9 and guide RNA molecules.
  • 100 ⁇ l of Anionic solution was added to each well: 100 ng/ ⁇ l DNA, 80 ng/ ⁇ l poly(D-Glutamic Acid) (PDE), 0.5 ng/ ⁇ l Ethidium Bromide. Condensing species were titrated at 12.5 ⁇ l for each data point.
  • the total concentration (m/v) of condensing agent in the solution was 0.4 ug/ ⁇ l, with different compositions of histone tail peptide (HTP)[H3K4(me3)] and poly(L-arginine) (PLR) denoted by their mass fraction in parenthesis.
  • HTP histone tail peptide
  • PLR poly(L-arginine)
  • a first solution (an anionic solution) was prepared by combining the appropriate amount of payload (in this case plasmid DNA (EGFP-N1 plasmid) with an aqueous mixture (an ‘anionic polymer composition’) of poly(D-glutamic Acid) and poly(L-glutamic acid). This solution was diluted to the proper volume with 10 mM Tris-HCl at pH 8.5.
  • a second solution (a cationic solution), which was a combination of a ‘cationic polymer composition’ and a ‘cationic polypeptide composition’, was prepared by diluting a concentrated solution containing the appropriate amount of condensing agents to the proper volume with 60 mM HEPES at pH 5.5.
  • the ‘cationic polymer composition’ was poly(L-arginine) and the ‘cationic polypeptide composition’ was 16 ⁇ g of H3K4(me3) (tail of histone H3, tri methylated on K4).
  • Precipitation of nanoparticle cores in batches less than 200 ⁇ l can be carried out by dropwise addition of the condensing solution to the payload solution in glass vials or low protein binding centrifuge tubes followed by incubation for 30 minutes at 4° C.
  • the two solutions can be combined in a microfluidic format using a standard mixing chip (e.g. Dolomite Micromixer) or a hydrodynamic flow focusing chip.
  • a standard mixing chip e.g. Dolomite Micromixer
  • optimal input flowrates can be determined such that the resulting suspension of nanoparticle cores is monodispersed, exhibiting a mean particle size below 100 nm.
  • the two equal volume solutions from above were prepared for mixing.
  • polymer/peptide solutions were added to one protein low bind tube (eppendorf) and were then diluted with 60 mM HEPES (pH 5.5) to a total volume of 100 ⁇ l (as noted above). This solution was kept at room temperature while preparing the anionic solution.
  • anionic condensing agents the anionic solutions were chilled on ice with minimal light exposure.
  • Each of the two solutions was filtered using a 0.2 micron syringe filter and transferred to its own Hamilton 1 ml Gastight Syringe (Glass, (insert product number). Each syringe was placed on a Harvard Pump 11 Elite Dual Syringe Pump. The syringes were connected to appropriate inlets of a Dolomite Micro Mixer chip using tubing, and the syringe pump was run at 120 ⁇ l/min for a 100 ⁇ l total volume. The resulting solution included the core composition (which now included nucleic acid payload, anionic components, and cationic components). The nanoparticle size (peak) was 128.8 nm, and the zeta potential (peak) was +10.5 mV (100%) (e.g., see FIG. 2 ).
  • the resulting suspension of nanoparticle cores was then combined with a dilute solution of sodium silicate in 10 mM Tris HCl (pH8.5, 10-500 mM) or calcium chloride in 10 mM PBS (pH 8.5, 10-500 mM), and allowed to incubate for 1-2 hours at room temperature.
  • the core composition was added to a diluted sodium silicate solution to coat the core with an acid labile coating of polymeric silica (an example of a sheddable layer).
  • Stabilized (coated) cores can be purified using standard centrifugal filtration devices (100 kDa Amicon Ultra, Millipore) or dialysis in 30 mM HEPES (pH 7.4) using a high molecular weight cutoff membrane.
  • the stabilized (coated) cores were purified using a centrifugal filtration device.
  • the collected coated nanoparticles (nanoparticle solution) were washed with dilute PBS (1:800) or HEPES and filtered again (the solution can be resuspended in 500 ⁇ l sterile dispersion buffer or nuclease free water for storage). Effective silica coating was demonstrated.
  • the stabilized cores had a size of 110.6 nm and zeta potential of ⁇ 42.1 mV (95%) ( FIG. 3 ).
  • a surface coat also referred to as an outer shell
  • surface functionalization was accomplished by electrostatically grafting ligand species (in this case Rabies Virus Glycoprotein fused to a 9-Arg peptide sequence as a cationic anchoring domain—‘RVG9R’) to the negatively charged surface of the stabilized (in this case silica coated) nanoparticles.
  • ligand species in this case Rabies Virus Glycoprotein fused to a 9-Arg peptide sequence as a cationic anchoring domain—‘RVG9R’
  • RVG9R cationic anchoring domain
  • the desired surface constituents were added and the solution was sonicated for 20-30 seconds prior to incubate for 1 hour. Centrifugal filtration was performed at 300 kDa (the final product can be purified using standard centrifugal filtration devices, e.g., 300-500 kDa from Amicon Ultra Millipore, or dialysis, e.g., in 30 mM HEPES (pH 7.4) using a high molecular weight cutoff membrane), and the final resuspension was in either cell culture media or dispersion buffer.
  • optimal outer shell addition yields a monodispersed suspension of particles with a mean particle size between 50 and 150 nm and a zeta potential between 0 and ⁇ 10 mV.
  • the nanoparticles with an outer shell had a size of 115.8 nm and a Zeta potential of ⁇ 3.1 mV (100%) ( FIG. 4 ).
  • HTT018B charge ratio (cations/anions) was 2; surface coat was poly(L-Arginine)
  • HTT019B charge ratio was 5; surface coat was poly(L-Arginine)
  • HTT020B charge ratio was 2
  • surface coat was N-acetyl Semax
  • HTT021B charge ratio was 5
  • surface coat was N-acetyl Semax
  • HTT022B charge ratio was 2
  • surface coat was N-acetyl Selank
  • HTT023B charge ratio was 5
  • surface coat was N-acetyl Selank
  • L3000GFP lipofectamine (non-nanoparticle) delivery of a nucleic acid encoding GFP (plasmid encoding GFP)
  • L3000CRISPR lipofectamine (non-nanoparticle) delivery of CRISPR/Cas components with no GFP or fluorescent tag.
  • Nanoparticles were generated.
  • the core components included a nucleic acid payload (CRISPR/Cas encoding nucleic acids: one plasmid encoding a Cas9 guide RNA and a second plasmid encoding a Cas9 protein) and poly(L-arginine) (a cationic polymer composition) that was tagged with a fluorophore (FITC) so that uptake could be assessed by fluorescent microscopy.
  • CRISPR/Cas encoding nucleic acids one plasmid encoding a Cas9 guide RNA and a second plasmid encoding a Cas9 protein
  • poly(L-arginine) a cationic polymer composition
  • FITC fluorophore
  • L3000GFP positive control
  • no nanoparticle was used and the delivered nucleic acid was a plasmid encoding GFP.
  • L3000CRISPR negative control
  • no nanoparticle was used and the delivered nu
  • Neural stem cells were seeded at a density of 10 5 cells per well (96-well plate) and grown in Neurobasal medium supplemented with fibroblast growth factor (FGF)(1:1000).
  • FGF fibroblast growth factor
  • the nanoparticles and Lipofectamine 3000 (0.75 ⁇ L reagent/ ⁇ g DNA) were introduced to cells 24 hr after seeding with 400 ng of DNA payload transfected per well.
  • the nanoparticle samples were applied to neural stem cells in culture and allowed to incubate for 4-24 hours before washing with PBS up to 3 times to remove any non-internalized particles. Uptake was determined by imaging with the appropriate laser excitation and filter selection. Cells were imaged with a Zeiss LSM780 using a 20 ⁇ objective.
  • quantitative uptake data can be obtained using high content imaging and flow cytometry. The three rows depict three different replicates.
  • samples HTT18B, HTT20B, and HTT22B were prepared with a charge ratio of 2, whereas, samples HTT19B, HTT21B, and HTT23B where prepared with a charge ratio of 5.
  • the data show that a charge ratio of 2 (for condensation of the core) resulted in higher internalization than a charge ratio of 5.
  • surface coatings (outer shells) of the heptapeptide adaptogens Selank and Semax promoted a higher degree of internalization than a surface coat including the cell penetrating peptide poly(L-Arginine) [9.7 kDa].
  • Nanoparticles that include CRISPR/Cas9 plasmids as the nucleic acid payload.
  • the nanoparticle core included poly(L-arginine) (a cationic polymer composition) that was tagged with a fluorophore (FITC) so that uptake could be assessed by fluorescent microscopy.
  • FITC fluorophore
  • the endosome and nucleus were stained using Lysotracker (Red) and Hoescht 3342 (blue) respectively.
  • Nanoparticles and Lipfectamine 3000 (0.75 ⁇ l reagent/ ⁇ g DNA) were introduced to cells 16 hours after seeding with 400 ng of DNA payload transfected per well. Cells were incubated with Hoescht 3342 and Lysotracker Red before imaging.
  • FIG. 6 panels A-B
  • Co-localization of the nanoparticle's fluorescent signal with that of the stained endosome and nucleus were quantitatively measured and extent of co-localization was denoted by the resulting Pearson product-moment coefficient ( FIG. 6 , panels C-D).
  • the core included (i) the cationic polypeptide composition indicated in the table, (ii) a nucleic acid payload, (iii) poly(L-arginine) [a cationic amino acid polymer], and (iv) poly(L-glutamic acid) [an anionic amino acid polymer].
  • the sheddable layer was a silica coat and the surface coat was as indicated in Table 2.
  • FIG. 6 panel C depicts Pearson product-moment correlation coefficients between nanoparticle polymers (the FITC tagged core polymer) and Hoescht DNA stain or between nanoparticle polymers (the FITC tagged core polymer) and Lyotracker. The correlation coefficients were calculated while generating the images in panels A and B. All values were normalized to the value of the negative control (Cas9 Lipofectamine 3000). The decreased Pearson correlation between Hoescht and FITC between the 2.5 hour and 5 hour time-points indicated nanoparticle polymer degradation, release, or diffusion to compartments other than the nucleus over time.
  • FIG. 6 panel C depicts Pearson product-moment correlation coefficients between nanoparticle polymers (the FITC tagged core polymer) and Hoescht DNA stain or between nanoparticle polymers (the FITC tagged core polymer) and Lyotracker. The correlation coefficients were calculated while generating the images in panels A and B. All values were normalized to the value of the negative control (Cas
  • panel D depicts Pearson product-moment correlation coefficients between nanoparticle polymers and endosomes (Lysotracker Red). The correlation coefficients were calculated while generating the images in panels A and B. All values were normalized to the value of the negative control (Cas9 Lipofectamine 3000). Comparison of the 2.5 hour and 5 hour time points indicated endosomal escape over time.
  • FIG. 7 depicts microscopy images of peripheral blood mononuclear cells (PBMCs) that were been transfected with nanoparticles, where the nucleic acid payload was mRNA encoding GFP.
  • the images demonstrate that mRNA expression was extended to 16 days with nanoparticles that include a core with, at a defined ratio, a polymer of D-isomers of an anionic amino acid and a polymer of L-isomers of an anionic amino acid.
  • the nanoparticle core included (i) an anionic polymer composition: 7 ⁇ g total of poly(glutamic acid) (i.e., D- and L- isomers combined totaled 7 ⁇ g); (ii) a cationic polymer composition: poly(L-arginine); (iii) a cationic polypeptide composition: H3K4(me3) [i.e., tail of histone H3, tri methylated on K4]; and (iv) a nucleic acid payload: mRNA encoding GFP.
  • the nanoparticle core was encapsulated by a silica coat (a sheddable layer) and the surface coat was poly(L-arginine) (PLR).
  • Example 6 Targeting Ligand that Provides for Targeted Binding to a Family B GPCR
  • FIG. 11 provides a schematic diagram of a family B GPCR, highlighting separate domains to consider when evaluating a targeting ligand, e.g., for binding to allosteric/affinity N-terminal domains and orthosteric endosomal-sorting/signaling domains.
  • a targeting ligand e.g., for binding to allosteric/affinity N-terminal domains and orthosteric endosomal-sorting/signaling domains.
  • Figure is adapted from Siu, Fai Yiu, et al., Nature 499.7459 (2013): 444-449). Such domains were considered when selecting a site within the targeting ligand exendin-4 for cysteine substitution.
  • a cysteine 11 substitution (5110) was identified as one possible amino acid modification for conjugating exendin-4 to an anchoring domain (e.g., cationic anchoring domain) in such a way that maintains affinity and also engages long endosomal recycling pathways that promote nucleic acid release and limit nucleic acid degradation.
  • an anchoring domain e.g., cationic anchoring domain
  • SEQ ID NO: 1 simulated Exendin-4
  • PDB 3IOL
  • FIG. 13 shows a tbFGF fragment as part of a ternary FGF2-FGFR1-HEPARIN complex (1fq9 on PDB).
  • CKNGGFFLRIHPDGRVDGVREKS (highlighted) (SEQ ID NO: 14) was determined to be important for affinity to FGFR1.
  • FIG. 14 shows that HFKDPK (SEQ ID NO: 5) was determined as a peptide to use for ligand-receptor orthosteric activity and affinity.
  • FIG. 15 shows that LESNNYNT (SEQ ID NO: 6) was also determined as a peptide to use for ligand-receptor orthosteric activity and affinity.
  • Table 3-Table 5 provide a guide for the components used in the experiments that follow (e.g., condensation data; physiochemical data; and flow cytometry and imaging data).
  • Nanoparticles without targeting ligands contained the non-conjugated cationic species poly(arginine) AA chain with length 10 (PLR10) or PEGylated poly(lysine) with AA chain length of 10. All cationic species in the table have L:D isomer ratios of 1:0.
  • HSC hematopoietic stem cells
  • BM bone marrow cells
  • Tcell T cells
  • blood whole blood
  • cynoBM cynomolgus bone marrow
  • targeting ligand sequences were designed in-house and custom manufactured by 3rd party commercial providers.
  • Peptide ligands were derived from native polypeptide sequences and in some cases, mutated to improve binding affinity. Computational analysis of binding kinetics and the determination of optimal mutations was achieved through the use of Rosetta software. In the case where targeting ligands were manufactured in-house, the method and materials were as follows:
  • payloads e.g., genetic material (RNA or DNA), genetic material-protein-nuclear localization signal polypeptide complex (ribonucleoprotein), or polypeptide
  • RNA or DNA genetic material
  • ribonucleoprotein genetic material-protein-nuclear localization signal polypeptide complex
  • polypeptide polypeptide
  • the payload was manufactured to be covalently tagged with or genetically encode a fluorophore.
  • pDNA payloads a Cy5-tagged peptide nucleic acid (PNA) specific to TATATA tandem repeats was used to fluorescently tag fluorescent reporter vectors and fluorescent reporter-therapeutic gene vectors.
  • PNA Cy5-tagged peptide nucleic acid
  • a timed-release component that may also serve as a negatively charged condensing species (e.g.
  • poly(glutamic acid)) was also reconstituted in a basic, neutral or acidic buffer.
  • Targeting ligands with a wild-type derived or wild-type mutated targeting peptide conjugated to a linker-anchor sequence were reconstituted in acidic buffer.
  • additional condensing species or nuclear localization signal peptides were included in the nanoparticle, these were also reconstituted in buffer as 0.03% w/v working solutions for cationic species, and 0.015% w/v for anionic species.
  • Experiments were also conducted with 0.1% w/v working solutions for cationic species and 0.1% w/v for anionic species. All polypeptides, except those complexing with genetic material, were sonicated for ten minutes to improve solubilization.
  • Each separately reconstituted component of the nanoparticle was then mixed in the order of addition that was being investigated.
  • Different orders of additions investigated include:
  • a cryovial containing 20M human primary Pan-T cells (Stemcell #70024) was thawed and seeded in 4 ⁇ 66 wells of 4 96-well plates at 200 ⁇ l and 75,000 cells/well (1.5E6 cells/ml).
  • Cells were cultured in antibiotic free RPMI 1640 media (Thermofisher #11875119) supplemented with 10% FBS and L-glutamine, and maintained by exchanging the media every 2 days.
  • HSC Hematopoietic Stem Cells
  • a cryovial containing 500 k human primary CD34+ cells (Stemcell #70002) was thawed and seeded in 48 wells of a 96-well plate, at 200 ⁇ l and 10-12 k cells per well.
  • the culture media consisted of Stemspan SFEM 11 (Stemcell #09605) supplemented with 10% FBS, 25 ng/ml TPO, 50 ng/ml Flt-3 ligand, and 50 ng/ml SCF and the cells were maintained by exchanging the media every 2 days.
  • a cryovial containing 1.25M Cynomolgus monkey bone marrow cells (IQ Biosciences # IQB-MnBM1) was thawed and 48 wells of a round bottom 96-well plate, were seeded at 200 ⁇ l and ⁇ 30 k cells/well.
  • the cells are cultured in antibiotic free RPMI 1640 media supplemented with 12% FBS, and maintained by exchanging the media every 2 days.
  • Nanoparticles were either directly transfected into 15 mL tubes, or 100 ⁇ l of blood was titrated into each well of a 96-well plate prior to nanoparticle transfection.
  • nanoparticles After forming stock solutions of nanoparticles, 10 ⁇ l of nanoparticles were added per well of 96-well plates and incubated without changes to cell culture conditions or supplementation of media (See Table 6). 96-well plates were maintained during live cell imaging via a BioTek Cytation 5 under a CO2 and temperature controlled environment.
  • HBB gRNA hemoglobin subunit beta
  • VWF von Willebrand factor
  • cationic species were added in order to reach the desired amine to phosphate (N:P) or amine to phosphate+carboxylate [N:(P+C)] ratios.
  • Representative cationic species included PLR10, PLR50, PLR150, anchor-linker peptides, various mutated targeting ligands conjugated to GGGGSGGGGS (SEQ ID NO: xx) linker conjugated to a charged poly(arginine) chain (i.e.
  • Fluorescence emissions from intercalated SYBR Gold in the GFP channel were recorded in a flat bottom, half area, 96 well-plate using a Synergy Neo2 Hybrid Multi-mode reader (Biotek, USA) or a CLARIOstar Microplate reader (BMG, Germany).
  • the hydrodynamic diameter and zeta potential of the nanoparticle formulations were investigated by nanoparticle tracking analysis using a ZetaView instrument (Particle Metrix, Germany). Samples are diluted 1:100 in PBS (1:12) before injection into the instrument. To obtain the measurement, the camera settings are adjusted to the optimal sensitivity and particles/frame ( ⁇ 100-150) before analysis.
  • a Cytation 5 high-content screening live-cell imaging microscope (BioTek, USA) was utilized to image transfection efficiency prior to evaluation by flow cytometry. Briefly, cells were imaged prior to transfection, in 15 m increments post-transfection for 4 h, and then in 2 h increments for the following 12 hours utilizing the GFP and/or Cy5 channels as well as bright field under a 10 ⁇ objective. Images were subsequently gathered as representative of continuous kinetics or discrete 1-18, 24, 36, or 48-hour time-points.
  • Attune multiparametric flow cytometry measurements were conducted on live cells using an Attune NxT Flow Cytometer (ThermoFisher, USA) after appropriate compensations among different channels have been applied. Representative populations of cells were chosen by selection of appropriate gates of forward and side scattering intensities. The detection of cell fluorescence was continued until at least 10000 events had been collected.
  • FIG. 19 -FIG. 44 Condensation Data
  • FIG. 19 (a) SYBR Gold exclusion assay showing fluorescence intensity variations as a function of positive to negative charge ratio (CR) in nanoparticles containing VWF-EGFP pDNA with PNA payload initially intercalated with SYBR Gold.
  • the carboxylate to phosphate (C:P) ratios shown in the legend are based on the nanoparticle's ratio of carboxylate groups on anionic polypeptides species (PLE100) to phosphate groups on the genetic material of the payload.
  • CR was increased via stepwise addition of cationic PLR150. The fluorescence decrease observed show that increasing the CR through addition of PLR150 causes SYBR to be displaced from the payload as the particle condenses. Additionally, condensation remains consistent across various c:p ratios. Blank solutions contain SYBR Gold in absence of the payload.
  • FIG. 20 SYBR Gold exclusion assay showing fluorescence intensity variations as a function of positive to negative charge ratio (CR*) in nanoparticles containing NLS-CAS9-NLS RNP complexed w/ HBB gRNA payload initially intercalated with SYBR Gold. Additionally, determination of CR* does not include the negatively charged portion of the gRNA shielded by complexation with cas9.
  • the carboxylate to phosphate (C:P) ratios shown in the legend are based on the nanoparticle's ratio of carboxylate groups on anionic polypeptides species (PLE100) to phosphate groups on the genetic material of the payload.
  • CR was increased via stepwise addition of cationic PLR150.
  • Blank solutions contain SYBR Gold in absence of the payload.
  • the fluorescence decrease observed show that increasing the CR through addition of PLR150 causes SYBR to be displaced from the payload as the particle condenses. Additionally, condensation remains consistent across various c:p ratios.
  • FIG. 21 SYBR Gold exclusion assay showing fluorescence intensity variations as a function of positive to negative charge ratio (CR) in nanoparticles containing gRNA HBB payloads initially intercalated with SYBR Gold.
  • the carboxylate to phosphate (C:P) ratios shown in the legend are based on the nanoparticle's ratio of carboxylate groups on anionic polypeptides species (PLE100) to phosphate groups on the genetic material of the payload.
  • CR was increased via stepwise addition of PLR150.
  • Blank solutions contain SYBR Gold in absence of the payload.
  • the fluorescence decrease observed show that increasing the CR through addition of PLR150 causes SYBR to be displaced from the payload as the particle condenses. Additionally, condensation with respect to CR remains consistent across various C:P ratios.
  • FIG. 22 (a)(b) SYBR Gold exclusion assay showing fluorescence intensity variations as a function of positive to negative charge ratio (CR) in nanoparticles containing HBB gRNA payload initially intercalated with SYBR Gold.
  • the carboxylate to phosphate (C:P) ratios shown in the legend are based on the nanoparticle's ratio of carboxylate groups on anionic polypeptides species (PLE100) to phosphate groups on the genetic material of the payload.
  • CR was increased via stepwise addition of cationic mutated cKit targeting ligand conjugated to a (GGGS)2 linker conjugated to positively charged poly(arginine) (internal ligand name: SCF_rmAc-cKit(4GS)2_9R_C).
  • SCF_rmAc-cKit(4GS)2_9R_C internal ligand name: SCF_rmAc-cKit(4GS)2_9R_C
  • FIG. 23 (a)(b) SYBR Gold exclusion assay showing fluorescence intensity variations as a function of positive to negative charge ratio (CR) in nanoparticles containing NLS-CAS9-NLS RNP complexed w/ HBB gRNA payload initially intercalated with SYBR Gold.
  • the carboxylate to phosphate (C:P) ratios shown in the legend are based on the nanoparticle's ratio of carboxylate groups on anionic polypeptides species (PLE100) to phosphate groups on the genetic material of the payload.
  • CR was increased via stepwise addition of cationic mutated cKit targeting ligand conjugated to a (GGGS)2 linker conjugated to positively charged poly(arginine) (internal ligand name: SCF_rmAc-cKit_(4GS)2_9R_C).
  • SCF_rmAc-cKit_(4GS)2_9R_C positively charged poly(arginine)
  • FIG. 24 (a)(b) SYBR Gold exclusion assay showing fluorescence intensity variations as a function of positive to negative charge ratio (CR) in nanoparticles containing VWF-EGFP pDNA with PNA payload initially intercalated with SYBR Gold.
  • the carboxylate to phosphate (C:P) ratios shown in the legend are based on the nanoparticle's ratio of carboxylate groups on anionic polypeptides species (PLE100) to phosphate groups on the genetic material of the payload.
  • CR was increased via stepwise addition of cationic mutated cKit targeting ligand conjugated to a (GGGS)2 linker conjugated to positively charged poly(arginine) (internal ligand name: SCF_rmAc-cKit_(4GS)2_9R_C).
  • SCF_rmAc-cKit_(4GS)2_9R_C positively charged poly(arginine)
  • FIG. 25 - FIG. 26 Condensation Curves with Histone H3K4Me as Cationic Material
  • FIG. 25 SYBR Gold exclusion assay showing fluorescence intensity variations as a function of positive to negative charge ratio (CR) in nanoparticles containing VWF-EGFP pDNA with PNA payload initially intercalated with SYBR Gold.
  • the carboxylate to phosphate (C:P) ratios shown in the legend are based on the nanoparticle's ratio of carboxylate groups on anionic polypeptides species (PLE100) to phosphate groups on the genetic material of the payload.
  • CR was increased via stepwise addition of cationic mutated Histone_H3K4(Me3) peptide [1-22] (internal peptide name mH3_K4Me3_1).
  • FIG. 26 (a) SYBR Gold exclusion assay showing fluorescence intensity variations as a function of positive to negative charge ratio (CR) in nanoparticles containing NLS-CAS9-NLS RNP complexed w/ HBB gRNA payload initially intercalated with SYBR Gold.
  • the carboxylate to phosphate (C:P) ratios shown in the legend are based on the nanoparticle's ratio of carboxylate groups on anionic polypeptides species (PLE100) to phosphate groups on the genetic material of the payload.
  • CR was increased via stepwise addition of cationic mutated Histone_H3K4(Me3) peptide [1-22] (internal peptide name mH3_K4Me3_1).
  • FIG. 27 - FIG. 30 Condensation Curves with Peptide CD45 aSiglec_(4GS)2_9R_C as Cationic Material
  • FIG. 27 SYBR Gold exclusion assay showing fluorescence intensity variations as a function of positive to negative charge ratio (CR) in nanoparticles containing VWF-EGFP pDNA with PNA payload initially intercalated with SYBR Gold.
  • the carboxylate to phosphate (C:P) ratios shown in the legend are based on the nanoparticle's ratio of carboxylate groups on anionic polypeptides species (PLE100) to phosphate groups on the genetic material of the payload.
  • CR was increased via stepwise addition of cationic mutated CD45 receptor targeting ligand conjugated to a (GGGS)2 linker conjugated to positively charged poly(arginine) (internal ligand name: CD45_mSiglec_(4GS)2_9R_C). Empty symbols represent blank solutions containing SYBR Gold in absence of the payload.
  • FIG. 28 SYBR Gold exclusion assay showing fluorescence intensity variations as a function of positive to negative charge ratio (CR) in nanoparticles containing Cy5-EGFP mRNA payload initially intercalated with SYBR Gold.
  • the carboxylate to phosphate (C:P) ratios shown in the legend are based on the nanoparticle's ratio of carboxylate groups on anionic polypeptides species (PLE100) to phosphate groups on the genetic material of the payload.
  • CR was increased via stepwise addition of cationic mutated CD45 receptor targeting ligand conjugated to a (GGGS)2 linker conjugated to positively charged poly(arginine) (internal ligand name: CD45_mSiglec_(4GS)2_9R_C).
  • CD45_mSiglec_(4GS)2_9R_C internal ligand name: CD45_mSiglec_(4GS)2_9R_C
  • FIG. 29 SYBR Gold exclusion assay showing fluorescence intensity variations as a function of positive to negative charge ratio (CR) in nanoparticles containing BLOCK-iT Alexa Fluor 555 siRNA payload initially intercalated with SYBR Gold.
  • the carboxylate to phosphate (C:P) ratios shown in the legend are based on the nanoparticle's ratio of carboxylate groups on anionic polypeptides species (PLE100) to phosphate groups on the genetic material of the payload.
  • CR was increased via stepwise addition of cationic mutated CD45 receptor targeting ligand conjugated to a (GGGS)2 linker conjugated to positively charged poly(arginine) (internal ligand name: CD45_mSiglec_(4GS)2_9R_C).
  • CD45_mSiglec_(4GS)2_9R_C internal ligand name: CD45_mSiglec_(4GS)2_9R_C
  • FIG. 30 (a) SYBR Gold exclusion assay showing fluorescence intensity variations as a function of positive to negative charge ratio (CR) in nanoparticles containing NLS-Cas9-EGFP RNP complexed to HBB gRNA payload initially intercalated with SYBR Gold.
  • the carboxylate to phosphate (C:P) ratios shown in the legend are based on the nanoparticle's ratio of carboxylate groups on anionic polypeptides species (PLE100) to phosphate groups on the genetic material of the payload.
  • CR was increased via stepwise addition of cationic mutated CD45 receptor targeting ligand conjugated to a (GGGS)2 linker conjugated to positively charged poly(arginine) (internal ligand name: CD45_mSiglec_(4GS)2_9R_C). Filled symbols represent blank solutions containing SYBR Gold in absence of the payload.
  • Hydrodynamic diameter and zeta potential for some formulations were measured at the condensation end-points and are reported in the following table.
  • Hydrodynamic diameter Zeta Potential Payload C P Cationic Peptide [nm] [mV]
  • RNP (NLS- 0 CD45_mSiglec_(4GS)2_9R_C 134 ⁇ 65 13 ⁇ 1 Cas9-EGFP and gRNA)
  • RNP (NLS- 10 CD45_mSiglec_(4GS)2_9R_C 166 ⁇ 75 19.2 ⁇ 1 Cas9-EGFP and gRNA
  • RNP NLS- 20 CD45_mSiglec_(4GS)2_9R_C 179 ⁇ 92 21 ⁇ 1 Cas9-EGFP and gRNA
  • FIG. 31 -FIG. 34 Inclusion Curves
  • FIG. 31 SYBR Gold inclusion assay showing fluorescence intensity variations as a function of stepwise SYBR addition to different nanoparticles formulations all containing 150 ng of BLOCK-iT Alexa Fluor 555 siRNA payload.
  • the delta change in fluorescence from 0 ⁇ l to 50 ⁇ l of SYBR indicates the stability of the nanoparticle formulations. The less stably condensed a formulation, the more likely SYBR Gold is to intercalate with the genetic payload.
  • Lipofectamine RNAiMAX is used here as a positive control. Tables 2-4.
  • FIG. 32 SYBR Gold inclusion assay showing fluorescence intensity variations as a function of stepwise SYBR addition to different nanoparticles formulations all containing 300 ng the HBB gRNA payload.
  • the delta change in fluorescence from 0 ⁇ l to 50 ⁇ l of SYBR indicates the stability of the nanoparticle formulations. The less stably condensed a formulation, the more likely SYBR Gold is to intercalate with the genetic payload.
  • Lipofectamine CRISPRMAX is used here as a positive control. Tables 2-4.
  • FIG. 33 SYBR Gold inclusion assay showing fluorescence intensity variations as a function of stepwise SYBR addition to different nanoparticles formulations all containing the Cy5 EGFP mRNA payload.
  • the delta change in fluorescence from 0 ⁇ l to 50 ⁇ l of SYBR indicates the stability of the nanoparticle formulations. The less stably condensed a formulation, the more likely SYBR Gold is to intercalate with the genetic payload.
  • Lipofectamine Messenger MAX is used here as a positive control. Tables 2-4.
  • FIG. 34 SYBR Gold inclusion assay showing fluorescence intensity variations as a function of stepwise SYBR addition to different nanoparticles formulations all containing 600 ng of VWF-EGFP pDNA with Cy5 tagged peptide nucleic acid (PNA) Binding Site payload.
  • the delta change in fluorescence from 0 ⁇ l to 50 ⁇ l of SYBR indicates the stability of the nanoparticle formulations. The less stably condensed a formulation, the more likely SYBR Gold is to intercalate with the genetic payload.
  • Lipofectamine 3000 is used here as a positive control. Tables 2-4.
  • FIG. 35 -FIG. 44 SYBR Exclusion/Condensation Assays on TC.001 (See Tables 2-4)
  • FIG. 35 SYBR Gold exclusion assay showing fluorescence intensity decrease by addition of cationic polypeptide CD45_mSiglec_(4GS)2_9R_C followed by PLE100 and by further addition of the cationic polypeptide to RNP.
  • the fluorescence background signal id due to GFP fluorescence from the RNP.
  • FIG. 36 SYBR Gold exclusion assay showing fluorescence intensity variations by addition of cationic polypeptide CD45_mSiglec_(4GS)2_9R_C followed by PLE100 and by further addition of the cationic polypeptide to siRNA and SYBR Gold.
  • FIG. 37 SYBR Gold exclusion assay showing fluorescence intensity variations by addition of cationic polypeptide histone peptide H2A followed by CD45_mSiglec_(4GS)2_9R_C and by further addition of PLE100 to RNP of NLS-Cas9-EGFP with HBB gRNA and SYBR Gold.
  • FIG. 38 SYBR Gold exclusion assay showing fluorescence intensity variations by addition of cationic polypeptide histone peptide H4 together with CD45_mSiglec_(4GS)2_9R_C and by further addition of PLE100 to RNP of NLS-Cas9-EGFP with HBB gRNA and SYBR Gold.
  • FIG. 39 SYBR Gold exclusion assay showing fluorescence intensity variations by addition of cationic polypeptide CD45_mSiglec_(4GS)2_9R_C fand by further addition of PLE100 to mRNA.
  • FIG. 40 SYBR Gold exclusion assay showing fluorescence intensity variations by addition histone H4 and by further addition of CD45-mSiglec-(4GS)2_9R_c and PLE100 to mRNA.
  • FIG. 41 SYBR Gold exclusion assay showing fluorescence intensity variations by addition histone H2A and by further addition of CD45-mSiglec-(4GS)2_9R_c and PLE100 to mRNA.
  • FIG. 42 SYBR Gold exclusion assay from intercalation with VWF_EGFP pDNA showing fluorescence intensity variations by addition of cationic polypeptide CD45_mSiglec_(4GS)2_9R_C followed by PLE100.
  • FIG. 43 SYBR Gold exclusion assay from intercalation with VWF_EGFP pDNA showing fluorescence intensity variations by addition of histone H4, followed by cationic polypeptide CD45_mSiglec_(4GS)2_9R_C followed by PLE100.
  • FIG. 44 SYBR Gold exclusion assay from intercalation with VWF_EGFP pDNA showing fluorescence intensity variations by addition of histone H4, followed by cationic polypeptide CD45_mSiglec_(4GS)2_9R_C followed by PLE100.
  • FIG. 45 -FIG. 83 Physicochemical Data
  • Nanoparticle tracking analysis utilizes laser scattering microscopy and image analysis to obtain measurements of particle size and zeta potential with high resolution.
  • Dispersity is a measure of sample heterogeneity and is determined by the distribution, where a low standard of deviation and single peak indicates particle uniformity.
  • Targeting ligands consisting of polypeptides with a ligand, (GGGGS)2 linker, and electrostatic anchor domain were synthesized by solid phase peptide synthesis and used to functionalize the silica surface (sheddable layer) of particles carrying pEGFP-N1 plasmid DNA payload.
  • the resulting particle size and zeta potential distributions were obtained by nanoparticle tracking analysis using a ZetaVIEW instrument (Particle Metrix, Germany).
  • GGGS glycine-serine linker
  • FIG. 46 Branched Histone Peptide Conjugate Pilot Particle.
  • Histone H3 peptide with a C-terminal Cysteine was conjugated to 48 kD poly(L-Lysine) with 10% side-chain thiol substitutions.
  • the final product purified by centrifugal filtration and molecular weight exclusion, was used to complex plasmid DNA (pEGFP-N1).
  • the resulting measurements portrayed in FIG. 46 , show a narrow size distribution. Size Distribution of H3-Poly(L-Lysine) conjugate in complex with plasmid DNA (pEGFP-N1)
  • FIG. 47 provides data related to project HSC.001.001.
  • FIG. 48 provides data related to project HSC.001.002, which used H3-poly(L-Lysine) conjugate complexed to PNA-tagged pDNA and an E-Selectin targeting peptide (ESELLg_mESEL_(4GS)2_9R_N).
  • FIG. 49 - FIG. 52 provide data for experiments in which various targeting ligands or stealth molecules were coated upon silica-coated particles and silica-coated nanodiamonds (for diagnostic enhanced fluorescent applications). Size and Zeta Potential distributions are presented with associated statistics.
  • Targeting ligands were ESELLg_mESEL_(4GS)2_9R_N, ESELLg_mESEL_(4GS)2_9R_C, CD45_mSiglec_(4GS)2_9R_C, and Cy5mRNA-SiO2-PEG, respectively.
  • Performance of nanoformulations and targeting ligands was significantly improved in all data that follows—elimination of silica layer and replacement with a charged anionic sheddable polypeptide matrix significantly enhanced transfection efficiencies of nanoparticles across all formulations, with a variety of payloads and ligand-targeting approaches.
  • the multilayering techniques used in the data above, as well as enhanced condensation with branched histone complexes and subsequent peptide matrix engineering (working examples are presented in Tcell.001, HSC.004, CYNOBM.002, and Blood.002) demonstrate the flexibility of the techniques (e.g., multilayering) and core biomaterials (e.g., see entirety of disclosure and subsequent experiments).
  • branched histones may be conjugated to linker-ligand domains or co-condensed with a plurality of embodiments and uses thereof.
  • FIG. 53 - FIG. 57 depict particles carrying Cy5-EGFP mRNA payload, complexed with a sheddable poly(glutamic acid) surface matrix and CD45 ligand.
  • Nanoparticles produced using this formulation were highly uniform in particle size and zeta potential. Particles with poly(glutamic acid) added after SIGLEC-derived peptide association with mRNA (BLOOD.002.88) were more stable and monodisperse than particles with poly(glutamic acid) added before SIGLEC-derived association with mRNA and poly(glutamic acid), indicating that a particular order of addition can be helpful in forming more stable particles.
  • particles formed from poly(glutamic acid) complexed with SIGLEC-derived peptides without a phosphate-containing nucleic acid were highly anionic monodispersed (BLOOD.002.92).
  • Particles formed from PLR50 with PLE100 added after PLR association with mRNA were highly stable, monodispersed and cationic (BLOOD.002.91).
  • PLK-PEG association with mRNA prior to PLE100 addition resulted in very small particles with heterogenous charge distributions.
  • FIG. 53 provides data from BLOOD.002.88. Nanoparticles had zeta potential of ⁇ 3.32+/ ⁇ 0.29 mV with 90% having diameters less than 180 nm. These nanoparticles resulted in 58.6% efficient Cy5_EGFP_mRNA uptake in whole blood according to flow cytometry data. The narrow and uniform peak is exemplary of excellent charge distributions and was reproducible in forming net anionic particles in TCELL.001.18. This demonstrates broad applicability of SIGLEC-derived targeting peptides for systemic delivery (e.g., see flow cytometry and imaging data below).
  • FIG. 54 provides data from BLOOD.002.89. Nanoparticles hadzeta potential of ⁇ 0.25+/ ⁇ 0.12 mV with 90% having diameters less than 176 nm. These nanoparticles resulted in 58.6% efficient Cy5_EGFP_mRNA uptake in whole blood respectively according to flow cytometry data. This demonstrates broad applicability of Siglec derived targeting peptide for systemic delivery (e.g., see flow cytometry and imaging data below).
  • FIG. 55 provides data from BLOOD.002.90. Nanoparticles had zeta potential of 2.54+/ ⁇ 0.03 mV with 90% having diameters less than 99 nm. These nanoparticles resulted in 79.9% efficient Cy5_EGFP_mRNA uptake in whole blood respectively according to flow cytometry data (e.g., see flow cytometry and imaging data below).
  • FIG. 56 provides data from BLOOD.002.91. Nanoparticles had zeta potential of 27.10 FWHM 18.40 mV with 90% having diameters less than 130 nm. These nanoparticles resulted in 96.7% efficient Cy5_EGFP_mRNA uptake in whole blood respectively according to flow cytometry data (e.g., see flow cytometry and imaging data below). Strongly positively charged zeta potentials led to high efficiencies and intensities of Cy5+ signal on whole blood cells. Briefly, in this embodiment, a larger dose of PLR50 (15 ⁇ l of PLR50 0.1% w/v solution) was added to 100 ⁇ l pH 5.5 30 mM HEPES with 2.5 ug Cy5 mRNA (TriLink). After 5 minutes at 37° C., 1.5 ⁇ l of PLE100 0.1% was added to the solution. In contrast, other experiments involved adding larger relative volumes (5-20% of total solution volume) of PLE100 to a preformed cationic polymer+anionic material core.
  • TriLink
  • FIG. 57 provides data from BLOOD.002.92.
  • Nanoparticles had zeta potential of ⁇ 22.16 FWHM 18.40 mV with 90% having diameters less than 130 nm. These nanoparticles did not result in detectable Cy5_EGFP_mRNA uptake in whole blood according to flow cytometry data, as they were not labeled with a fluorophore (e.g., see flow cytometry and imaging data below).
  • the effective condensation of these nanoparticles without a payload (vehicle) also has implications in non-genetic material payload delivery, such as conjugation of the charged polymer to a small molecule or chemotherapeutic agent.
  • FIG. 58 - FIG. 73 depict results from experiments performed to characterize representative particles containing CRISPR ribonucleoprotein (RNP) (TCELL.001.01-TCELL.001.15), mRNA (TCELL.001.16-TCELL.001.30), plasmid DNA (TCELL.001.31-TCELL.001.45) and siRNA (TCELL.001.46-TCELL.001.60) and patterned with identical ligands in corresponding groups.
  • RNP CRISPR ribonucleoprotein
  • FIG. 58 provides data from TCELL.001.1. Nanoparticles had zeta potential of ⁇ 3.24+/ ⁇ 0.32 mV with 90% having diameters less than 77 nm. These nanoparticles resulted in 99.16% and 98.47% efficient CRISPR-GFP-RNP uptake in viable CD4+ and CD8a+ pan T cells respectively according to flow cytometry data (e.g., see flow cytometry and imaging data below).
  • TCELL.001.1 was subsequently coated in PLE100+mRNA prior to addition of charged polymers or charged anchor-linker-ligands in CYNOBM.002.82-CYNOBM.002.85.
  • FIG. 59 provides data from TCELL.001.3.
  • Nanoparticles had zeta potential of ⁇ 0.98+/ ⁇ 0.08 mV with 90% having diameters less than 65 nm. Despite ideal size ranges, these nanoparticles resulted in 11.6% and 13.2% efficient CRISPR-GFP-RNP uptake in viable CD4+ and CD8a+ pan T cells, respectively, according to flow cytometry data in contrast to the strongly anionic similarly-sized particles in TCELL.001.1 that achieved ⁇ 99% efficiency in the same cell populations.
  • the relationship of particle size and stable negative zeta potential and methods and uses thereof are shown to be predicable constraints through the experiments described herein.
  • An ideal nanoparticle has a majority of particles ⁇ 70 nm with zeta potentials of ⁇ 5 mV, and the sheddable anionic coating methods described herein as well as multistage-layering sheddable matrices for codelivery described in CYNOBM.002 achieve stable and extremely efficient transfection of sensitive primary cells from human and cynomolgus blood, bone marrow, and specific cells within the aforementioned.
  • TCELL.001.3 The reduced efficiency of TCELL.001.3 is a marked contrast to the results of TCELL.01.27, where the same ligands achieved stable condensation of mRNA at an altered amine-to-phosphate-to-carboxylate ratio than the one used for this particular CRISPR formulation (e.g., see flow cytometry and imaging data below).
  • FIG. 60 provides data from TCELL.001.13. Nanoparticles have zeta potential of 2.19+/ ⁇ 0.08 mV with 90% having diameters less than 101 nm. See flow cytometry/imaging data below for the efficiency of CRISPR-GFP-RNP uptake in viable CD4+ and CD8a+ pan T cells.
  • FIG. 61 provides data from TCELL.001.14. Nanoparticles have zeta potential of ⁇ 9.37+/ ⁇ 0.16 mV with 90% having diameters less than 111 nm. These nanoparticles resulted in 25.7% and 28.6% efficient CRISPR-GFP-RNP uptake in viable CD4+ and CD8a+ pan T cells respectively according to flow cytometry data. (e.g., see flow cytometry and imaging data below).
  • FIG. 62 provides data from TCELL.001.16.
  • FIG. 63 provides data from TCELL.001.18.
  • the size and zeta potential of these particles demonstrate average particle sizes of 80.9 nm with zeta potentials of ⁇ 20.26+/ ⁇ 0.15 mV and 90% of particles with 39.2-129.8 nm diameters, indicating strong particle stability at a 1.35 carboxylate-to-phosphate (C:P) and 0.85 amine-to-phosphate ratio wherein poly(glutamic acid) was added following inclusion of the cationic anchor-linker-ligand.
  • C:P carboxylate-to-phosphate
  • amine-to-phosphate ratio wherein poly(glutamic acid) was added following inclusion of the cationic anchor-linker-ligand.
  • FIG. 64 provides data from TCELL.001.28.
  • FIG. 65 provides data from TCELL.001.29.
  • FIG. 66 provides data from TCELL.001.31.
  • FIG. 67 provides data from TCELL.001.33.
  • FIG. 68 provides data from TCELL.001.43.
  • FIG. 69 provides data from TCELL.001.44.
  • FIG. 70 provides data from TCELL.001.46.
  • FIG. 71 provides data from TCELL.001.48.
  • FIG. 72 provides data from TCELL.001.58.
  • FIG. 73 provides data from TCELL.001.59.
  • FIG. 74 - FIG. 83 depict results characterizing the formulations used in cynomolgus bone marrow cells.
  • FIG. 74 provides data from CYNOBM.002.82. Particles successfully deleted the BCL11a erythroid enhancer in whole bone marrow erythroid progenitor cells as evidenced by fetal hemoglobin protein expression in 3% of live cells. CYNOBM.002.82 nanoparticles had zeta potential of 2.96+/ ⁇ 0.14 mV with 90% having diameters less than 132 nm and 50% of particles with diameters less than 30 nm.
  • CYNOBM.002.75 with an identical core template consisting of PLR10, PLE100, PDE100 and Cas9 RNP but without an mRNA co-delivery component or additional layer of PLR50, exhibited ⁇ 20%, ⁇ 14%, and ⁇ 100% efficient CRISPR-GFP-RNP uptake in viable CD3+, CD45+, and CD34+ bone marrow subpopulations, respectively, and 18.0% overall bone marrow viable subpopulation targeting according to flow cytometry data.
  • FIG. 75 provides data from CYNOBM.002.83. Particles successfully deleted the BCL11a erythroid enhancer in whole bone marrow erythroid progenitor cells as evidenced by fetal hemoglobin protein expression in 1.9% of live cells, with none of these cells being CD34+. The nanoparticles had a zeta potential of ⁇ 2.47+/ ⁇ 0.33 mV with 90% having diameters less than 206 nm, leading to improved transfection efficiency vs. CYNOBM.002.03 with the same IL2-mimetic peptide coating.
  • the large charge distribution with tails at approximately ⁇ 50 mV and +25 mV were indicative of a polydisperse particle population with a variance of particle stabilities, similarly to CYNOBM.002.83, and in contrast to CYNOBM.002.84 which has a stable anionic single-peak zeta potential of ⁇ 18 mV and corresponding increase in cellular viability compared to other CRISPR+mRNA co-delivery particle groups (CYNOBM.002.82-CYNOBM.002.85).
  • next-best nanoparticle group in terms of overall cynomolgus bone marrow co-delivery was CYNOBM.002.86, which demonstrated similar highly net-negatively charged zeta potential of ⁇ 20 mV and a corresponding high efficiency of transfection, CD34 clonal expansion, and fetal hemoglobin production from BCL11a erythroid enhancer knockout.
  • These nanoparticles resulted in ⁇ 100% efficient CRISPR-GFP-RNP+Cy5_EGFP_mRNA uptake in viable CD34+ bone marrow cells, within mixed cell populations, as well as 8.1% of whole bone marrow viable subpopulations according to flow cytometry data.
  • the flow cytometry data indicates induction of selective CD34+ proliferation in cynomolgus bone marrow cells suggesting successful clonal expansion of BCL11a erythroid progenitor knockout populations within CD34 ⁇ erythroid progenitor cells. (e.g., see flow cytometry and imaging data below). The results also implicate successful targeting in endothelial cells, osteoblasts, osteoclasts, and/or other cells of the bone marrow.
  • FIG. 76 provides data from CYNOBM.002.84. Particles successfully deleted the BCL11a erythroid enhancer in whole bone marrow erythroid progenitor cells as evidenced by fetal hemoglobin protein expression in 9.5% of live whole bone marrow cells and no positive fetal hemoglobin measurements in CD34+, CD45 or CD3+ subpopulations despite moderate transfection efficiencies, as measured by Cy5-mRNA+ and CRISPR-GFP-RNP+ gates in each selective subpopulation. CYNOBM.002.84 nanoparticles had zeta potential of ⁇ 18.07+/ ⁇ 0.71 mV with 90% having diameters less than 205 nm. The high net-negative charge indicates stable particle formation.
  • FIG. 77 provides data from CYNOBM.002.85. Nanoparticles had zeta potential of ⁇ 12.54+/ ⁇ 0.25 mV with 90% having diameters less than 186 nm. These nanoparticles resulted in ⁇ 33%, ⁇ 23%, and ⁇ 100% efficient CRISPR-GFP-RNP+Cy5_EGFP_mRNA uptake in viable CD3+, CD45+, and CD34+ bone marrow cells, respectively, according to flow cytometry data. (e.g., see flow cytometry and imaging data below). The results may implicate successful targeting in endothelial, osteoblasts, osteoclasts, and other cells of the bone marrow. Particle sizes and charge distributions were consistent with subsequent CYNOBM.002 groups and their expected biological performance in cynomolgus bone marrow CRISPR and/or mRNA delivery.
  • FIG. 78 provides data from CYNOBM.002.86.
  • Nanoparticles had zeta potential of ⁇ 20.02+/ ⁇ 0.10 mV with 90% having diameters less than 120 nm. These nanoparticles resulted in 20.1% efficient codelivery of CRISPR-GFP-RNP+Cy5_EGFP_mRNA in viable cynomolgus bone marrow, with ⁇ 68%, 70%, and ⁇ 97% efficient CD3+, CD45+, and CD34+ respective targeting according to flow cytometry data. (e.g., see flow cytometry and imaging data below). The results may implicate successful targeting in endothelial, osteoblasts, osteoclasts, and other cells of the bone marrow. A highly negatively charged zeta potential and of 90% of particles counts ⁇ 200 nm predicts high efficiency.
  • FIG. 79 provides data from CYNOBM.002.76. Nanoparticles had zeta potential of ⁇ 12.02+/ ⁇ 0.59 mV with 90% having diameters less than 135 nm. These nanoparticles resulted in 18.4%, 10.3%, and ⁇ 100% efficient CRISPR-GFP-RNP uptake in viable CD3+, CD45+, and CD34+ bone marrow cells, respectively, according to flow cytometry data (e.g., see flow cytometry and imaging data below).
  • particles exhibit limited toxicity as expected from a histone-mimetic particle with highly negative zeta potential 10th-50th percentile particle sizes of 25.8-80.6 nm with no large aggregates as seen in CYNOBM.002.78, which exhibits similar zeta potential distributions and sizes with the addition of a large volume peak at ⁇ 500 nm.
  • FIG. 80 provides data from CYNOBM.002.77. Nanoparticles had 90% of their diameters below 254 nm with a large portion in the 171-254 nm range. (e.g., see flow cytometry and imaging data below). Additionally, the 10th-50th percentile particles by number were 70-172 nm, indicating a reasonable size distribution within this population. Consistent with other studies where a large number of particles >200 nm existed in solution and/or had a large, distributed zeta potential and/or a non-anionic zeta potential, these particles lead to significant cell death. These nanoparticles resulted in high uptake percentages overall, but a large number of cells (>90%) being dead.
  • the particles resulted in negligible uptake at the limits of detection of CRISPR-GFP-RNP in viable CD3+, CD45+, and CD34+ bone marrow cells, and 3.8% CRISPR uptake within whole bone marrow viable subpopulations according to flow cytometry data.
  • 90% of CYNOBM.002.83 (a CRISPR & mRNA codelivery variant) particles with the same surface coating were below 200 nm with the number average being 121 nm.
  • FIG. 81 provides data from CYNOBM.002.78. Nanoparticles had zeta potential of ⁇ 11.72+/ ⁇ 0.79 mV with 90% having diameters less than 223 nm. (e.g., see flow cytometry and imaging data below).
  • CYNOBM.002.84 a CRISPR & mRNA codelivery variant particles with the same surface coating were below 200 nm with the number average being 125 nm, though the zeta potential of CYNOBM.002.84 is significantly more negative ( ⁇ 18.07 mV vs.-11.72 mV), indicating enhanced stability with an anionic sheddable interlayer step intermediate to initial Cas9 RNP charge homogenization with PLR10 and subsequent coating with ligands or additional, optionally molecular weight staggered polymers or polypeptides.
  • the differential physicochemical properties of these monodelivery vs. co-delivery (or interlayer vs. direct conjugation of ligands to RNP) nanoparticles and their respective size ranges is strongly correlated to transfection efficiency and toxicity.
  • FIG. 82 provides data from CYNOBM.002.79. Nanoparticles had diameters less than 200 nm. These nanoparticles resulted in very low (3.7%) GFP-RNP uptake in bone marrow overall, but the cells retained exceptional viability (70.0% vs. 71.6% for negative controls) in the culture. Despite very low overall uptake, the particles demonstrated selective uptake for ⁇ 9.0% of viable CD3+ cells, 4.4% of viable CD45+ cells, and ⁇ 100% of viable CD34+ cells according to flow cytometry data, which is at the limits of detection for cell counts in the CD34+ subpopulation. (e.g., see flow cytometry and imaging data below). The results implicate specific targeting of CD34+ hematopoietic stem cells within mixed cell populations.
  • FIG. 83 provides data from CYNOBM.002.80. Nanoparticles had zeta potential of 1.36+1-1.69 mV. These nanoparticles resulted in 8% transfection efficiency and ⁇ 100% efficient CRISPR-GFP-RNP uptake in viable CD34+ bone marrow cells according to flow cytometry data, which is at the limits of detection for cell counts. (e.g., see flow cytometry and imaging data below). The results may implicate successful targeting in endothelial, osteoblasts, osteoclasts, and other cells of the bone marrow. The even peak at ⁇ 0 mV with wide surfaces is indicative of a zwitterionic particle surface. A high degree of cellular viability indicates that particles were well tolerated with this size and that a c-Kit-receptor-derived particle surface is likely to mimic presentation of native stem cell population surface markers within the bone marrow during cell-cell interactions.
  • FIG. 84 - FIG. 120 Flow Cytometry and Imaging Data
  • FIG. 84 Untransfected controls for CynoBM.002 samples in cynomolgus bone marrow.
  • Microscope images Top: digital phase contrast; middle: GFP; bottom: merge.
  • Flow cytometry data with viability, CD34, CD3, and CD45 stains.
  • FIG. 85 Lipofectamine CRISPRMAX delivery of NLS-Cas9-EGFP BCL11a gRNA RNPs attains 2.5% transfection efficiency in viable cells and causes significant toxicity, with percentage of CD45 and CD3 relative subpopulations significantly decreased compared to negative controls in cynomolgus bone marrow. Lipofectamine CRISPRMAX does not exhibit cell-selectivity as exemplified by 7.4% efficient targeting of remaining CD3+ cells and negligible remaining populations of CD45+ and CD34+ cells. Microscope images—Top: digital phase contrast; middle: GFP; bottom: merge.
  • FIG. 86 CynoBM.002 RNP-Only controls show NLS-Cas9-EGFP BCL11a gRNA RNPs attaining negligible transfection efficiencies in cynomolgus bone marrow without a delivery vector, but with both payloads pre-combined prior to transfection.
  • a high degree of colocalization despite no delivery vector and minimal events is indicative of association of the ribonucleoprotein complex with mRNA, and exemplary of anionic functionalization of CRISPR RNPs. (In this instance, the mRNA acts as a loosely-associated sheddable coat for the RNP and could be further layered upon with cationic materials). Calculating colocalization coefficient.
  • X % CRISPR uptake in live cells:
  • FIG. 87 CynoBM.002.82 demonstrated that non-specifically-targeted NLS-Cas9-EGFP achieves 11.3% efficient mRNA delivery and 11.4% efficient CRISPR delivery to cynomolgus bone marrow with a 98.9% colocalization coefficient.
  • Subcellular localization demonstrated that noon-specifically targeted NLS-Cas9-EGFP BCL11a gRNA RNPs co-localize with Cy5 mRNA and attain high transfection efficiencies.
  • a high degree of colocalization determines that discrete particles were loaded with both payloads.
  • Cas9 can be seen neatly localized in a separate compartment from the mRNA, wherein the mRNA forms a ringed structure around the nuclear-associated Cas9.
  • Microscope images Top: digital phase contrast; middle: Cy5 mRNA; bottom: merge. and top: Cas9-GFP RNP; bottom: Cy5 mRNA colocalized with Cas9-GFP RNP.
  • CYNOBM.002.82 had zeta potential of 2.96+/ ⁇ 0.14 mV with 90% having diameters less than 132 nm and 50% of particles with diameters less than 30 nm. These nanoparticles resulted in 45.5%, 56.0%, and 97.3% efficient CRISPR-GFP-RNP+Cy5_EGFP_mRNA uptake in viable CD3+, CD45+, and CD34+ bone marrow subpopulations, respectively, despite only 11.4% overall bone marrow viable subpopulation targeting.
  • Cas9-mRNA Colocalization Coefficient 94.8%.
  • Viable CD34+ and CRISPR+ 97.2% of Viable CD34+. Fetal Hemoglobin Positive: 3.022% of viable cells
  • FIG. 88 CynoBM.002.83 achieves 8.1% efficient mRNA delivery and 8.1% efficient CRISPR delivery to cynomolgus bone marrow with a 93.0% colocalization coefficient.
  • Subcellular localization demonstrated that homovalently-targeted IL2-derived peptides associated with NLS-Cas9-EGFP BCL11a gRNA RNPs co-localize with Cy5 mRNA and attain high transfection efficiencies.
  • a high degree of colocalization determines that discrete particles were loaded with both payloads.
  • Cas9 can be seen neatly localized in a separate compartment from the mRNA, wherein the mRNA forms a ringed structure around the nuclear-associated Cas9.
  • Microscope images Top: digital phase contrast; middle: Cy5 mRNA; bottom: merge. and top: Cas9-GFP RNP; bottom: Cy5 mRNA colocalized with Cas9-GFP RNP.
  • FIG. 89 CYNOBM.002.84 particles successfully delete the BCL11a erythroid enhancer in whole bone marrow erythroid progenitor cells as evidenced by fetal hemoglobin protein expression in 9.5% of live whole bone marrow cells and no positive fetal hemoglobin measurements in CD34+, CD45 or CD3+ subpopulations despite moderate transfection efficiencies, as measured by Cy5-mRNA+ and CRISPR-GFP-RNP+ gates in each selective subpopulation. Subcellular localization demonstrated that homovalently-targeted E-selectin-derived peptides associated with NLS-Cas9-EGFP BCL11a gRNA RNPs co-localize with Cy5 mRNA and attain high transfection efficiencies.
  • a high degree of colocalization determines that discrete particles were loaded with both payloads. Additionally, Cas9 can be seen neatly localized in a separate compartment from the mRNA, wherein the mRNA forms a ringed structure around the nuclear-associated Cas9. This indicates cytosolic (mRNA) vs. nuclear (CRISPR) localization of the two payloads.
  • FIG. 90 CynoBM.002.85 achieved 5.2% efficient mRNA delivery and 5.3% efficient CRISPR delivery to cynomolgus bone marrow with a 87.2% colocalization coefficient. Despite 5.3% efficient CRISPR delivery to viable cells, CynoBM.002.85 did not lead to a concomitant increase in fetal hemoglobin positive cells as seen in other codelivery embodiments. Subcellular localization demonstrated that homovalently-targeted SCF-derived peptides associated with NLS-Cas9-EGFP BCL11a gRNA RNPs co-localize with Cy5 mRNA and attain high transfection efficiencies. A high degree of colocalization determined that discrete particles were loaded with both payloads.
  • Cas9 could be seen neatly localized in a separate compartment from the mRNA, wherein the mRNA forms a ringed structure around the nuclear-associated Cas9. This indicates cytosolic (mRNA) vs. nuclear (CRISPR) localization of the two payloads.
  • FIG. 91 CynoBM.002.86 achieved 20.1% efficient mRNA delivery and 21.8% efficient CRISPR delivery to cynomolgus bone marrow with a 98.6% colocalization coefficient.
  • Subcellular localization demonstrated that heterotrivalently-targeted IL2-, E-selectin- and SCF-derived NLS-Cas9-EGFP BCL11a gRNA RNPs co-localized with Cy5 mRNA and attain high transfection efficiencies.
  • a high degree of colocalization determined that discrete particles were loaded with both payloads. Additionally, Cas9 could be seen neatly localized in a separate compartment from the mRNA, wherein the mRNA forms a ringed structure around the nuclear-associated Cas9.
  • Microscope images Top: digital phase contrast; middle: Cy5 mRNA; bottom: merge. and top: Cas9-GFP RNP; bottom: Cy5 mRNA colocalized with Cas9-GFP RNP.
  • FIG. 92 CynoBM.002.75 demonstrated that non-specifically-targeted NLS-Cas9-EGFP BCL11a gRNA RNPs with sheddable anionic polypeptide coats attain 18.0% transfection efficiency in viable cynomolgus bone marrow.
  • 20% of viable CD3+ T-cells were CRISPR+ in the mixed population cynomolgus bone marrow culture model herein, in contrast to 97-99% of viable CD4 and CD8a T-cells in human primary Pan T-cells being CRISPR+ in TCELL.001.
  • Particle sizes of an identical formulation were smaller and more uniform in TCELL1, which was synthesized via fluid-handling robotics as opposed to by hand. See above data for additional qualitative and quantitative commentary and data comparisons. Top: digital phase contrast; middle: GFP; bottom: merge.
  • FIG. 93 CynoBM.002.76 demonstrated that dual-histone-fragment-associated and non-specifically-targeted NLS-Cas9-EGFP BCL11a gRNA RNPs attain 13.1% transfection efficiency and limited toxicity versus negative controls in cynomolgus bone marrow. 18%, 10%, and 0% of CD3+, CD45+ and CD34+ viable subpopulations were CRISPR+. See above data for additional physicochemical characteristics and observations. Top: digital phase contrast; middle: GFP; bottom: merge.
  • FIG. 94 CynoBM.002.77 demonstrated that homovalently-targeted IL2-derived peptides associated with NLS-Cas9-EGFP BCL11a gRNA RNPs attain 3.8% transfection efficiency and enhanced viability over negative controls in cynomolgus bone marrow.-90% of transfected cells were dead. Ultimately, the particles resulted in negligible uptake at the limits of detection of CRISPR-GFP-RNP in viable CD3+, CD45+, and CD34+ bone marrow cells, indicating that the remaining 3.8% of live CRISPR+ cells were not from those subpopulations. Size data supports a causative role for toxicity in large particle polydispersity and ⁇ 999 nm 90th volume percentile particle sizes. See above data for additional physicochemical properties. Top: digital phase contrast; middle: GFP; bottom: merge.
  • FIG. 95 CynoBM.002.78 demonstrated that homovalently-targeted E-selectin-derived peptides associated with NLS-Cas9-EGFP BCL11a gRNA RNPs attain ⁇ 71% transfection efficiency overall (including dead cells), with only 4.5% of live cells remaining transfected in cynomolgus bone marrow. This is indicative of particle toxicity and may be correlated to a large size distribution, despite 50% of the particles by number being 33.1-113.1 nm. The >250 nm particles, comprising the majority of particle mass and volume in solution, likely led to the reduced viability of this experiment. CD45+ and CD3+ subpopulation densities were manifold reduced in this embodiment as well. See above data for more detailed physicochemical characteristics and qualitative observations comparing nanoparticle groups from the same transfection. Top: digital phase contrast; middle: GFP; bottom: merge.
  • FIG. 96 CynoBM.002.79 demonstrated that homovalently-targeted SCF-derived peptides associated with NLS-Cas9-EGFP BCL11a gRNA RNPs attain 3.7% transfection efficiencies and excellent viability over negative controls in cynomolgus bone marrow. These nanoparticles resulted in very low (3.7%) GFP-RNP uptake in bone marrow overall, but the cells retained exceptional viability (69.0% vs. 71.6% for negative controls) in the culture.
  • the particles demonstrated selective uptake for ⁇ 5% of viable CD3+ cells, ⁇ 4% of viable CD45+ cells, and ⁇ 100% of viable CD34+ cells (the latter which were at the limits of detection in number).
  • the high degree of cellular viability coupled with a strongly negative zeta potential and significantly more CD45+ cells than other groups is implicative of a SCF-mimetic particle surface's multifactorial role in establishing stem cell niche targeting and proliferation and/or survival techniques. See above data for additional physicochemical parameters. Top: digital phase contrast; middle: GFP; bottom: merge.
  • FIG. 97 CynoBM.002.80 demonstrated that homovalently-targeted c-Kit-(CD117)-derived peptides associated with NLS-Cas9-EGFP BCL11a gRNA RNPs attain 8.097% transfection efficiencies. Transfection efficiencies were 3.3%, 2.4%, and at the limits of detection for CD3+, CD45+ and CD34+ viable subpopulations, respectively, indicating low selectivity for CD3+ and CD45+ cells. See above data for more quantitative and qualitative data. Top: digital phase contrast; middle: GFP; bottom: merge. (cont.): flow cytometry data.
  • FIG. 98 CynoBM.002.81 demonstrated that heterotrivalently-targeted IL2-, E-selectin- and SCF-derived NLS-Cas9-EGFP BCL11a gRNA RNPs attain 5% transfection efficiency in cynomolgus bone marrow with ⁇ 10% of transfected cells being live CD34+ cells despite only 0.48% of cells being CD34+. This indicates nearly 100% efficient selective transfection of CD34+ cells. Top: digital phase contrast; middle: GFP; bottom: merge.
  • FIG. 99 Qualitative images of CynoBM.002 RNP-Only control show NLS-Cas9-EGFP BCL11a gRNA RNPs attaining mild positive signal in cynomolgus bone marrow without a delivery vector. Top: digital phase contrast; middle: GFP; bottom: merge.
  • FIG. 100 HSC.004 (nanoparticles 69-74, see Table 5) High-Content Screening. Fluorescence microscopy images (Cy5 mRNA) of HSC.004 Cy5 mRNA delivery 12-15 h post-transfection in Primary Human CD34+ Hematopoietic Stem Cells.
  • Cy5 mRNA Fluorescence microscopy images
  • SCF peptides and E-selectin As well as homovalent targeting with E-selectin but not SCF peptides, achieves higher transfection efficiencies than Lipofectamine MessengerMAX.
  • HSC.001.69 A1-A6; HSC.001.70: B1-B6; HSC.001.71: C1-C6; HSC.001.72: D1-D6; HSC.001.73: E1-E6; HSC.001.74: F1-F6; HSC.004
  • Lipofectamine MessengerMAX Dose 1: G1-G2 & G4-G5; TC.001 Lipofectamine MessengerMAX Dose 2: H1-H2 & H4-H5; TC.001 Negative: G3, G6, H3, H6
  • FIG. 101 TCELL.001 (nanoparticles 1-15, see Table 5) High-Content Screening. Robotic formulations were performed for TC.001.1-TC.001.60, representing 15 ligands across 4 payloads (CRISPR RNP, mRNA, siRNA and pDNA). Shown are embodiments of T-cell CRISPR delivery and qualitative transfection efficiencies—thumbnail images of 12-15 h post-transfection composite microscopy of TCELL.001 CRISPR-EGFP RNP delivery to Primary Human Pan T-cells.
  • FIG. 102 TCELL.001 Lipofectamine CRISPRMAX. Lipofectamine CRISPRMAX attained 4.7% and 4.8% efficient delivery of NLS-Cas9-EGFP RNP in viable CD4+ and CD8a+ subpopulations, respectively, of human primary Pan T-cells at 24 h post-transfection. Overall, 12.5% of CRISPR+ cells and 65.9% of overall cells were viable.
  • FIG. 103 TCell.001.1 demonstrated 99.163% efficient and 98.447% efficient non-specifically-targeted CRISPR-GFP Ribonucleoprotein uptake in viable CD4+ and CD8a+ subpopulations, respectively, of human primary Pan T-cells at 24 h post-transfection. Overall, 60.2% of CRISPR+ cells and 57.2% of overall cells were viable.
  • FIG. 104 TCell.001.2, a non-specifically-targeted PEGylated control, demonstrated 5.5% efficient and 6.9% efficient CRISPR-GFP Ribonucleoprotein uptake in viable CD4+ and CD8a+ subpopulations, respectively, of human primary Pan T-cells at 24 h post-transfection. Overall, 5.6% of CRISPR+ cells and 40.5% of overall cells were viable.
  • FIG. 105 TCell.001.3 demonstrated that homovalently-targeted sialoadhesin-derived peptides associated with CRISPR-GFP Ribonucleoprotein generate 11.6% and 13.2% efficient uptake in viable CD4+ and CD8a+ subpopulations, respectively, of human primary Pan T-cells at 24 h post-transfection. Overall, 40.0% of CRISPR+ cells and 79.2% of overall cells were viable.
  • FIG. 106 TCell.001.4 demonstrated that homovalently-targeted CD80-derived peptides associated with CRISPR-GFP Ribonucleoprotein generate 6.8% and 8.8% efficient uptake in viable CD4+ and CD8a+ subpopulations, respectively, of human primary Pan T-cells at 24 h post-transfection. Overall, 12.9% of CRISPR+ cells and 60.2% of overall cells were viable.
  • FIG. 107 TCell.001.5 demonstrated that homovalently-targeted CD80-derived peptides associated with CRISPR-GFP Ribonucleoprotein generate 10.3% and 10.9% efficient CRISPR-GFP Ribonucleoprotein uptake in viable CD4+ and CD8a+ subpopulations, respectively, of human primary Pan T-cells at 24 h post-transfection. Overall, 48.3% of CRISPR+ cells and 85.1% of overall cells were viable.
  • FIG. 108 TCell.001.6 demonstrated that homovalently-targeted CD86-derived peptides associated with CRISPR-GFP Ribonucleoprotein generate 1.7% and 2.9% efficient uptake in viable CD4+ and CD8a+ subpopulations, respectively, of human primary Pan T-cells at 24 h post-transfection. Overall, 6.8% of CRISPR+ cells and 69.1% of overall cells were viable.
  • FIG. 109 TCell.001.7 demonstrated that homovalently-targeted CD86-derived peptides associated with CRISPR-GFP Ribonucleoprotein generate 1.6% and 2.1% efficient uptake in viable CD4+ and CD8a+ subpopulations, respectively, of human primary Pan T-cells at 24 h post-transfection. Overall, 10.3% of CRISPR+ cells and 76.4% of overall cells were viable.
  • FIG. 110 TCell.001.8 demonstrated that homovalently-targeted CD86-derived peptides associated with CRISPR-GFP Ribonucleoprotein generate 14.5% and 16.0% efficient uptake in viable CD4+ and CD8a+ subpopulations, respectively, of human primary Pan T-cells at 24 h post-transfection. Overall, 39.1% of CRISPR+ cells and 76.3% of overall cells were viable.
  • FIG. 111 TCell.001.9 demonstrated that homovalently-targeted 4-1BB-derived peptides associated with CRISPR-GFP Ribonucleoprotein generate 3.6% and 3.2% efficient uptake in viable CD4+ and CD8a+ subpopulations, respectively, of human primary Pan T-cells at 24 h post-transfection. Overall, 27.5% of CRISPR+ cells and 87.8% of overall cells were viable.
  • FIG. 112 TCell.001.10 demonstrated that homovalently-targeted 4-1BB-derived peptides associated with CRISPR-GFP Ribonucleoprotein generate 5.8% and 5.4% efficient uptake in viable CD4+ and CD8a+ subpopulations, respectively, of human primary Pan T-cells at 24 h post-transfection. Overall, 30.8% of CRISPR+ cells and 84.2% of overall cells were viable.
  • FIG. 113 TCell.001.11 demonstrated that homovalently-targeted CD3-Ab-derived peptides associated with CRISPR-GFP Ribonucleoprotein generate 12.9% and 12.4% efficient uptake in viable CD4+ and CD8a+ subpopulations, respectively, of human primary Pan T-cells at 24 h post-transfection. Overall, 50.0% of CRISPR+ cells and 77.6% of overall cells were viable.
  • FIG. 114 TCell.001.12 demonstrated that homovalently-targeted CD3-Ab-derived peptides associated with CRISPR-GFP Ribonucleoprotein generate 9.0% and 9.5% efficient uptake in viable CD4+ and CD8a+ subpopulations, respectively, of human primary Pan T-cells at 24 h post-transfection. Overall, 38.9% of CRISPR+ cells and 80.7% of overall cells were viable.
  • FIG. 115 TCell.001.13 demonstrated that homovalently-targeted IL2-derived peptides associated with CRISPR-GFP Ribonucleoprotein generate 25.7% and 28.6% efficient uptake in viable CD4+ and CD8a+ subpopulations, respectively, of human primary Pan T-cells at 24 h post-transfection. Overall, 40.3% of CRISPR+ cells and 68.1% of overall cells were viable.
  • FIG. 116 TCell.001.14 demonstrated that homovalently-targeted IL2-derived peptides associated with CRISPR-GFP Ribonucleoprotein generate 24.9% and 25.8% efficient uptake in viable CD4+ and CD8a+ subpopulations, respectively, of human primary Pan T-cells at 24 h post-transfection. Overall, 45.9% of CRISPR+ cells and 70.1% of overall cells were viable.
  • FIG. 117 TCell.001.15, a dodecavalently-targeted 12-ligand variant, does not lead to endocytic uptake or CRISPR delivery. Overall, 59.8% of overall cells were viable.
  • FIG. 118 TCELL.001 Negative Controls. Representative results from one of 9 wells of negative (non-transfected) control. Overall, 81.4%, 84.7%, and 82.5% of total cells were viable 52 h after cell seeding (24 h post-transfection).
  • FIG. 119 Blood.002 attains 60%-97% mRNA delivery efficiency in the lymphocyte gate of whole human blood through utilizing a SIGLEC derivative for glycosylated cell surface marker targeting; shown is Cy5-tagged EGFP mRNA assayed via an Attune NxT flow cytometer.
  • Ligand targeting is a significant enhancer of cellular signal versus a PEGylated control. See above data for additional physicochemical properties predictive of nanoparticle behavior.
  • Blood.002 Control Untransfected.
  • Blood.002.88 CD45- and Neu5Ac-targeting SIGLEC derivative (cationic anchor-linker-ligand peptide added before anionic polymer).
  • Blood.002.89 CD45- and Neu5Ac-targeting SIGLEC derivative (cationic anchor-linker-ligand peptide added after anionic polymer).
  • Blood.002.90 PEGylated control (cationic anchor-PEG added before anionic polymer).
  • Blood.002.91 Non-specifically-targeted variant.
  • Blood.002.92 CD45- and Neu5Ac-targeting SIGLEC derivative without payload (anchor-linker-ligand is directly conjugated to anionic polymer, negative fluorescent control).
  • FIG. 120 TCell.001.27 demonstrated that homovalently-targeted SIGLEC-derived peptides direct 45% efficient Cy5 mRNA uptake in viable CD8a+ and CD4+ subpopulations of human primary Pan T-cells at 5 h post-transfection, as measured via flow cytometry.
  • the size and zeta potential of these particles demonstrated average particle sizes of 171 nm with zeta potentials of ⁇ 25.5+/ ⁇ 0.15 mV, indicating strong particle stability at a 1.35 carboxylate-to-phosphate (C:P) and 0.85 amine-to-phosphate ratio wherein poly(glutamic acid) is added following inclusion of the cationic anchor-linker-ligand.
  • C:P carboxylate-to-phosphate
  • 0.85 amine-to-phosphate ratio wherein poly(glutamic acid) is added following inclusion of the cationic anchor-linker-ligand.
  • Top-right bright field
  • middle-right Cy5 mRNA
  • bottom-right merge
  • Top bright field of negative control
  • bottom Cy5 channel of negative control.
  • FIG. 121 Rationale for Ribonucleoprotein and Protein Delivery.
  • Charge density plots of CRISPR RNP allow for determining whether an anionic or cationic peptide/material should be added to form a stable charged layer on the protein surface.
  • exposed nucleic acid (anionic) and anionic charge pockets serve as strong electrostatic anchoring sites for charged cations prior to addition of charged anions, or as their own ligand-linker anionic anchors.
  • Scale bar charge.
  • FIG. 122 Rationale for Ribonucleoprotein and Protein Delivery.
  • Charge density plots of Sleeping Beauty Transposons allow for determining whether an anionic or cationic peptide/material should be added to form a stable charged layer on the protein surface.
  • cationic charge pockets serve as strong electrostatic anchoring sites for charged anions, either as their own ligand-linker-anionic anchor domains, or prior to addition of charged cations. Scale bar: charge.
  • FIG. 123 (1) Exemplary anionic peptides (9-10 amino acids long, approximately to scale to 10 nm diameter CRISPR RNP) anchoring to cationic sites on the CRISPR RNP surface prior to (2) addition of cationic anchors as (2a) anchor-linker-ligands or standalone cationic anchors, with or without addition of (2b) subsequent multilayering chemistries, co-delivery of multiple nucleic acid or charged therapeutic agents, or layer stabilization through cross-linking.
  • anionic peptides (9-10 amino acids long, approximately to scale to 10 nm diameter CRISPR RNP) anchoring to cationic sites on the CRISPR RNP surface prior to (2) addition of cationic anchors as (2a) anchor-linker-ligands or standalone cationic anchors, with or without addition of (2b) subsequent multilayering chemistries, co-delivery of multiple nucleic acid or charged therapeutic agents, or layer stabilization through cross-linking.
  • FIG. 124 Rationale for Payload Co-delivery with Charged Protein Core Templates.
  • core templates which may include Cas9 RNP or any homogenously or zwitterionically charged surface.
  • a method for homogenizing the charge of a zwitterionic surface utilizing a variety of polymers is shown.
  • a ⁇ 10 nm core particle consisting of CRISPR-Cas9 RNP bound to gRNA is shown with zwitterionic domains.
  • a cationic polymer or anionic polymer may be added to homogenize the surface charge prior to addition of oppositely charged polymers.
  • Charged core template embodiments encompass any charged surface including a charged dendrimer or oligosaccharide-dendrimer, recombinant or synthetic histone dimer/trimer/tetramer/octamer, nanodiamond, gold nanoparticle, quantum dot, MRI contrast agent, or combination thereof with the above.
  • the negatively charged coating may be layered upon by with cationic polymer or anchor-linker-ligand, wherein the anchor is cationic.’ ‘amino sugar’. ‘charged glycosaminoglycan’. ‘pDNA’. ‘CODELIVERY’. ‘exposed gRNA’. ‘net negative sheddable polymer coat’. ‘glycan’. ‘cationic protein domain on cas9’. ‘ ⁇ 10 nm cas9 RNP’. ‘cationic protein domain on cas9’. ‘PLR’. ‘PDE (5-100)’. ‘PLE(5-100)’. ‘anionic protein domain on cas9’. ‘mRNA’. ‘branched cationic polymer on glycopeptide’. ‘histone’. ‘siRNA’.
  • the negatively charged coating may also be domain of an anionic anchor-linker-ligand or a standalone anionic matrix composition. Staggered mw of consistent polymers increases colloidal stability and gene editing efficiency.
  • FIG. 125 Peptide Engineering—Novel IL2-Mimetic Fragment for IL2R Targeting.
  • Interleukin-2 (left) bound to the Interleukin-2 Receptor (right) (PDB: 1Z92)
  • the sequence ASN(33)-PRO(34)-LYS(35)-LEU(36)-THR(37)-ARG(38)-MET(39)-LEU(40)-THR(41)-PHE(42)-LYS(43)-PHE(44)-TYR(45) is selected from IL2 (PDB 1Z92), correlating to the areas of active binding to the IL2 receptor alpha chain.
  • FIG. 126 PEPTIDE ENGINEERING—A Novel Antibody-Derived “Active Binding Pocket” Engineering Proof of Concept with CD3.
  • the sequence THR(30)-GLY(31)-ASN(52)-PRO(53)-TYR(54)-LYS(55)-GLY(56)-VAL(57)-SER(58)-THR(59)-TYR(101)-TYR(102)-GLY(103)-ASP(104) is selected from a CD3 antibody (PDB 1XIW), correlating to the areas of active binding to CD3 epsilon and delta chains.
  • PDB 1XIW CD3 antibody
  • the order of the amino acids is rearranged in order to reflect binding kinetics of a 2-dimensional plane of peptides in the binding pocket which no longer have tertiary structure maintained by the larger protein.
  • This dimensional reduction results in: THR(59)-SER(58)-VAL(57)-GLY(56)-LYS(55)-TYR(54)-PRO(53)-ASN(52)-THR(30)-GLY(31)-TYR(101)-TYR(102)-GLY(103)-ASP(104).
  • FIG. 127 PEPTIDE ENGINEERING—A Novel SIGLEC Derivative for CD45 Glycosylation Targeting.
  • a sialoadhesin fragment proximal to sialoadhesin in the rendering was utilized for targeting glycosylated CD45 and other complex cell-surface glycoproteins. It generates successful targeting of T-cells with CRISPR RNP in TCELL.001.3, as well as mRNA in whole blood lymphocyte gates in BLOOD.002.1-BLOOD.002.2.
  • the sequence for the ligand is SNRWLDVK (SEQ ID NO: xx).
  • FIG. 128 PEPTIDE ENGINEERING—A Novel SCF Fragment for c-Kit Targeting. Dashed circles—signal peptide domains of Stem Cell Factor (RCS PDB 1SCF) represent dimeric domains necessary for c-Kit activity. Effect of ligand presentation on cellular uptake due to particular nanoparticle surface size+ SCF coating densities can be compared and contrasted between CynoBM.002.79 ( ⁇ 5% efficiency) and CynoBM.002.85 ( ⁇ 56% efficiency).
  • RCS PDB 1SCF Stem Cell Factor
  • a contrast is displayed with qualitative imagery of human CD34+ hematopoietic stem cell transfections, where E-selectin+ SCF Fragment (HSC.004.73) achieves high efficiencies, but the SCF Fragment on its own does not (HSC.004.74).
  • the marked difference in behavior is suggestive of a particular role of the dimeric peptide in generating endocytic cues and subsequent nuclear targeting of nucleic acid and/or ribonucleoprotein materials.
  • the sequence for the ligand is EKFILKVRPAFKAV (SEQ ID NO: xx) (mSCF); and EKFILKVRPAFKAV (SEQ ID NO: xx) (rmSCF).
  • FIG. 129 PEPTIDE ENGINEERING—A Novel cKit Receptor Fragment for Membrane-Bound SCF Targeting. Rational design of a stem cell factor targeting peptide derived from c-Kit to mimic behavior of hematopoietic stem cell rolling behavior on endothelial and bone marrow cells and increase systemic transfection efficiency (see CynoBM.002.80). Sequence evaluated for folding: Name SCFN, Sequence: RRRRRRRRRGGGGSGGGGSEGICRNRVTNNVKDVTKLVANLPK (SEQ ID NO: xx). Sequences were evaluated with Rosetta and NAMD simulation packages—Rosetta Results: A shortened sequence was placed into Rosetta for ab initio folding (GGSEGICRNRVTNNVKDVTKLVANLPK)(SEQ ID NO: xx).
  • FIG. 130 PEPTIDE ENGINEERING—cKit Receptor Fragment (Continued). Molecular dynamics simulations with anchor segment of anchor-linker-ligands held in place to allow for simulating entropically favorable conformation as would be presented on the nanoparticle surface. Each result contains the same scoring factor which means it's difficult to determine if any of these structures would be preferred. Also Rosetta does not do folding dynamics so it is highly possible that these sequences will not fold into a helix-like structure.
  • NAMD results Because Rosetta doesn't do folding dynamics, it was checked if the full sequences would quickly fold into a secondary structure. Simulations were performed in NAMD using replica exchange molecular dynamics (REMD) on 16 or 32 replicas between 300-500 K and simulated to 10 ns on each replica. The anchor section (poly-R) was fixed as linear to simulate bound protein to particle. Lowest energy snapshots are shown.
  • REMD replica exchange molecular dynamics
  • FIG. 131 PEPTIDE ENGINEERING—cKit Receptor Fragment (Continued). Stabilization of a random coiled peptide with strong ligand-linker self-folding into a stable helical peptide for effective ligand presentation through modification of key hydrophobic domains with amino isobutyric acid.
  • Blue chains represented a more ordered helix present in KIT, ranging from residues 71 to 94:SNYSIIDKLVNIVDDLVECVKENS.
  • FIG. 132 PEPTIDE ENGINEERING—cKit Receptor Fragment (Continued)
  • Full anchor-linker-KIT7194_AIB1 construct RRRRRRRRR-GGGGSGGGGS-SNYS AibADK AibANAibA DD AibAEAibAKENS.
  • FIG. 133 PEPTIDE ENGINEERING—cKit Receptor Fragment. Stable conformation of SCF_mcKit(Ac)_(4GS)2_9R_N following modification of key hydrophobic residues with amino isobutyric acid.

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