Engineered nanoparticles for targeted internalization by human blood cells

Abstract

Engineered nanoparticles for genetic therapies in human blood cells are described and can be used to safely and efficiently treat a variety of genetic, infectious, and malignant diseases. Targeting of human stem cells is achieved through the binding of CD133, CD117, CD90, with linked targeting moieties that result in rapid internalization of the engineered nanoparticles following binding.

Description

F053-6005PCT / 24-085-WO-PCT ENGINEERED NANOPARTICLES FOR TARGETED INTERNALIZATION BY HUMAN BLOOD CELLS CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 549,350 filed February 2, 2024, which is incorporated herein by reference in its entirety as if fully set forth herein. REFERENCE TO SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is provided in XML format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the file containing the Sequence Listing is F053-6005PCT_Seq.xml. The file is 201,046 bytes, was created February 3, 2025, and is being submitted electronically via Patent Center. FIELD OF THE DISCLOSURE

[0003] The current disclosure provides engineered nanoparticles for targeted internalization by human blood cells, such as human stem cells and progenitor cells. The disclosed targeted nanoparticles can be used to safely and efficiently treat a variety of genetic, infectious, and malignant diseases. BACKGROUND OF THE DISCLOSURE

[0004] Gene therapy has great potential to treat genetic, infectious, and malignant diseases. For example, retrovirus-mediated gene addition into hematopoietic stem cells (HSC) and hematopoietic stem and progenitor cells (HSPC) has demonstrated curative outcomes for several genetic diseases including inherited immunodeficiencies (e.g., X-linked and adenosine deaminase deficient severe combined immunodeficiency (SCID)), hemoglobinopathies, Wiskott- Aldrich syndrome and metachromatic leukodystrophy. Additionally, this treatment approach has also improved outcomes for poor prognosis diagnoses such as glioblastoma.

[0005] Despite the potential of gene therapy, its success depends largely on the efficient and selective delivery of gene editing components to target cell types. Nanoparticles, such as polyplexes and lipoplexes, have been proposed as a method to simplify the delivery of all gene- editing components to cells. For instance, WO2020 / 118110 provides nanoparticles that provide high gene editing efficiency. These nanoparticles include layered gene-editing reagents loaded onto a nanoparticle core. However, for gene therapy to be more therapeutically effective, the nanoparticles need to be specifically and efficiently delivered to the target cells or tissues. One method of targeted delivery is the use of targeting moieties that specifically bind antigens on the target cells.F053-6005PCT / 24-085-WO-PCT

[0006] While the use of targeting moieties to direct therapeutic agents to specific cell types has been established, translating this strategy into therapeutically effective nanoparticle delivery systems is not straightforward. For instance, two targeting moieties that bind the same antigen with similar binding efficiencies may be associated with different nanoparticle performance, such as cellular uptake and / or gene editing efficacy. The difference in performance may be due to interactions between targeting moieties and gene-editing components, changes in the binding kinetics of the targeting moiety after nanoparticle conjugation (e.g., due to structural changes of the targeting moiety), or how the targeting moiety is conjugated to the nanoparticle (e.g., by direct conjugation, by a particular linker, etc.). Furthermore, the amount of targeting moieties on a nanoparticle can affect targeting efficacy. While increasing the number of targeting moieties on a nanoparticle can improve interaction with the targeted cell type, too many targeting moieties can lead to aggregation of the nanoparticles by promoting crosslinking between the particles. Aggregation can compromise nanoparticle stability and hinder efficient delivery. Too few targeting moieties can result in insufficient binding to the target cells, thereby reducing therapeutic efficacy. In addition, experimental conditions (e.g., pH, salt concentration) during nanoparticle synthesis can affect the stability and interactions between various components of the nanoparticle, presenting further challenges in engineered targeted nanoparticles for gene therapy. SUMMARY OF THE DISCLOSURE

[0007] Implementations described herein provide nanoparticles and synthesis methods thereof that address challenges related to selective and efficient delivery of gene-editing reagents to targeted cell types. In particular embodiments, nanoparticles including the targeting moiety and gene-editing components have a modification on the outermost surface providing endosomal escape and targeting moiety addition for delivery to a select cell type. In particular embodiments, the targeting moiety can be attached to an endosomal polymer shell via a heterofunctional chemical tether using bioinert linkers such as a poly(ethylene glycol) (PEG) or an oligo(ethylene glycol) (OEG) chain. The addition of the targeting moiety provides enhanced delivery of the nanoparticle payload to the select cell type, specifically via internalization. In particular embodiments, the targeting moiety is linked to the surface of the nanoparticle through a linker.

[0008] In particular embodiments, targeting moiety linking chemistry uses a PEG spacer based hetero-functional linker. In certain examples, three chemical schema can be used and altered to link to the targeting moiety to the nanoparticle on either end as all rely on two groups for attachment, primary amines (N-hydroxysuccinimide (NHS) / trifluoromethylphenyl (TFP) ester chemistry) and sulfhydryls (maleimide chemistry).

[0009] Chemical schema 1 uses primary amine reactive groups to target both the targeting moietyF053-6005PCT / 24-085-WO-PCT and the nanoparticle. This attaches either click chemistry (e.g., dibenzocyclooctyne (DBCO) to azide strain promoted azide-alkyne click (SPAAC) or other SPAAC or copper-catalyzed azide- alkyne click (CuAAC) based reactions. Alternative linkers, such as biotin-streptavidin with similar covalent-like modifications can also be used under this schema.

[0010] The second schema involves reduction of the targeting moiety to expose thiols for maleimide reaction. This attaches a click chemical or other linker to the targeting moiety in a site selective manner, increasing targeting moiety activity retention post modification.

[0011] The third schema can use enzymatic reactions to attach azide to a targeting moiety (or other galactose carbohydrate modified protein / group) for further click chemistry.

[0012] Using these methods, active groups for conjugation can be attached to the nanoparticle through modification of either the targeting moiety or the nanoparticle in any order.

[0013] In particular embodiments, a linker length is 4 or 12 PEG repeats for use as a spacer (e.g., 2-5 nm in length).

[0014] Particular embodiments utilize a DBCO-PEG4-TFP ester on the nanoparticle, and enzymatic azide conjugation onto targeting moieties.

[0015] The pH used for modification can be based on the surface charge of the nanoparticle being conjugated to, with a reduction to pH 6.0 being preferred for a 2.1 generation nanoparticle using Cas9.

[0016] Particular embodiments utilize 500-1000 linkers per nanoparticle. Further, free linkers should be quenched before nanoparticle purification, for example by adding excess free azide. Failure to quench can result in hydrophobic / crosslinking aggregation during nanoparticle.

[0017] The engineered nanoparticles can be used for therapies where a loss-of-function mutation is needed, but importantly, can also provide components needed for gene addition or correction of a specific mutation. The described approaches are safe (e.g., limited to no off-target toxicity), reliable, scalable, easy to manufacture, synthetic, plug-and-play (e.g., the same basic platform can be used to deliver different therapeutic nucleic acids), and compatible with routine in vivo administration (through, for example, a syringe). The nanoparticles can also be used in ex vivo cell manufacturing. BRIEF DESCRIPTION OF THE FIGURES

[0018] Some of the drawings submitted herewith may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0019] FIGs.1A, 1B. (1A) Proportion of hCD45+ cells in murine peripheral blood post cord blood humanization in mice (NSGW) by flow analysis. Cells were stained for murine and human CD45F053-6005PCT / 24-085-WO-PCT and gated for live and singlet cells. (n=12) (1B) Inductively coupled plasma mass spectrometery (ICP-MS) derived gold concentration after aqua regia (NO3:HCl) microwave digestion of femurs post-necropsy using targeting group conjugated (aCD117 / 133) AuNP, quantified as percent injected dose. (n=3, ***p<0.001).

[0020] FIGs. 2A-2D. Schematic description of general CRISPR gold nanoparticle (AuNP) for hematopoietic stem and progenitor cell editing. (2A) CRISPR AuNP have distinct active chemical groups required fully or in part for cellular editing. (2B) Nanoparticle synthesis pathways covering two generation schema to achieve CRISPR AuNP. (2C) HSPCs (isolated by CD34+ positive selection by FHCC Center for Excellence in Hematology) in vitro treatment methodology. Scientific readouts primarily covering gene editing via Sanger sequencing and TIDE analysis, as well as cellular viability using a Countess II (Invitrogen).

[0021] FIG.3. (Left) Diagram description of micropinocytosis / passive internalization of CRISPR AuNP in generic cells. (Right) Occurrence of this untargeted mechanism in all cells challenges the uptake in the low circulating fraction HSPCs.

[0022] FIG.4. The bone marrow fraction of HSPCs is the highest of all tissues, being their primary biological niche. Interosseous delivery can improve uptake into desired cells, however, off target cell numbers remain high, limiting efficacy of passive uptake.

[0023] FIG.5. Diagram of a nanoparticle and targeted HSPCs with surface markers for specific, activated internalization. Targeting group represented as an antibody. Additional examples of rapidly endocytosing groups include aptamers and growth factors (e.g., stem cell factor (SCF) cytokine).

[0024] FIGs.6A-6C. Active uptake qualitative confocal analysis for HSPC targeting moieties. (6A) Experimental schematic for confocal based analysis in HSPCs after 3 hrs of internalization of select targeting groups. Requiring poly L-lysine to maintain stable imaging of normally suspended cells. (6B) Targeting groups tested and not selected for further investigation. An antibody targeting CD34 acted as a negative, non-internalizing control. Rejected targeting groups either showed poor affinity (ex: aCD90:DG3), surface binding only (ex: aCD133:293C3) or were outcompeted by other clones (ex: aCD133:AC141 vs aCD133:Clone7). (6C) Identification of strong uptake agents including the IgGs: anti-human CD90:5E10, CD117:104D2 and CD133:clone7. CD34 included as a negative uptake control.

[0025] FIG. 7. Purification schema of HSPCs from whole bone marrow for specificity quantification using flow cytometry. Process isolates nucleated white blood cells (including HSPCs and other differentiated progenitor / terminal cells) for targeting group competition.

[0026] FIG.8. Flow cytometry gating strategy for isolating HSPCs (CD34+,CD38low) labeled forF053-6005PCT / 24-085-WO-PCT targeting group from total white blood cells (WBCs).

[0027] FIG.9. Flow cytometry analysis of targeting group positive total white blood cell fraction vs HSPC fraction. (δ=DNA aptamer, λ=growth factor, α=antibody targeting moiety).

[0028] FIG. 10. Ratiometric fraction of HSPC specific binding vs total WBS binding. While all targeting groups bind HSPCs strongly, only antibodies reveal a 10-100x enhancement in specificity.

[0029] FIG.11. Cartoon depiction of electrostatic anchoring to produce stable antibody-CRISPR AuNP. This method was the method used in FIG.1B.

[0030] FIG.12. The effect of differing linker lengths on CRISPR-AuNP stability is illustrated by dynamic light scatter (DLS, Zetasizer nano ZS), revealing a slight decrease in Z-average diameter and a stable PDI below 0.3 (0 is perfectly sized / monodisperse, 1 perfect chaos in particle size). (n=3).

[0031] FIGs.13A, 13B. DLS stability of AuNP after antibody (aCD117) conjugation using varying linker lengths and differing pH for conjugation. Particles were purified of free linkers and antibodies via centrifugation and resuspended into pH 7.4 HEPES buffer before analysis (n=3). (13A) Z-average diameter remains stable after purification, excluding the largest spacer length and lowest pH. This low pH is a restriction of the Cas9 nuclease stability, being a key design parameter for surface modified gold nanoparticles. (13B) The PDI post antibody purification shows at or near 0.35 PDI limit, with increasing proximity to Cas9 instability (pH 5.0) revealing an increase in interparticle variability.

[0032] FIGs. 14A, 14B. Effect of pH and linker length on AuNP particle size and antibody modification level. (14A) DLS determined size of particles, (14B) Denaturing poly-acrylamide gel electrophoresis (SDS-PAGE) using linker length of 4 at pH 6 (the selected pH above). The relative modified fraction of heavy chain bound to Cas9 vs the antibody’s light chain, used as a loading control.

[0033] FIGs. 15A-15C. SDS-PAGE fluorescent quantification of linker bound RNP, PEI, or full AuNP with a dye or antibody. Conjugations were carried out using Cyanine3-Azide and aCD117- Alexa647 as reporters. (15A) A representative image of free Cas9 to dye linking at varying linker ratios (In order from Lane 6-12; 200, 50, 20, 10, 4, 2, 0). Higher linker ratios reveal over conjugation results in poor solubility (wells 6 and 7, upper band). (15B) Beta mercaptoethanol released CRISPR AuNP after Ab binding under differing pH / PEG spacer linking conditions. Low pH increases the conjugation ratio of antibody, however, below pH 5, insolubility occurs. Additionally, the shorter spacer length (PEG4) produced equal or higher conjugation to longer lengths. Wells 2 and 8 contained a non-fluorescent antibody. (15C) Quantified fraction ofF053-6005PCT / 24-085-WO-PCT conjugation between CRISPR AuNP surface bound Cas9 and fluorescently modified Ab. Note: 0 represents purely electrostatic antibody binding.

[0034] FIG. 16. Schematic of local intraosseous injection utilized for FIG 1B. Mice were humanized within 48 hrs after birth using 150 cGy γ-rays in a cesium-137 irradiator, followed by injection of cord derived CD34+ cells. After 12 wk of engraftment tracking in peripheral blood (2 wk intervals) the mice were injected with CRISPR AuNP derivatives. The mice were then tracked biweekly over 12 weeks for peripheral blood editing levels by sanger sequencing, for acute toxicity via behavioral observation and weight, then necropsy for tissue editing and biodistribution.

[0035] FIG. 17. Cutting efficiency changes using differing trifluoromethylphenyl (TFP) ester- polyethylene glycol (PEG)4- dibenzocyclooctyne (DBCO) linker ratios. Linker modification shows decreased activity of free (non-AuNP bound) Cas9 targeting hB2M DNA fragments (250 bp of Exon 2 DNA) in an in vitro DNA cutting assay. (n=3).

[0036] FIG.18. Linker addition to RNP on Au surface holds trend under the conditions as FIG. 17. Overall cutting was decreased in this assay due to poor release with dithiothreitol (DTT) vs β- mercaptoethanol (βME). (n=1)

[0037] FIG.19. Depiction of key covalent biding parameters to an AuNP coated with RNP alone. (Left) An RNP only surface is highly negatively charged repulsing the antibody, this repulsion can be reduced through pH decrease or the addition of salt. (Right) The length of the PEG spacer within a TFP-DBCO heterolinker can increase the binding distance, thereby reducing steric interference and charge repulsion effects.

[0038] FIG.20. Chemical groups utilized in strain promoted azide-alkyne cycloaddition chemistry (SPAAC) based conjugation between the TFP-PEGx-DBCO linker and the enzymatically functionalized antibody-azide. (Top) DBCO-PEG4-TFP, (Middle) Azide modified antibody using the SiteClick Azido Modification Kit. (Bottom) 1,2,3-triazole formation during click reaction.

[0039] FIG.21. Antibody conjugation at 100 linkers per Au core for CRISPR AuNP, quantification by fluorescence tracking of aCD117-A647-Azide. Detection level near noise floor. (n=2)

[0040] FIG. 22. DLS melting curves measuring Cas9 RNP’s thermal stability in differing pH solutions (25 mM Citrate buffer). Shift in melting temperature to lower level at lower pH reveals the instability of RNP at lower pH.

[0041] FIG. 23. Effect of both reduced pH and linker length on the retention of trRNA, a major component in Cas9 RNP activity. The loss of this group strongly represents loss of activity of Cas9 on particle surface. Loss begins to occur at pH 6, increasing with decreasing pH.

[0042] FIG.24. Zetapotential measurements in pH 7.4 HEPES at 10 mM of AuNP modified with varying amounts of PEI, representing the charge variation.F053-6005PCT / 24-085-WO-PCT

[0043] FIG 25. Antibody per Au core on a CRISPR-AuNP, determined by fluorescence standard curve analysis from antiCD117-Alexa647 after conjugation. The CRISPR-AuNP contain PEI so representative conjugation does not distinguish between PEI and / or Cas9 binding. (n=2)

[0044] FIG.26. Sequences supporting the disclosure. DETAILED DESCRIPTION

[0045] Gene therapy has great potential to treat genetic, infectious, and malignant diseases. For example, retrovirus-mediated gene addition into hematopoietic stem cells (HSC) and hematopoietic stem and progenitor cells (HSPC) has demonstrated curative outcomes for several genetic diseases including inherited immunodeficiencies (e.g., X-linked and adenosine deaminase deficient severe combined immunodeficiency (SCID)), hemoglobinopathies, Wiskott- Aldrich syndrome and metachromatic leukodystrophy. Additionally, this treatment approach has also improved outcomes for poor prognosis diagnoses such as glioblastoma.

[0046] While the use of targeting moieties to direct therapeutic agents to specific cell types has been established, translating this strategy into effective nanoparticle delivery systems is not straightforward. For instance, two targeting moieties that bind the same antigen may be associated with different nanoparticle performance, such as cellular uptake and / or gene editing efficacy. The difference in performance may be due to interactions between targeting moieties and gene-editing components, changes in the binding kinetics of the targeting moiety after nanoparticle conjugation (e.g., due to structural changes of the targeting moiety), or how the targeting moiety is conjugated to the nanoparticle (e.g., by direct conjugation, by a particular linker, etc.). Furthermore, the amount of targeting moieties on a nanoparticle can affect targeting efficacy. While increasing the number of targeting moieties on a nanoparticle can improve interaction with the targeted cell type, too many targeting moieties can lead to aggregation of the nanoparticles by promoting crosslinking between the particles. Aggregation can compromise nanoparticle stability and hinder efficient delivery. Too few targeting moieties can result in insufficient binding to the target cells, thereby reducing therapeutic efficacy. In addition, experimental conditions (e.g., pH, salt concentration) during nanoparticle synthesis can affect the stability and interactions between various components of the nanoparticle, presenting further challenges in engineered targeted nanoparticles for gene therapy.

[0047] Implementations described herein provide nanoparticles and synthesis methods thereof that address challenges related to selective and efficient delivery of gene-editing reagents to targeted cell types. In particular embodiments, nanoparticles including the targeting moiety and gene-editing components have a modification on the outermost surface providing endosomal escape and targeting moiety addition for delivery to a select cell type. In particular embodiments,F053-6005PCT / 24-085-WO-PCT the targeting moiety can be attached to an endosomal polymer shell via a heterofunctional chemical tether using bioinert linkers such as a poly(ethylene glycol) (PEG) or an oligo(ethylene glycol) (OEG) chain. The addition of the targeting moiety provides enhanced delivery of the nanoparticle payload to the select cell type, specifically via internalization. In particular embodiments, the targeting moiety is linked to the surface of the nanoparticle through a linker.

[0048] In particular embodiments, targeting moiety linking chemistry uses a PEG spacer based hetero-functional linker. In certain examples, three chemical schema can be used and altered to link to the targeting moiety to the nanoparticle on either end as all rely on two groups for attachment, primary amines (N-hydroxysuccinimide (NHS) / trifluoromethylphenyl (TFP) ester chemistry) and sulfhydryls (maleimide chemistry).

[0049] Chemical schema 1 uses primary amine reactive groups to target both the targeting moiety and the nanoparticle. This attaches either click chemistry (e.g., dibenzocyclooctyne (DBCO) to azide strain promoted azide-alkyne click (SPAAC) or other SPAAC or Copper-catalyzed Azide- alkyne click (CuAAC) based reactions.

[0050] SPAAC refers to a Cu-free variation of click chemistry that is generally biocompatible with cells. In particular embodiments, SPAAC utilizes a substituted cyclooctyne having an internal alkyne in a strained ring system. Ring strain together with electron-withdrawing substituents in the cyclooctyne promote a [3+2] dipolar cycloaddition with an azide functional group. SPAAC can be used for bioconjugation and crosslinking by attaching azide and cyclooctyne moieties to molecules. For a description of SPAAC, see, e.g., Baskin et al. (2007) Proc Natl Acad Sci USA 104(43):16793-16797, Agard et al. (2006) ACS Chem. Biol.1: 644-648, Codelli et al. (2008) J. Am. Chem. Soc. 130:11486-11493, Gordon et al. (2012) J. Am. Chem. Soc. 134:9199-9208, Jiang et al. (2015) Soft Matter 11(30):6029-6036, Jang et al. (2012) Bioconjug Chem. 23(11):2256-2261, Ornelas et al. (2010) J Am Chem Soc.132(11):3923-3931.

[0051] Heterobifunctional crosslinking agents can be used to attach suitable azide and alkyne moieties to molecules for performing SPAAC. In particular, reactions with N-hydroxysuccinimide (NHS) can be used for bioconjugation of proteins, which have multiple primary amines available as targets for coupling with NHS-activated reagents. Exemplary alkyne-NHS-crosslinker agents include dibenzycyclooctyne-N-hydroxysuccinimide (DBCO-NHS), bicyclononyne-N- hydroxysuccinimide (BCN-NHS), and dibenzocyclooctyne-sulfo-N-hydroxysuccinimide (DBCO- sulfo-NHS). Exemplary azide-NHS crosslinker agents include azide-polyethylene glycol (PEG)- NHS crosslinkers with PEG polymers of various lengths (azide-PEG n-NHS). The length of the PEG polymer can be used to control the spacing between the NHS and azide moieties. The PEG spacer arms may range from one PEG unit to many PEG units in length (generally 3-25 units).F053-6005PCT / 24-085-WO-PCT The spacer arm may also include other types of chemical backbones, such as an aliphatic backbone. Heterobifunctional crosslinking agents suitable for performing SPAAC are commercially available from a number of companies, including JenKem Technology USA (Plano, Tex.), Sigma-Aldrich, Inc. (St. Louis, Mo.), BroadPharm (San Diego, Calif.), Quanta BioDesign (Plain City, Ohio), Thermo Fisher Scientific Inc. (Waltham, Mass.), and Nanocs Inc. (New York, N.Y.).

[0052] In some embodiments, the azide-alkyne cycloaddition reaction is copper-catalyzed azide- alkyne cycloaddition (CuAAC). In particular embodiments, a CuAAC reaction includes a copper reaction reagent, and may contain a reducing reagent and an accelerating ligand. The copper reaction reagent catalyzes the activation of the alkyne-labeled molecule for combination with the azide-labeled molecule. Examples of copper reaction reagents include copper sulfate (CuS04), tetrakis(acetonitrile)copper(I)hexafluorophosphate ((Cu(CH3CN4)PF6), tetrakis(acetonitrile)copper(I) triflate ((Cu(CH3CN)4OTf) copper acetate (C4H6Cu04), copper bromide (BrCu), and copper iodide (Cul).

[0053] In particular embodiments, a reducing reagent catalyzes the reduction of Cu(II) to Cu(I). Examples of reducing reagents include sodium ascorbate (CekFNaOe), hydrazine (N2H4), tris(2- carboxyethyl)phosphine (TCEP), dithiotreitol (DTT), and beta-mercaptoethanol.

[0054] In particular embodiments, an accelerating ligand increases the speed of the reaction and also protects the molecules from oxidation. Examples of accelerating ligands include 2-[4-({bis [(-tert-butyl- lH-1,2,3 triazol-4-yl)methyl] amino} methyl)-H-1,2, 3-triazol-l-yl] acetic acid (BTTAA), (l-(4- methoxybenzyl)-l-H-l,2,3-triazol-4-yl)methanol (MBHTM), and tris-hydroxypropyltriazolylmethylamine (THPTA).

[0055] Alternative linkers, such as biotin-streptavidin with similar covalent-like modifications can also be used under this schema.

[0056] The second schema involves reduction of the targeting moiety to expose thiols for maleimide reaction. This attaches a click chemical or other linker to the targeting moiety in a site selective manner, increasing targeting moiety activity retention post modification.

[0057] Sulfhydryls, also called thiols, exist in proteins in the side-chain of cysteines. Pairs of cysteine sulfhydryl groups are often linked via disulfide bonds (–S–S–) within or between polypeptide chains and contribute to forming a protein structure. Typically, only free or reduced sulfhydryl groups (–SH) [rather than sulfur atoms in disulfide bonds] are available for reaction with thiol-reactive compounds. Examples of sulfhydryl-reactive chemical groups include haloacetyls, maleimides, aziridines, acryloyls, arylating agents, vinyl sulfones, pyridyl disulfides, TNB-thiols and disulfide reducing agents.

[0058] Particular embodiments utilize methods for crosslinking that include thiol-ene click chemistry. In particular embodiments, conjugation using thiol-ene click chemistry involves reacting a thiol group with an alkene group via Michael addition. The thiol-ene click reaction canF053-6005PCT / 24-085-WO-PCT be optionally augmented by light (i.e., photo-click reaction). For a description of the use of thiol- ene click chemistry for crosslinking, see, e.g., Grim et al. (2015) J. Control Release 219:95-106; Scanlan et al. (2014) Molecules 19(11):19137-151; Hoyle et al. (2010) Angew Chem. Int. Ed. Engl.49(9):1540-1573; van Dijk et al. (2009) Bioconjug Chem.20(11):2001-2016.

[0059] The third schema can use enzymatic reactions to attach azide to a targeting moiety (or other galactose carbohydrate modified protein / group) for further click chemistry.

[0060] Using these methods, active groups for conjugation can be attached to the nanoparticle through modification of either the targeting moiety or the nanoparticle in any order.

[0061] In particular embodiments, a linker length is 4 or 12 PEG repeats for use as a spacer (e.g., 2-5 nm in length).

[0062] Particular embodiments utilize a DBCO-PEG4-TFP ester on the nanoparticle, and enzymatic azide conjugation onto targeting moieties.

[0063] The pH used for modification can be based on the surface charge of the nanoparticle being conjugated to, with a reduction to pH 6.0 being preferred for a 2.1 generation nanoparticle using Cas9.

[0064] Particular embodiments utilize 500-1000 linkers per nanoparticle. Further, free linkers should be quenched before nanoparticle purification, for example by adding excess free azide. Failure to quench can result in hydrophobic / crosslinking aggregation during nanoparticle.

[0065] Implementations described herein provide nanoparticles and synthesis methods thereof that address challenges related to selective and efficient delivery of gene editing reagents to human blood cells. Implementations described herein provide targeting moieties that can be attached to nanoparticles and effectively bind to target cell types resulting in internalization of the nanoparticle by the target cell type. For instance, the present disclosure describes identification of antibody clones targeted to a particular antigen that show increased binding and internalization to target cell types compared to other antibody clones targeted to the same particular antigen. Implementations described herein provide linkers that can attach targeting moieties to nanoparticles without compromising the functionality of the gene-editing components loaded on the nanoparticle or of the targeting moiety. In particular embodiments, a linker includes a poly(ethylene glycol) (PEG) spacer with 4 to 12 ethylene glycol units. In particular embodiments, the linker is attached to a azide group or a thiol group on the targeting moiety. In particular embodiments, the targeting moiety is modified (e.g., by an enzymatic reaction or by a reduction) to display the azide group or the thiol group. In particular embodiments, the linker is attached to an amine group of a gene-editing component. Implementations described herein provide amounts of linkers and / or targeting moieties that promote effective binding to and internalization by targetF053-6005PCT / 24-085-WO-PCT cells while avoiding crosslinking or aggregation between nanoparticles. In particular embodiments, 250 to 1500 linkers are attached to each nanoparticle. In particular embodiments, 400 to 1200 linkers are attached to each nanoparticle. In particular embodiments, 500 to 1000 linkers are attached to each nanoparticle. In particular embodiments, the linker is attached at a ratio of 500 to 1000 linkers per nanoparticle to overcome steric restriction of the surface and lower diffusion rates. In particular embodiments, the linker is attached at a ratio of 1000 linkers per nanoparticle. In particular embodiments, the pH is equal to the isoelectric point of the targeting moiety during attachment of the linker and / or the targeting moiety to the nanoparticle. In particular embodiments, the pH is above 5.5 during attachment of the linker and / or the targeting moiety to the nanoparticle to stabilize the gene-editing components (e.g., the nuclease). In particular embodiments, unbound linkers are quenched (e.g., by adding excess azide) after attachment of the linker and / or the targeting moiety to avoid hydrophobic and / or crosslinking aggregation and retain nanoparticle functionality. The current disclosure provides significant advances in the ability to perform genetic therapies for a variety of genetic, infectious, and malignant diseases by using nanoparticles engineered for endosomal uptake into blood stem cells.

[0066] Nanoparticles described herein include targeting moieties that are used to target the nanoparticle to a specific cell type within a population of cells so that activity of the gene editing system can be controlled. For example, the activity and destination of the gene editing system may be controlled by a targeting moiety that selectively delivers the nanoparticle to a selected blood stem cell type (also referred to as targeted cell type). In particular embodiments, a targeting moiety can include a protein, an aptamer, or small molecule. In particular embodiments, the protein includes an antibody, a peptide, or a ligand. The targeting moiety directs the nanoparticle to a select cell type. In particular embodiments, the select cell type includes a blood stem and progenitor cell. In particular embodiments, the selected cell type includes a hematopoietic stem cell (HSC). In particular embodiments, the selected cell type includes a hematopoietic stem and progenitor cell (HSPC). In particular embodiments, the targeting moiety binds CD133, CD117, or CD90. In particular embodiments, the antibody includes an anti-CD133 antibody, an anti-CD117 antibody, and / or an anti-CD90 antibody. In particular embodiments, the antibody includes αCD133:7, αCD117:104D2 and / or αCD90:5E10. In particular embodiments, the aptamer includes an anti-CD133 aptamer. In particular embodiments, the anti-CD133 aptamer includes A15 and / or B19. In particular embodiments, the ligand includes stem cell factor (SCF). In particular embodiments, the protein includes human luteinizing hormone. In particular embodiments, the small molecule includes degarelix acetate.

[0067] In particular embodiments, the gene-editing components include a cutting element,F053-6005PCT / 24-085-WO-PCT targeting element, and / or a donor template. In particular embodiments, the donor template includes dsDNA. In particular embodiments, the donor template includes ssDNA. In particular embodiments, the donor template includes a dsDNA template including a therapeutic gene and / or homology arms to a target sequence.

[0068] Nanoparticles including the targeting moiety and gene-editing components have a modification on the outermost surface providing endosomal escape and targeting moiety addition for delivery to a select cell type. In particular embodiments, the targeting moiety can be directly attached to a cutting element (e.g., nuclease) of the gene-editing components or attached to an endosomal polymer shell via a heterofunctional chemical tether using bioinert linkers such as a polyethylene glycol (PEG) or oligoethylene glycol (OEG) chain. The addition of the targeting moiety provides enhanced delivery of the nanoparticle payload to the select cell type, specifically via endosomal uptake. In particular embodiments, the targeting moiety is linked to the surface of the nanoparticle through a linker. In particular embodiments, nanoparticles are internalized into target cells in less than 12 hours following administration to a subject. In particular embodiments, nanoparticles are internalized into target cells in less than 10 hours, less than 8 hours, or less than 5 hours following administration to a subject. In particular embodiments, nanoparticles are internalized into target cells in less than 3 hours following administration to a subject. In particular embodiments, administration is intraosseous.

[0069] Aspects of the current disclosure are now described in more supporting detail as follows: (I) Targeting Moieties; (II) Nanoparticles; (III) Targeted Gene Editing Systems; (IV) Conjugation of Components to Nanoparticles; (V) Nanoparticle Compositions and Cell Formulations; (VI) Methods of Use; (VII) Reference Levels Derived from Control Populations; (VIII) Kits; (IX) Exemplary Embodiments; (X) Experimental Examples; and (XI) Closing Paragraphs. These headings are provided for organizational purposes only and do not limit the scope or interpretation of the disclosure.

[0070] (I) Targeting Moieties. Targeting moieties can be used to target the nanoparticle to a specific cell so that activity of the gene editing system can be controlled. For example, the activity and destination of the gene editing system can be controlled by a targeting moiety that has binding affinity for a cell surface protein or other localized cellular component.

[0071] In particular embodiments, a targeting moiety can include a surface-anchored targeting moiety that selectively binds the nanoparticle to a selected cell and initiates cellular uptake. In particular embodiments, cellular uptake can be mediated by receptor-induced endocytosis. In particular embodiments, a targeting moiety can include any substance that binds to another substance to form a complex capable of supporting selective delivery. In particular embodiments,F053-6005PCT / 24-085-WO-PCT a targeting moiety can include a protein, an aptamer, or a small molecule. In particular embodiments, a protein can include an antibody, a peptide, or a ligand. In particular embodiments, the selected cell type includes a hematopoietic stem cell (HSC). In particular embodiments, the selected cell type includes a hematopoietic stem and progenitor cell (HSPC).

[0072] “Selective delivery” to a selected cell type within a heterogenous mixture of cells means that at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of administered nanoparticles are taken up by the intended cell type. In particular embodiments, when a gain of function genetic modification is intended, selective delivery can be enhanced by including regulatory elements that restrict expression of inserted constructs to the intended / selected cell type. For example, selective delivery can be enhanced by using the CD45 promoter, WASP promoter or IFN-beta promoter for HSCs; or the murine stem cell virus promoter or the distal lck promoter for HSCs or T cells.

[0073] “Antibodies” are one example of binding domains and include whole antibodies or binding fragments of an antibody, e.g., Fv, Fab, Fab', F(ab')2, Fc, and single chain Fv fragments (scFvs) or any biologically effective fragments of an immunoglobulin that bind specifically to a motif expressed by an HSPC or lymphocyte. Antibodies or antigen binding fragments include all or a portion of polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, bispecific antibodies, mini bodies, and linear antibodies.

[0074] A single chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy and light chains of immunoglobulins connected with a short linker peptide. Fv fragments include the VL and VH domains of a single arm of an antibody but lack the constant regions. Although the two domains of the Fv fragment, VL and VH, are coded by separate genes, they can be joined, using, for example, recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (single chain Fv (scFv)). For additional information regarding Fv and scFv, see e.g., Bird, et al., Science 242 (1988) 423-426; Huston, et al., Proc. Natl. Acad. Sci. USA 85 (1988) 5879-5883; Plueckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York), (1994) 269-315; WO1993 / 16185; U.S. Patent No.5,571,894; and U.S. Patent No.5,587,458.

[0075] A Fab fragment is a monovalent antibody fragment including VL, VH, CL and CH1 domains. A F(ab')2 fragment is a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region. Diabodies include two epitope-binding sites that may be bivalent. See, for example, EP 0404097; WO1993 / 01161; and Holliger, et al., Proc. Natl. Acad.F053-6005PCT / 24-085-WO-PCT Sci. USA 90 (1993) 6444-6448. Dual affinity retargeting antibodies (DART™; based on the diabody format but featuring a C-terminal disulfide bridge for additional stabilization (Moore et al., Blood 117, 4542-51 (2011))) can also be formed. Antibody fragments can also include isolated CDRs. For a review of antibody fragments, see Hudson, et al., Nat. Med.9 (2003) 129-134.

[0076] Antibodies from human origin or humanized antibodies have lowered or no immunogenicity in humans and have a lower number of non-immunogenic epitopes compared to non-human antibodies. Antibodies and their fragments will generally be selected to have a reduced level or no antigenicity in human subjects.

[0077] Antibodies that specifically bind a motif expressed by an HSPC, HSC, or lymphocyte can be prepared using methods of obtaining monoclonal antibodies, methods of phage display, methods to generate human or humanized antibodies, or methods using a transgenic animal or plant engineered to produce antibodies as is known to those of ordinary skill in the art (see, for example, U.S. Patent Nos.6,291,161 and 6,291,158). Phage display libraries of partially or fully synthetic antibodies are available and can be screened for an antibody or fragment thereof that can bind to an HSPC or lymphocyte motif. For example, binding domains may be identified by screening a Fab phage library for Fab fragments that specifically bind to a target of interest (see Hoet et al., Nat. Biotechnol.23:344, 2005). Phage display libraries of human antibodies are also available. Additionally, traditional strategies for hybridoma development using a target of interest as an immunogen in convenient systems (e.g., mice, HuMAb mouse®, TC mouse™, KM- mouse®, llamas, chicken, rats, hamsters, rabbits, etc.) can be used to develop binding domains. In particular embodiments, antibodies specifically bind to motifs expressed by a selected lymphocyte and do not cross react with nonspecific components or unrelated targets. Once identified, the amino acid sequence or nucleic acid sequence coding for the antibody can be isolated and / or determined.

[0078] In particular embodiments, the antibody includes a binding domain that binds CD133, CD117, or CD90. In particular embodiments, the antibody includes αCD133:clone7, αCD117:104D2 and αCD90:5E10. In particular embodiments, the antibody that binds CD133 includes αCD133:7, 5E3, MA5-18323, RM1002, RM1029, EPR20980-45, or BLR093G. In particular embodiments, the antibody that binds CD133 includes αCD133:7. In particular embodiments, the antibody that binds CD117 includes αCD117:104D2, MA5-42433 (Thermo Fisher Scientific, Waltham, MA), YR145 (Abcam, Cambridge, England), MAB332, AF332, or AF3267. In particular embodiments, the antibody that binds CD117 includes αCD117:104D2. In particular embodiments, the antibody that binds CD90 includes αCD90:5E10, F15-42-1, EPR28145-53, 7E1B11, or AF-9. In particular embodiments, the antibody that binds CD90F053-6005PCT / 24-085-WO-PCT includes αCD90:5E10.

[0079] In particular embodiments, variable heavy and variable light chain pairs of clone 7 include: AQAAELDIVLTQSPAIMSASPGEKVTISCSASSSVGYMYWYQQKPGSSPKPWIYRPSNLASGV PARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELK (SEQ ID NO: 77) and RSSLEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETGEP SYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSSAKTT PPSVTSGQ (SEQ ID NO: 78); AQAAELDIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLASGVP ARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELK(SEQ ID NO: 79) and RSSLEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETGEP SYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSSAKTT PPSVTSGQAGQ (SEQ ID NO: 80); AQAAELDIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRPSNLASGV PARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELK(SEQ ID NO: 81) and RSSPEVMLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETGEP SYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSSAKTT PPSVTSGQAGQ (SEQ ID NO: 82); AQAAELDIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGQPPRLLIYLVSNLESGVP ARFSGSGSGTDFTLNIHPVEEEDAATYYCQQYHSYPPTFGAGTKLEIK(SEQ ID NO:83) and RSSLEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETGEP SYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSSAKTT PPSVTSGQAGQ (SEQ ID NO: 80); AQAAELDIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRPSNLASGV PARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELK (SEQ ID NO: 81) and RSSLEVHLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETGEP SYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSSAKTT PPSVTSGQAGQ (SEQ ID NO: 86); AQAAELDIVLSQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSPPKPWIYRTSNLASGV PARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELKSS (SEQ ID NO:87) and RSSLEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETGEP SYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDCGDYFDYWGQGTTLTVSSAKTT PPSVTSGQAGQ (SEQ ID NO:88);F053-6005PCT / 24-085-WO-PCT AQAAELDIVLSQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLASGV PARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELK (SEQ ID NO:89) and LEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETGEPSYA DDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSSAKTTPPS VTSGQAGQ (SEQ ID NO:90); AQAAELDIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRPSNLASGV PARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELKSSGD(SEQ ID NO:91) and RSSLEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETGEP SYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSSAKTT APSVTSGQAGQ (SEQ ID NO:92); AQAAELDIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRPSNLASGV PARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELK (SEQ ID NO:81) and RSSLEVQLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETGEP SYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSSAKTT APSVTSGQAGQ (SEQ ID NO:94);or MDIVLSQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLASGVPARFS GSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELK (SEQ ID NO:95) and RSSLEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETGEP SYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSS (SEQ ID NO:96).

[0080] In particular embodiments, the heavy and light chain pairs are utilized within a traditional antibody format and as, for example, an IgG isotype (e.g., IgG1). The heavy and light chain pairs can also be formed into scFv as follows: AQAAELDIVLTQSPAIMSASPGEKVTISCSASSSVGYMYWYQQKPGSSPKPWIYRPSNLASGV PARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELKSSGGGGSGGGGGG SSRSSLEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETG EPSYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSSAK TTPPSVTSGQ (SEQ ID NO:97); AQAAELDIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLASGVP ARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELKSSGGGGSGGGGGGS SRSSLEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETGE PSYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSSAKT TPPSVTSGQAGQ (SEQ ID NO:98);F053-6005PCT / 24-085-WO-PCT AQAAELDIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRPSNLASGV PARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELKSSGGGGSGGGGGG SSRSSPEVMLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETG EPSYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSSAK TTPPSVTSGQAGQ (SEQ ID NO:99); AQAAELDIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGQPPRLLIYLVSNLESGVP ARFSGSGSGTDFTLNIHPVEEEDAATYYCQQYHSYPPTFGAGTKLEIKSSGGGGSGGGGGGS SRSSLEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETGE PSYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSSAKT TPPSVTSGQAGQ (SEQ ID NO:100);SSRSSLEVHLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETG EPSYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSSAK TTPPSVTSGQAGQ (SEQ ID NO:101); AQAAELDIVLSQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSPPKPWIYRTSNLASGV PARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELKSSGGGGSGGGGGG SSRSSLEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETG EPSYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDCGDYFDYWGQGTTLTVSSAK TTPPSVTSGQAGQ (SEQ ID NO:102); AQAAELDIVLSQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLASGV PARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELKSSGGGGSGGGGGG SSRSSLEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETG EPSYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSSAK TTPPSVTSGQAGQ (SEQ ID NO:103); AQAAELDIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRPSNLASGV PARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELKSSGDGGSGGGGGG SSRSSLEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETG EPSYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSSAK TTAPSVTSGQAGQ (SEQ ID NO:104);PARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELKSSGGGGSGGGGGG SSRSSLEVQLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETGF053-6005PCT / 24-085-WO-PCT EPSYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSSAK TTAPSVTSGQAGQ (SEQ ID NO:105);or MDIVLSQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLASGVPARFS GSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELKSSGGGGSGGGGGGSSRS SLEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETGEPSY ADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSS (SEQ ID NO:106).

[0081] Antibody 104D2 is commercially available (Dianova (#117PE-100T)). This antibody binds the extracellular domain of CD117 (c-kit) and such binding does not interfere with the ability of CD117 to bind stem cell factor (SCF).

[0082] The following tables provide sequences and information for the 5E10 antibody:

[0083] Table 1. Heavy Chain CDR coding sequences. CDR SEQUENCE Position in VH SEQ ID Antibody r VL NO:F053-6005PCT / 24-085-WO-PCT CDR SEQUENCE Position in VH SEQ ID Antibody or VL NO:Antibody CDR SEQUENCE Position in VH SEQ IDF053-6005PCT / 24-085-WO-PCT Antibody CDR SEQUENCE Position in VH SEQ ID or VL NO:, in encoded by the sequence: CAGGTCCAACTGCTGCAGCCTGGGGCTGAGCTGGTGAGGCCTGGGGCCTCAGTGAGGCT GTCCTGCAAGACTTCTGGCTACACCTTCACCAGTTACTGGATAAACTGGGTGAAACAGAGG CCTGGACAAGGCCTTGAGTGGATCGGAAAAATTTTTCCTTCTGACAGTCATACTAATTACAA TCAAAAATTCAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATG CAGCTCATCAGCCCGACATCTGAGGACTCTGCGGTCTATTACTGTACGAGGGACTTCGATA CCCAGTTCTATGCTATGGAATACTGGGGTCAGGGAACCTCAGTCACCGTCTCCTCAG (SEQ ID NO: 140) and a variable light chain encoded by the sequence: CAAATTGTTCTCACCCAGTCTCCAACAATCATGTCTGCATCTCCAGGGCAGAAAGTCACCA TAAACTGCAGTGCCATCTCAAGTGTAAATTGCATGCACTGGTACCAGCAGAAGCCAGGATC CTCCCCCAAACTCTGGATTTATGCAACATCCAAACTGACTCTTGGAGTCCCTGCTTGCTTCA GTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAGTCAACAGCATGGTGACTGAAGATG CCACCTCTTATTTCTGTCATCAGTGGAGTAGTTACCCACCAACGTTCGGTGCTGGGACCAA GCTGGAGCTGAAA (SEQ ID NO: 141).

[0087] In particular embodiments, a CD90 5E10 antibody includes a variable heavy chain including the sequence: QVQLLQPGAELVRPGASVRLSCKTSGYTFTSYWINWVKQRPGQGLEWIGKIFPSDSHTNYNQ KFKDKATLTVDKSSSTAYMQLISPTSEDSAVYYCTRDFDTQFYAMEYWGQGTSVTVSS (SEQ ID NO: 142) and a variable light chain including the sequence: QIVLTQSPTIMSASPGQKVTINCSAISSVNCMHWYQQKPGSSPKLWIYATSKLTLGVPACFSGSF053-6005PCT / 24-085-WO-PCT GSGTSYSLTVNSMVTEDATSYFCHQWSSYPPTFGAGTKLELK (SEQ ID NO: 143).

[0088] Aptamers may be designed to facilitate selective delivery, including delivery across the cellular membrane, to intracellular compartments, or into the nucleus. Such a structure can include, either in addition to the one or more aptamer(s) or without such one or more aptamer(s), moiety(ies) so as to render the guide deliverable, inducible or responsive to (for example activatable or inactivatable by) a selected effector, for example responds to normal or pathological physiological conditions, including pH, hypoxia, O2 concentration, temperature, protein concentration, enzymatic concentration, lipid structure, light exposure, mechanical disruption (e.g., ultrasound waves), magnetic fields, electric fields, or electromagnetic radiation. Methods of making aptamers and conjugating such aptamers to the surface of a nanoparticle are known in the art, see for example Huang et al. Anal. Chem., 2008, 80 (3), pp 567–572. In particular embodiments, an aptamer includes a peptide aptamer and / or a nucleic acid aptamer (e.g., DNA or RNA).

[0089] In particular embodiments, peptide aptamers refer to a peptide loop (which is specific for a target protein) attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to an antibody. The variable loop length is typically 8 to 20 amino acids (e.g., 8 to 12 amino acids), and the scaffold may be any protein which is stable, soluble, small, and non-toxic (e.g., thioredoxin-A, stefin A triple mutant, green fluorescent protein, eglin C, and cellular transcription factor Spl). Peptide aptamer selection can be made using different systems, such as the yeast two-hybrid system (e.g., Gal4 yeast-two-hybrid system) or the LexA interaction trap system.

[0090] Nucleic acid aptamers are single-stranded nucleic acid (DNA or RNA) ligands that function by folding into a specific globular structure that dictates binding to target proteins or other molecules with high affinity and specificity, as described by Osborne et al., Curr. Opin. Chem. Biol.1:5-9, 1997; and Cerchia et al., FEBS Letters 528:12-16, 2002. In particular embodiments, aptamers are small (15 kDa; or between 15-80 nucleotides or between 20-50 nucleotides). Aptamers are generally isolated from libraries consisting of 1014-1015 random oligonucleotide sequences by a procedure termed SELEX (systematic evolution of ligands by exponential enrichment; see, for example, Tuerk et al., Science, 249:505-510, 1990; Green et al., Methods Enzymology. 75-86, 1991; and Gold et al., Annu. Rev. Biochem., 64: 763-797, 1995). Further methods of generating aptamers are described in, for example, US Patent Nos. 6,344,318; 6,331,398; 6,110,900; 5,817,785; 5,756,291; 5,696,249; 5,670,637; 5,637,461; 5,595,877; 5,527,894; 5,496,938; 5,475,096; and 5,270,16. Spiegelmers are similar to nucleic acid aptamers except that at least one β-ribose unit is replaced by β-D-deoxyribose or a modified sugar unitF053-6005PCT / 24-085-WO-PCT selected from, for example, β-D-ribose, α-D-ribose, β-L-ribose.

[0091] In particular embodiments, an RNA aptamer sequence has binding affinity for an aptamer ligand on or in the cell. In particular embodiments, the aptamer ligand is on the cell, for example so that it is at least partially available on the extra-cellular face or side of the cell membrane. For example, the aptamer ligand may be a cell-surface protein. The aptamer ligand may therefore be one part of a fusion protein, one other part of the fusion protein having a membrane anchor or membrane-spanning domain. In particular embodiments, the aptamer ligand is in the cell. For example, the aptamer ligand may be internalized within a cell, i.e., within (beyond) the cell membrane, for example in the cytoplasm, within an organelle (including mitochondria), within an endosome, or in the nucleus. In particular embodiments, an aptamer can include a donor template sequence, which can include an HDR template and a therapeutic nucleic acid sequence.

[0092] In particular embodiments, the targeting moiety that binds CD133 is an aptamer. The aptamer can be Aptamer A15 or B19 from Tocris Biosciences. In particular embodiments, aptamer A15 refers to an RNA aptamer with 15 bases and the formula Ci82H2i9F9N580io4Pi6. This aptamer has a molecular weight of 5549.58, and sequence modifications: 2- fluoropyrimidines, 3'- inverted deoxythymidine cap, 5'-fluorescent DY647 tag. See also Shigdar et al (2013) RNA aptamers targeting cancer stem cell marker CD133. Cancer Lett.33084 PMID: 23196060. In particular embodiments, the A15 aptamer has the sequence: CCCTCCTACATAGGG (SEQ ID NO: 144); or CCCUCCUACAUAGGG (SEQ ID NO: 145).

[0093] In particular embodiments, aptamer B19 refers to an RNA aptamer with 19 bases and the formula C221 H263F10N73O131 P20. This aptamer has a molecular weight of 6847.32, and sequence modifications: 2-fluoropyrimidines, 3'-inverted deoxythymidine cap, 5'-fluorescent DY647 tag. See also Shigdar et al (2013) RNA aptamers targeting cancer stem cell marker CD133. Cancer Lett.33084 PMID: 23196060. In particular embodiments, the B19 aptamer has the sequence: AGACCUUAUACCUGAGAUUACACGCUCUUCGAGCACGUCGAC (SEQ ID NO: 146).

[0094] In particular embodiments, the protein includes a ligand. In particular embodiments, the ligand includes stem cell factor (SCF).

[0095] In particular embodiments, stem cell factor includes the sequence: MKKTQTWILTCIYLQLLLFNPLVKTEGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLP SHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPE PRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAASSLRND SSSSNRKAKNPPGDSSLHWAAMALPALFSLIIGFAFGALYWKKRQPSLTRAVENIQINEEDNEIS MLQEKEREFQEV (SEQ ID NO: 1) or a portion of the sequence as set forth in SEQ ID NO: 1.F053-6005PCT / 24-085-WO-PCT

[0096] In particular embodiments, the protein includes human luteinizing hormone.

[0097] In particular embodiments, the small molecule includes degarelix acetate.

[0098] In particular embodiments, degarelix acetate is an antagonist of the luteinizing hormone- releasing hormone (LHRH) receptor.

[0099] In particular embodiments, nanoparticles include 500 to 1000 targeting moieties per nanoparticle. In particular embodiments, nanoparticles include 600 to 900 targeting moieties per nanoparticle. In particular embodiments, nanoparticles include 700 to 800 targeting moieties per nanoparticle. In particular embodiments, nanoparticles include 100 to 500 targeting moieties per nanoparticle In particular embodiments, nanoparticles include 300 to 400 targeting moieties per nanoparticle. In particular embodiments, nanoparticles include 400 to 500 targeting moieties per nanoparticle. In particular embodiments, targeting moieties result in selective delivery of nanoparticles to selected cell types. In particular embodiments, selective delivery is exclusive to a selected cell population. In particular embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of administered nanoparticles are delivered to a selected cell population.

[0100] In particular embodiments, HSCs are targeted for selective delivery of nanoparticles. In particular embodiments, HSCs can be targeted for selective delivery with binding domains (binding domains on antibody, aptamer, glycoprotein, a peptide, a ligand, or small molecule) that selectively bind CD34, CD133, CD117, and / or CD90. In particular embodiments, HSC are CD38- cells. In particular embodiments, HSC can be targeted for selective delivery with one or more binding domains that selectively bind known antigens expressed on the surface of HSCs and HSPCs: CD34, CD46, CD90, CD133, CD164, Sca-1 and / or CD117. In particular embodiments, HSC and / or HSPC can be targeted for selective delivery with binding domains that bind CD117, CD133, or CD90.

[0101] Mature T cells can be targeted for selective delivery with binding domains that selectively bind CD3. Activated T-cells can be targeted for selective delivery with binding domains that selectively bind 4-1BB (CD137), CD69, and / or CD25. Helper T cells can be targeted for selective delivery with binding domains that selectively bind CD4. Cytotoxic T-cells can be targeted for selective delivery with binding domains that selectively bind CD8. “Central memory” T-cells (or “TCM”) can be targeted for selective delivery with binding domains that selectively bind CD62L, CCR7, CD25, CD127, CD45RO, and / or CD95. “Effector memory” T-cell (or “TEM”) can be targeted for selective delivery with binding domains that selectively bind granzyme B and / or perforin. Regulatory T cells (“TREG”) can be targeted for selective delivery with binding domains that selectively bind CD25, CTLA-4, GITR, GARP and / or LAP. “Naive” T-cells can be targeted for selective delivery with binding domains that selectively bind CD62L, CCR7, CD28, CD127 and / orF053-6005PCT / 24-085-WO-PCT CD45RA.

[0102] Natural killer cells (also known as NK cells, K cells, and killer cells) can be targeted for selective delivery with binding domains that selectively bind CD8, CD16 and / or CD56.

[0103] Macrophages (and their precursors, monocytes) can be targeted for selective delivery with binding domains that selectively bind CD11b, F4 / 80; CD68; CD11c; IL-4Rα; and / or CD163.

[0104] Immature dendritic cells (i.e., pre-activation) can be targeted for selective delivery with binding domains that selectively bind: CD1a, CD1b, CD1c, CD1d, CD21, CD35, CD39, CD40, CD86, CD101, CD148, CD209, and / or DEC-205.

[0105] B cells can be targeted for selective delivery with binding domains that selectively bind CD5, CD19, CD20, CD21, CD22, CD35, CD40, CD52, and / or CD80.

[0106] According to the present disclosure, two targeting moieties that bind the same antigen may be associated with different nanoparticle performance. For instance, two antibodies (e.g., two distinct antibody clones) that bind the same antigen may be associated with different levels of cellular uptake by a targeted cell type, different levels of cellular uptake by a non-targeted cell type, different nanoparticle stability, different binding kinetics, or different gene-editing efficacy. In particular embodiments, the targeting moiety can affect the structure and / or functionality of one or more gene editing components of the nanoparticle. In particular embodiments, a linker attached between the targeting moiety and the nanoparticle can affect the structure and / or functionality of one or more gene editing components of the nanoparticle.

[0107] (II) Nanoparticles. Particular embodiments utilize colloidal metal nanoparticles. A colloidal metal includes any water-insoluble metal particle or metallic compound dispersed in liquid water. A colloid metal can be a suspension of metal particles in aqueous solution. Any metal that can be made in colloidal form can be used, including gold, silver, copper, nickel, aluminum, zinc, calcium, platinum, palladium, and iron. In particular embodiments, gold nanoparticles are used, e.g., prepared from HAuCl4. In particular embodiments, the nanoparticles are non- gold nanoparticles that are coated with gold to make gold-shell nanoparticles.

[0108] Methods for making colloidal metal nanoparticles, including gold colloidal nanoparticles from HAuCl4, are known to those having ordinary skill in the art. For example, the methods described herein as well as those described elsewhere (e.g., US 2001 / 005581; 2003 / 0118657; and 2003 / 0053983) can be used to make nanoparticles.

[0109] In particular exemplary embodiments, AuNPs were synthesized in three different size ranges (15, 50, 100 nm) by an optimized Turkevich and seeding-growth methods (Shahbazi, et al., Nanomedicine (Lond), 2017.12(16): p.1961-1973; Shahbazi, et al., Nanotechnology, 2017. 28(2): p. 025103; Turkevich, et al. Discussions of the Faraday Society, 1951. 11(0): p. 55-75;F053-6005PCT / 24-085-WO-PCT Perrault & Chan, Journal of the American Chemical Society, 2009.131(47): p.17042-17043). In the first step, seed AuNPs of 15 nm were synthesized by bringing 100 mL of 0.25 mM gold (III) chloride trihydrate solution to the boiling point and adding 1 mL of 3.33% trisodium citrate dihydrate solution. Synthesis of nanoparticles was carried out in high stirring speeds over 10 min. Prepared nanoparticles were cooled down to 4 °C and used in the following growth step.

[0110] In order to prepare AuNPs in 50 nm and 100 nm size ranges, two different 100 mL of 0.25 mM gold (III) chloride trihydrate solutions were prepared and in mild stirring conditions 2440 µL and 304 µL of seed AuNPs were added separately to synthesize 50 nm and 100 nm AuNPs, respectively. To these solutions was added 1 mL of 15 mM trisodium citrate dihydrate solution and the mixture was brought to the highest stirring speed. Then, 1 mL of 25 mM hydroquinone solution was added, and synthesis was continued over 30 min for 50 nm AuNPs and 5 h for 100 nm AuNPs. Finally, synthesized nanoparticles were purified by centrifuging at 5000×g and dispersing in ultra-pure water.

[0111] While AuNPs are particularly described, nanoparticles encompassed in the present disclosure may be provided in different forms, e.g., as solid nanoparticles (e.g., metal such as silver, gold, iron, titanium), non-metal, lipid-based solids, polymers, suspensions of nanoparticles, or combinations thereof. Metal, dielectric, and semiconductor nanoparticles may be prepared, as well as hybrid structures (e.g., core–shell nanoparticles). Nanoparticles made of semiconducting material may also be labeled quantum dots if they are small enough (typically sub 10 nm) that quantization of electronic energy levels occurs. Such nanoscale particles are used in biomedical applications as drug carriers or imaging agents and may be adapted for similar purposes in the present disclosure.

[0112] Lipidoid compounds are also particularly useful in the administration of gene editing system components. In particular embodiments, aminoalcohol lipidoid compounds are combined with gene editing system components to be delivered to a cell or a subject to form microparticles, nanoparticles, liposomes, or micelles. The gene editing system components to be delivered by the particles, liposomes, or micelles may be a polynucleotide, protein, peptide, or small molecule. Aminoalcohol lipidoid compounds may be combined with other aminoalcohol lipidoid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form particles.

[0113] The size of an AuNP can be selected to affect biodistribution within the human body. Nanoparticles suitable for use in the present disclosure can be any shape and can range in size from 5 nm - 550 nm in size, e.g., from 5 nm - 10 nm, 5 - 50 nm, 5 nm - 75 nm, 5 nm - 40 nm, 10 nm - 30, or 20 nm - 30 nm. Nanoparticles can also have a size in the range of from 10 nm - 15F053-6005PCT / 24-085-WO-PCT nm, 15 nm - 20 nm, 20 nm - 25 nm, 25 nm - 30 nm, 30 nm - 35 nm, 35 nm - 40 nm, 40 nm - 45 nm, or 45 nm - 50 nm, 50 nm - 55 nm, 55 nm - 60 nm, 60 nm - 65 nm, 65 nm - 70 nm, 70 nm - 75 nm, 75 nm - 80 nm, 80 nm - 85 nm, 85 nm - 90 nm, 90 nm - 95 nm, 95 nm - 100 nm, 100 nm - 105 nm, 105 nm - 110 nm, 110 nm - 115 nm, 115 nm - 120 nm, 120 nm - 125 nm, 125nm - 130 nm, 130nm - 135 nm, 135 nm - 140 nm, 140 nm - 145 nm, 145 nm - 150 nm, 100 nm - 500 nm, 100 nm - 150 nm, 150 nm - 200 nm, 200 nm - 250 nm, 250 nm - 300 nm, 300 nm - 350 nm, 350 nm - 400 nm, 400 nm - 450 nm, or 450 nm - 500 nm. In particular embodiments, nanoparticles greater than 550 nm are excluded. This is because particles or aggregated particles of >600 nm are not amenable to cellular uptake.

[0114] (III) Targeted Gene Editing Systems. Within the teachings of the current disclosure, any gene editing system capable of precise sequence targeting and modification can be used. These systems typically include a targeting element for precise targeting and a cutting element for cutting the targeted genetic site. Guide RNA is one example of a targeting element while various nucleases provide examples of cutting elements. Targeting elements and cutting elements can be separate molecules or linked, for example, by a nanoparticle. Alternatively, a targeting element and a cutting element can be linked together into one dual purpose molecule. When insertion of a nucleic acid sequence (e.g., therapeutic nucleic acid sequence) is intended, the systems also include a homology-directed repair template (which can include homology arms) associated with the nucleic acid sequence. As detailed further below, however, different gene editing systems can adopt different components and configurations while maintaining the ability to precisely target, cut, and modify selected genomic sites. Identification of sites for precise genetic engineering targeting with gene editing systems greatly increases the safety of genetic therapies.

[0115] When a therapeutic use need only de-activate a problematic gene, the gene editing components include a targeting element and a cutting element (although other components may be included as necessary or helpful for a particular purpose). When a therapeutic use adds or corrects a gene, the gene editing components include a targeting element, a cutting element, and a donor template.

[0116] Particular embodiments utilize zinc finger nucleases (ZFNs) as gene editing agents. ZFNs are a class of site-specific nucleases engineered to bind and cleave DNA at specific positions. ZFNs are used to introduce double strand breaks (DSBs) at a specific site in a DNA sequence which enables the ZFNs to target unique sequences within a genome in a variety of different cells. Moreover, subsequent to double-stranded breakage, homology-directed repair (HDR) or non-homologous end joining (NHEJ) takes place to repair the DSB, thus enabling genome editing.F053-6005PCT / 24-085-WO-PCT

[0117] ZFNs are synthesized by fusing a zinc finger DNA-binding domain to a DNA cleavage domain. The DNA-binding domain includes three to six zinc finger proteins which are transcription factors. The DNA cleavage domain includes the catalytic domain of, for example, FokI endonuclease. The FokI domain functions as a dimer requiring two constructs with unique DNA binding domains for sites on the target sequence. The FokI cleavage domain cleaves within a five or six base pair spacer sequence separating the two inverted half-sites.

[0118] For additional information regarding ZFNs, see Kim, et al. Proceedings of the National Academy of Sciences of the United States of America 93, 1156-1160 (1996); Wolfe, et al. Annual review of biophysics and biomolecular structure 29, 183-212 (2000); Bibikova, et al. Science 300, 764 (2003); Bibikova, et al. Genetics 161, 1169-1175 (2002); Miller, et al. The EMBO journal 4, 1609-1614 (1985); and Miller, et al. Nature biotechnology 25, 778-785 (2007)].

[0119] Particular embodiments can use transcription activator like effector nucleases (TALENs) as gene editing agents. TALENs refer to fusion proteins including a transcription activator-like effector (TALE) DNA binding protein and a DNA cleavage domain. TALENs are used to edit genes and genomes by inducing DSBs in the DNA, which induce repair mechanisms in cells. Generally, two TALENs must bind and flank each side of the target DNA site for the DNA cleavage domain to dimerize and induce a DSB. The DSB is repaired in the cell by NHEJ or HDR if an exogenous double-stranded donor DNA fragment is present.

[0120] As indicated, TALENs have been engineered to bind a target sequence of, for example, an endogenous genome, and cut DNA at the location of the target sequence. The TALEs of TALENs are DNA binding proteins secreted by Xanthomonas bacteria. The DNA binding domain of TALEs include a highly conserved 33 or 34 amino acid repeat, with divergent residues at the 12th and 13th positions of each repeat. These two positions, referred to as the Repeat Variable Diresidue (RVD), show a strong correlation with specific nucleotide recognition. Accordingly, targeting specificity can be improved by changing the amino acids in the RVD and incorporating nonconventional RVD amino acids.

[0121] Examples of DNA cleavage domains that can be used in TALEN fusions are wild-type and variant FokI endonucleases. For additional information regarding TALENs, see Boch, et al. Science 326, 1509-1512 (2009); Moscou, & Bogdanove, Science 326, 1501 (2009); Christian, et al. Genetics 186, 757-761 (2010); and Miller, et al. Nature biotechnology 29, 143-148 (2011).

[0122] Particular embodiments utilize MegaTALs as gene editing agents. MegaTALs have a single chain rare-cleaving nuclease structure in which a TALE is fused with the DNA cleavage domain of a meganuclease. Meganucleases, also known as homing endonucleases, are single peptide chains that have both DNA recognition and nuclease function in the same domain. InF053-6005PCT / 24-085-WO-PCT contrast to the TALEN, the megaTAL only requires the delivery of a single peptide chain for functional activity.

[0123] The CRISPR nuclease system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. CRISPRs are DNA loci containing short repetitions of base sequences. In the context of a prokaryotic immune system, each repetition is followed by short segments of spacer DNA belonging to foreign genetic elements that the prokaryote was exposed to. This CRISPR array of repeats interspersed with spacers can be transcribed into RNA. The RNA can be processed to a mature form and associate with a Cas (CRISPR-associated) nuclease. A CRISPR-Cas system includes a guide RNA having a sequence that can hybridize to the foreign genetic elements and Cas nuclease can then recognize and cut these exogenous genetic elements in the genome.

[0124] A CRISPR-Cas system does not require the generation of customized proteins to target specific sequences, but rather a single Cas enzyme can be programmed by a short guide RNA molecule (gRNA) to recognize a specific DNA target. The CRISPR-Cas systems of bacterial and archaeal adaptive immunity show extreme diversity of protein composition and genomic loci architecture. The CRISPR-Cas system loci have more than 50 gene families and there are no strictly universal genes, indicating fast evolution and extreme diversity of loci architecture. So far, there is comprehensive Cas gene identification of 395 profiles for 93 Cas proteins. Classification includes signature gene profiles plus signatures of locus architecture. A new classification of CRISPR-Cas systems is proposed in which these systems are broadly divided into two classes, Class 1 with multi-subunit effector complexes and Class 2 with single-subunit effector modules exemplified by the Cas9 protein. Efficient gene editing in human CD34+ cells using electroporation of CRISPR / Cas9 mRNA and single-stranded oligodeoxyribonucleotide (ssODN) as a donor template for HDR has been demonstrated. De Ravin et al. Sci Transl Med. 2017; 9(372): eaah3480. Novel effector proteins associated with Class 2 CRISPR-Cas systems may be developed as powerful genome engineering tools and the prediction of putative novel effector proteins and their engineering and optimization is important. In addition to the Class 1 and Class 2 CRISPR-Cas systems, more recently a putative Class 2, Type V CRISPR-Cas class exemplified by Cpf1 has been identified Zetsche et al. (2015) Cell 163(3): 759-771.

[0125] Guide RNA (gRNA) is one example of a targeting element. In its simplest form, gRNA provides a sequence that targets a site within a genome based on complementarity (e.g., crRNA). As explained below, however, gRNA can also include additional components. For example, in particular embodiments, gRNA can include a targeting sequence (e.g., crRNA) and a component to link the targeting sequence to a cutting element. This linking component can be tracrRNA. InF053-6005PCT / 24-085-WO-PCT particular embodiments, as described below, gRNA including crRNA and tracrRNA can be expressed as a single molecule referred to as single gRNA (sgRNA). gRNA can also be linked to a cutting element through other mechanisms such as through a nanoparticle or through expression or construction of a dual or multi-purpose molecule. In particular embodiments, targeting elements (e.g., gRNA) can include one or more modifications (e.g., a base modification, a backbone modification), to provide the nucleic acid with a new or enhanced feature (e.g., improved stability).

[0126] As indicated, examples of cutting elements include nucleases. CRISPR-Cas loci have more than 50 gene families and there are no strictly universal genes, indicating fast evolution and extreme diversity of loci architecture. Exemplary Cas nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csxl2), Cas10, Cpf1, C2c3, C2c2 and C2c1Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csxl4, Csx10, Csx16, CasX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4.

[0127] There are three main types of Cas nucleases (type I, type II, and type III), and 10 subtypes including 5 type I, 3 type II, and 2 type III proteins (see, e.g., Hochstrasser and Doudna, Trends Biochem Sci, 2015:40(l):58-66). Type II Cas nucleases include Cas1, Cas2, Csn2, and Cas9. These Cas nucleases are known to those skilled in the art. For example, the amino acid sequence of the Streptococcus pyogenes wild-type Cas9 polypeptide is set forth, e.g., in NBCI Ref. Seq. No. NP 269215, and the amino acid sequence of Streptococcus thermophilus wild-type Cas9 polypeptide is set forth, e.g., in NBCI Ref. Seq. No. WP_011681470.

[0128] In particular embodiments, Cas9 refers to an RNA-guided double-stranded DNA-binding nuclease protein or nickase protein. Wild-type Cas9 nuclease has two functional domains, e.g., RuvC and HNH, that cut different DNA strands. Cas9 can induce double-strand breaks in genomic DNA (target DNA) when both functional domains are active. The Cas9 enzyme, in some embodiments, includes one or more catalytic domains of a Cas9 protein derived from bacteria such as Corynebacter, Sutterella, Legionella, Treponema, Filif actor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, and Campylobacter. In some embodiments, the Cas9 is a fusion protein, e.g., the two catalytic domains are derived from different bacterial species.

[0129] As indicated previously, the CRISPR / Cas system has been engineered such that, in certain cases, crRNA and tracrRNA can be combined into one molecule called a single gRNA (sgRNA). In this engineered approach, the sgRNA guides Cas to target any desired sequenceF053-6005PCT / 24-085-WO-PCT (see, e.g., Jinek et al. (2012) Science 337:816-821; Jinek et al. (2013) eLife 2:e00471; Segal (2013) eLife 2:e00563). Thus, the CRISPR / Cas system can be engineered to create a double- strand break at a desired target in a genome of a cell, and harness the cell's endogenous mechanisms to repair the induced break by homology-directed repair (HDR); homology- independent targeted integration (HITI)-associated microhomology-mediated end joining (MMEJ), HITI-associated non-homologous end joining (NHEJ); or complete NHEJ depending on whether a genetic construct is provided for insertion and the length of any provided homology regions (e.g., as used herein HDR occurs if a region of homology is > 75 bp and HITI occurs if a region of homology is < 75 bp).

[0130] Additional information regarding CRISPR-Cas systems and components thereof are described in, US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, US8932814, US8945839, US8993233 and US8999641 and applications related thereto; and WO2014 / 018423, WO2014 / 093595, WO2014 / 093622, WO2014 / 093635, WO2014 / 093655, WO2014 / 093661, WO2014 / 093694, WO2014 / 093701, WO2014 / 093709, WO2014 / 093712, WO2014 / 093718, WO2014 / 145599, WO2014 / 204723, WO2014 / 204724, WO2014 / 204725, WO2014 / 204726, WO2014 / 204727, WO2014 / 204728, WO2014 / 204729, WO2015 / 065964, WO2015 / 089351, WO2015 / 089354, WO2015 / 089364, WO2015 / 089419, WO2015 / 089427, WO2015 / 089462, WO2015 / 089465, WO2015 / 089473 and WO2015 / 089486, WO2016205711, WO2017 / 106657, WO2017 / 127807 and applications related thereto.

[0131] Particular embodiments utilize CRISPR gene editing. In particular embodiments, CRISPR gene editing can occur with CRISPR guide RNA (gRNA) and / or a CRISPR nuclease (e.g., Cpf1 or Cas9). In particular embodiments, a targeting element includes gRNA. In particular embodiments, a cutting element includes a nuclease.

[0132] The Cpf1 nuclease particularly can provide added flexibility in target site selection by means of a short, four base pair recognition sequence (TTTN), known as the protospacer- adjacent motif or PAM. Cpf1’s cut site is at least 18 bp away from the PAM sequence, thus the enzyme can repeatedly cut a specified locus after indel (insertion and deletion) formation, potentially increasing the efficiency of HDR. Successful HDR results in mutation of the PAM sequence such that no further cutting occurs. Moreover, staggered DSBs with sticky ends permit orientation-specific donor template insertion, which is advantageous in non-dividing cells.

[0133] Particular embodiments adopt features that increase the efficiency and / or accuracy of HDR. For example, Cpf1 has a short single gRNA and cuts target DNA in staggered form with 5′ 2-4 nucleotide (nt) overhangs called sticky ends. Sticky ends are favorable for HDR, Kim et al.F053-6005PCT / 24-085-WO-PCT (2016) Nat Biotechnol. 34(8): 863-8. Moreover, donor templates should be released from the nanoparticles before the genome cut by the RNP occurs to promote HDR. Accordingly, in particular embodiments disclosed herein donor templates are found farther from the surface of the nanoparticle than targeting elements and cutting elements. The current disclosure also unexpectedly found that delivery of gene-editing components on a gold nanoparticle increases the efficiency and / or accuracy of HDR. Accordingly, particular embodiments deliver gene-editing components utilizing gold nanoparticles.

[0134] Particular embodiments can utilize engineered variant Cpf1s. For example, US 2018 / 0030425 describes engineered Cpf1 nucleases from Lachnospiraceae bacterium ND2006 and Acidaminococcus sp. BV3L6 with altered and improved target specificity. Particular variants include Lachnospiraceae bacterium ND2006 with mutations (i.e., replacement of the native amino acid with a different amino acid, e.g., alanine, glycine, or serine), at one or more of the following positions: S203, N274, N278, K290, K367, K532, K609, K915, Q962, K963, K966, K1002, and / or S1003. Particular Cpf1 variants can also include Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) with mutations (i.e., replacement of the native amino acid with a different amino acid, e.g., alanine, glycine, or serine (except where the native amino acid is serine)), at one or more of the following positions: N178, S186, N278, N282, R301, T315, S376, N515, K523, K524, K603, K965, Q1013, Q1014, and / or K1054. In particular embodiments, engineered Cpf1 variants include eCfp1. Other Cpf1 variants are described in US 2016 / 0208243 and WO / 2017 / 184768.

[0135] Exemplary gRNA sequences that can be used within the current disclosure include: UAAUUUCUACUCUUGUAGAUUUCGGACCCGUGCUACAACUU (SEQ ID NO: 2); UAAUUUCUACUCUUGUAGAUAUAGAAUAGCCUCAUAUUUUA (SEQ ID NO: 3); UAAUUUCUACUCUUGUAGAUGAGCUGUUGGCAUCAUGUUCCUG (SEQ ID NO: 4); UAAUUUCUACUCUUGUAGAUUCCAAACCUCCUAAAUGAUAC (SEQ ID NO: 5); and UAAUUUCUACUCUUGUAGAUCACCCGAUCCACUGGGGAGCA (SEQ ID NO: 6).

[0136] Relevant target sites for genetic engineering include (with PAM sites italicized): TTTGTGTCCCCGTTTTGGTTGGTAAAC (SEQ ID NO: 7); TTTAAAAATCAATACCGATAATAATGA (SEQ ID NO: 8); TTTCTTAATATGAATATTAATATCGGT (SEQ ID NO: 9); TTTCCGTATCTGGAAGGGGCATCTTGG (SEQ ID NO: 10); TTTCCTTAGGACCGGAAGGATTACAGC (SEQ ID NO: 11); TTTGCCTAAAAGGCACTATGTCAAATG (SEQ ID NO: 12); TTTGGAGCTGTTGGCATCATGTTCCTG (SEQ ID NO: 13); TTTGATTCTTTTCTATCTCAGGACAGA (SEQ ID NO: 14);F053-6005PCT / 24-085-WO-PCT TTTATAGACATCCCACACTGTAGTTCT (SEQ ID NO: 15); TTTATTAATTTGAGAACCAACATAAGG (SEQ ID NO: 16); TTTATTTTCTTTTTGGTAAGAAGGAAC (SEQ ID NO: 17); TTTCACACACACACACACACACACACA (SEQ ID NO: 18); TTTGGAGCTGTTGGCATCATGTTCCTG (SEQ ID NO: 19); TTTATCCAAACCTCCTAAATGATAC (SEQ ID NO: 20); TTTACACCCGATCCACTGGGGAGCA (SEQ ID NO: 21); and TTTTTGATTCTTTTCTATCTCAGGACA (SEQ ID NO: 22).

[0137] These target sites reflect genomic safe harbors (GSH) within HSPC.

[0138] As indicated, in particular embodiments, gene editing systems to provide a genetic therapy within a targeted site will include guide RNA and a nuclease. In particular embodiments, donor templates can be used, especially when performing a gain-of-function therapy or a precise loss- of-function therapy. In particular embodiments, gene editing systems include a homology-directed repair template and a therapeutic nucleic acid sequence.

[0139] Particular embodiments include contacting a cell with a gene editing system capable of inserting a donor template at a target cell targeted genetic site. In particular embodiments, the gene editing system includes gRNA capable of hybridizing to a target sequence within the targeted genetic site, and a nucleic acid encoding a nuclease enzyme such as Cpf1 or Cas9.

[0140] All nucleic acid-based components of gene editing systems can be single stranded, double stranded, or may have mix of single stranded and double stranded regions. For example, guide RNA or a donor template may be a single-stranded DNA, a single-stranded RNA, a double- stranded DNA, or a double-stranded RNA. In particular embodiments utilizing nanoparticles described herein, the end of a nucleic acid farthest from the nanoparticle surface may be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues can be added to the 3' terminus of a linear molecule and / or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad Sci USA 84:4959-4963; Nehls et al. (1996) Science 272:886- 889. Additional methods for protecting exogenous polynucleotides from degradation include addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. Chemically modified mRNA can be used to increase intracellular stability, while asymmetric homology arms and phosphorothioate modification can be incorporated into the ssODN to improve HDR efficiency. In particular embodiments utilizing nanoparticles described herein, nucleic acids may be protected from electrostatic (charge-based) repulsions by, for example,F053-6005PCT / 24-085-WO-PCT addition of a charge shielding spacer. In particular embodiments, a charge shielding spacer can include an 18 atom oligoethylene glycol (OEG) spacer added to one or both ends. In particular embodiments, a charge shielding spacer can include a 10 - 26 atom oligoethylene glycol (OEG) spacer added to one or both ends.

[0141] Donor templates can be of any length, e.g., 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more, etc.

[0142] In particular embodiments, a homology-directed repair template is designed to serve as a template in homologous recombination, such as within or near a target sequence nicked or cleaved by an enzyme (e.g., nuclease) of a gene editing system. A homology-directed repair template polynucleotide may be of any suitable length, such as 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, 2000, 3000, 4000, 5000, or more nucleotides. In particular embodiments, the homology-directed repair template polynucleotide is complementary to a portion of a polynucleotide including the target sequence. When optimally aligned, a homology-directed repair template polynucleotide overlaps with one or more nucleotides of a target sequence (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides).

[0143] In particular embodiments, the homology-directed repair template can include sufficient homology to a genomic sequence at the cleavage site, e.g.70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequences flanking the cleavage site, e.g., within 50 bases or less of the cleavage site, e.g., within 30 bases, within 15 bases, within 10 bases, within 5 bases, or immediately flanking the cleavage site, to support HDR between it and the genomic sequence to which it bears homology. 25, 50, 100, or 200 nucleotides, or more than 200 nucleotides of sequence homology between a homology-directed repair template and a targeted genomic sequence (or any integral value between 10 and 200 nucleotides, or more) can support HDR. Homology arms or flanking sequences are generally identical to the genomic sequence, for example, to the genomic region in which the double stranded break (DSB) occurs. However, absolute identity is not required.

[0144] In particular embodiments, the donor template includes a heterologous therapeutic nucleic acid sequence flanked by two regions of homology, such that homology-directed repair between the target DNA region and the two flanking sequences results in insertion of the heterologous therapeutic nucleic acid sequence at the target region.

[0145] In some examples, homology arms or flanking sequences of homology-directed repair (HDR) templates are asymmetrical.

[0146] In particular embodiments, nanoparticles can deliver HDR factors that promote HDR. HDRF053-6005PCT / 24-085-WO-PCT factors that could be incorporated include: genes associated with end resection in homology- driven repair pathways: CtIP, Mre11, Rad50, Nbs1; genes associated with additional end resection in homology-driven repair pathways: BLM, EXO1, RPA; genes associated with homology-directed repair (HDR): BLM, EXO1, RPA, BRCA1, BRCA2, RAD51 (required), RAD54, (27M) RAD51 (required). These molecules can include oligos (mRNA) or proteins for any of the factors involved in HDR to ensure that cells receiving gene editing machinery also have these factors present. Alternatively, or in combination, small interfering RNAs (siRNAs, short- hairpin RNAs or microRNAs) that would reduce expression of NHEJ pathways could also be included. HSPC from various healthy donors do not readily express transcripts (mRNA) for many DNA repair factors (data not shown). Therefore, in particular embodiments, these missing factors can be provided to improve the efficiency of HDR.

[0147] As indicated, in particular embodiments, donor templates include a therapeutic nucleic acid sequence. Therapeutic nucleic acid sequences can include a corrected gene sequence; a complete gene sequence and / or one or more regulatory elements associated with expression of the gene. A corrected gene sequence can be a portion of a gene requiring correction or can provide a complete replacement copy of a gene. A corrected gene sequence can provide a complete copy of a gene, without necessarily replacing an existing defective gene. One of ordinary skill in the art will recognize that removal of a defective gene when providing a corrected copy may or may not be required. When inserting a gene within a genetic safe harbor, a therapeutic nucleic acid sequence should include a coding region and all regulatory elements required for its expression.

[0148] Examples of therapeutic genes and gene products include skeletal protein 4.1, glycophorin, p55, the Duffy allele, globin family genes, WAS, phox, dystrophin, pyruvate kinase, CLN3, ABCD1, arylsulfatase A, SFTPB, SFTPC, NLX2.1, ABCA3, GATA1, ribosomal protein genes, TERT, TERC, DKC1, TINF2, CFTR, LRRK2, PARK2, PARK7, PINK1, SNCA, PSEN1, PSEN2, APP, SOD1, TDP43, FUS, ubiquilin 2, C9ORF72, α2β1, αvβ3, αvβ5, αvβ63, BOB / GPR15, Bonzo / STRL-33 / TYMSTR, CCR2, CCR3, CCR5, CCR8, CD4, CD46, CD55, CXCR4, aminopeptidase-N, HHV-7, ICAM, ICAM-1, PRR2 / HveB, HveA, α-dystroglycan, LDLR / α2MR / LRP, PVR; PRR1 / HveC, laminin receptor, 101F6, 123F2, 53BP2, abl, ABLI, ADP, aFGF, APC, ApoAI, ApoAIV, ApoE, ATM, BAI-1, BDNF, Beta*(BLU), bFGF, BLC1, BLC6, BRCA1, BRCA2, CBFA1, CBL, C-CAM, CFTR, CNTF, COX-1, CSFIR, CTS-1, cytosine deaminase, DBCCR-1, DCC, Dp, DPC-4, E1A, E2F, EBRB2, erb, ERBA, ERBB, ETS1, ETS2, ETV6, Fab, FancA, FancB, FancC, FancD1, FancD2, FancE, FancF, FancG, FancI, FancJ, FancL, FancM, FancN, FancO, FancP, FancQ, FancR, FancS, FancT, FancU, FancV, andF053-6005PCT / 24-085-WO-PCT FancW, FCC, FGF, FGR, FHIT, fms, FOX, FUS 1, FUS1, FYN, G-CSF, GDAIF, Gene 21, Gene 26, GM-CSF, GMF, gsp, HCR, HIC-1, HRAS, hst, IGF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, ING1, interferon α, interferon β, interferon γ, IRF-1, JUN, KRAS, LCK, LUCA-1, LUCA-2, LYN, MADH4, MADR2, MCC, mda7, MDM2, MEN-I, MEN-II, MLL, MMAC1, MYB, MYC, MYCL1, MYCN, neu, NF-1, NF-2, NGF, NOEY1, NOEY2, NRAS, NT3, NT5, OVCA1, p16, p21, p27, p53, p57, p73, p300, PGS, PIM1, PL6, PML, PTEN, raf, Rap1A, ras, Rb, RB1, RET, rks-3, ScFv, scFV ras, SEM A3, SRC, TAL1, TCL3, TFPI, thrombospondin, thymidine kinase, TNF, TP53, trk, T-VEC, VEGF, VHL, WT1, WT-1, YES, zac1, iduronidase, IDS, GNS, HGSNAT, SGSH, NAGLU, GUSB, GALNS, GLB1, ARSB, HYAL1, F8, F9, HBB, CYB5R3, γC, JAK3, IL7RA, RAG1, RAG2, DCLRE1C, PRKDC, LIG4, NHEJ1, CD3D, CD3E, CD3Z, CD3G, PTPRC, ZAP70, LCK, AK2, ADA, PNP, WHN, CHD7, ORAI1, STIM1, CORO1A, CIITA, RFXANK, RFX5, RFXAP, RMRP, DKC1, TERT, TINF2, DCLRE1B, and SLC46A1.

[0149] In particular embodiments, a therapeutic nucleic acid sequence includes a coding sequence for a therapeutic expression product (e.g., protein, RNA) and all associated regulatory elements (e.g., promoters, etc.) to result in expression of the gene product.

[0150] In particular embodiments, a therapeutic nucleic acid sequence (e.g., a gene) can be selected for incorporation into a targeted genetic site to provide for in vivo selection of the genetically modified cell. For example, in vivo selection using a cell-growth switch allows a minor population of genetically modified cells to be inducibly amplified. A strategy to achieve in vivo selection has been to employ drug selection while coexpressing a transgene that conveys chemoresistance, such as O6-methylguanine-DNA-methyltransferase (MGMT). An alternate approach is to confer an enhanced proliferative potential upon gene-modified HSC through the delivery of the homeobox transcription factor HOXB4. In particular embodiments, a suicide gene can be incorporated into the genetically modified cell so that such population of cells can be eliminated, for example, by administration of a drug that activities the suicide gene. See, for example, Cancer Gene Ther. 2012 Aug;19(8):523-9; PLoS One. 2013;8(3):e59594. and Molecular Therapy — Oncolytics (2016) 3, 16011.

[0151] (IV) Conjugation of Components to Nanoparticles. As indicated, a variety of active components can be conjugated to the nanoparticles disclosed herein for targeted gene editing. In particular embodiments, active components include targeting moieties and gene-editing components. In particular embodiments, active components include nucleic acids. In particular embodiments, active components include proteins.

[0152] Nucleic acids can be conjugated directly or indirectly, and covalently or noncovalently, to the surface of the nanoparticle. For example, a nucleic acid may be covalently bonded at one endF053-6005PCT / 24-085-WO-PCT of the nucleic acid to the surface of the nanoparticle.

[0153] Nucleic acids conjugated to the nanoparticle can have a length of from 10 nucleotides (nt) - 1000 nt, e.g., 1 nt - 25 nt, 25 nt - 50 nt, 50 nt - 100 nt, 100 nt - 250 nt, 250 nt - 500 nt, 500 nt - 1000 nt or greater than 1000 nt. In particular embodiments, nucleic acids modified by conjugation to a linker do not exceed 50 nt or 40 nt in length.

[0154] When conjugated indirectly through, for example, an intervening linker, any type of molecule can be used as a linker. For example, a linker can be an aliphatic chain including at least two carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more carbon atoms), and can be substituted with one or more functional groups including a ketone, ether, ester, amide, alcohol, amine, urea, thiourea, sulfoxide, sulfone, sulfonamide, and / or disulfide.

[0155] In particular embodiments the linker includes a disulfide at the free end (e.g., the end not conjugated to the guide RNA) that couples the nanoparticle surface. In particular embodiments, the disulfide is a C2-C10 disulfide, that is it can be an aliphatic chain terminating in a disulfide that includes 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, although it is envisioned that longer aliphatic chains can be used. In particular embodiments, the disulfide is a 3 carbon disulfide (C3 S-S). Linkers can have either sulfhydryl groups (SH) or disulfide groups (S-S) or a different number of sulfur atoms. In particular embodiments, a thiol modification can be introduced without using a linker. In particular embodiments, a nuclease enzyme is delivered as a protein pre-conjugated with its guide RNA (a ribonucleoprotein (RNP) complex). In this formulation, the guide RNA molecule is bound to the nanoparticle and the nuclease enzyme, by default, can be also bound.

[0156] In particular embodiments, cyclic disulfides can be used to stably modify the surface of the particle. Cyclic disulfides can crosslink thiol pairs while minimizing dead-end Cys modifications.

[0157] Commonly used linkers include linker sequence with the amino acids glycine and serine (Gly-Ser linkers). In particular embodiments, the linker sequence includes sets of glycine and serine repeats such as from one to ten repeats of (GlyxSery)n, wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). Particular examples include (Gly4Ser)n(SEQ ID NO: 23), (Gly3Ser)n(Gly4Ser)n(SEQ ID NO: 24), (Gly3Ser)n(Gly2Ser)n(SEQ ID NO: 25), or (Gly3Ser)n(Gly4Ser)1(SEQ ID NO: 26). In particular embodiments, the linker is (Gly4Ser)4(SEQ ID NO: 27), (Gly4Ser)3(SEQ ID NO: 28), (Gly4Ser)2(SEQ ID NO: 29), (Gly4Ser)1(SEQ ID NO: 23), (Gly3Ser)2(SEQ ID NO: 31), (Gly3Ser)1(SEQ ID NO: 32), (Gly2Ser)2(SEQ ID NO: 33) or (Gly2Ser)1, GGSGGGSGGSG (SEQ ID NO: 34), GGSGGGSGSG (SEQ ID NO: 35), or GGSGGGSG (SEQ ID NO: 36).F053-6005PCT / 24-085-WO-PCT

[0158] Linkers can include tag cassettes. Tag cassettes that bind cognate binding molecules include, for example, His tag (HHHHHH; SEQ ID NO: 37), Flag tag (DYKDDDDK; SEQ ID NO: 38), Xpress tag (DLYDDDDK; SEQ ID NO: 39), Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO: 40), Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 41), Polyglutamate tag, HA tag (YPYDVPDYA; SEQ ID NO: 42), Myc tag (EQKLISEEDL; SEQ ID NO: 43), Strep tag (which refers the original STREP® tag (WRHPQFGG; SEQ ID NO: 44), STREP® tag II (WSHPQFEK SEQ ID NO: 45 (IBA Institut fur Bioanalytik, Germany); see, e.g., US 7,981,632), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 46), Softag 3 (TQDPSRVG; SEQ ID NO: 47), and V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 48).

[0159] In particular embodiments, CRISPR components are attached to the nanoparticle. For example, Li et al. (Engineering CRISPR–Cpf1 gRNAs and mRNAs to maximize genome editing efficiency.2017.1: p.0066) indicated that the 5’ end of Cpf1 gRNA is not safe for any modification because such modifications result in the abrogation of the gRNA binding to Cpf1 nuclease. Disclosed herein is a modification to the 3’ end of gRNA that does not compromise cutting efficiency. In particular embodiments, in the first step of conjugation to a nanoparticle the 3’ end of the gRNA is modified with an 18-atom hexa-ethyleneglycol spacer (18 spacer) and 3 carbon disulfide (C3 S-S) to attach the gRNA to the surface of AuNPs.

[0160] Based on the foregoing, in particular embodiments, for example when the nanoparticle includes gold, a linker can be any thiol- containing molecule. Reaction of a thiol group with Au results in a covalent sulfide (-S-) bond. AuNPs have high affinity to thiol (–SH) and dithiol (S–S) groups and semi-covalent bonds occur between the surface of AuNP and sulfur groups (Hakkinen, Nat Chem, 2012. 4(6): p. 443-455). In particular embodiments, thiol groups can be added to nucleic acids to facilitate attachment to the surface of AuNPs. This approach can improve nucleic acid uptake and stability (see, e.g., Mirkin, et al., A Nature, 1996.382(6592): p. 607-609). In the field, thiols are also known as sulfhydryl-, suylfanyl groups. Further a molecule that contains a primary sulfur group (a thiol) is known as a mercaptan.

[0161] In particular embodiments, the targeting moiety is associated with the nanoparticle by electrostatic anchoring. In particular embodiments, the targeting moiety is associated with the nanoparticle through a covalent chemical linker. In particular embodiments, the linker includes a functional group on each end. In some examples, the two functional groups are different (e.g., the linker is a heterofunctional linker). In particular examples, one or both functional groups are click chemistry reagents. Click chemistry refers to reactions that are highly efficient (e.g., reactions with a yield above 70%, above 80%, above 90%, or above 95%) under mild conditions (e.g., room temperature, neutral pH). For instance, a functional group may include a reagent for a SPAACF053-6005PCT / 24-085-WO-PCT (strain promoted azide-alkyne cycloadditions) reaction or a CuAAC (Copper-catalyzed Azide- alkyne cycloadditions) reaction. In some examples, a functional group includes streptavidin, biotin, or avidin. In particular embodiments, a functional group binds an azide group, an amine group, or a thiol group. In particular embodiments, a functional group includes a phenylboronic acid-lactrobionic acid conjugate. In particular embodiments, the linker may be associated to the targeting moiety and / or the nanoparticle via interactions between an aptamer and homologous DNA and / or RNA sequences. In particular embodiments, the linker may be associated to the targeting moiety and / or the nanoparticle via imine / hydrazone chemistry. In particular embodiments, the linker is a self-immolating linker, a UV-sensitive linker, tetrazine, or a modified carbohydrate.

[0162] In particular embodiments, a functional group includes a dibenzocyclooctyne (DBCO) group. In particular embodiments, the targeting moiety is attached to an azide by an enzymatic reaction. For instance, an antibody may be modified, via an enzymatic reaction, to include a galactose molecule with an azide group. The DBCO group may react with an azide on the targeting moiety in a click chemistry reaction. In particular embodiments, a functional group includes a trifluoromethylphenyl (TFP) ester. TFP esters can bind amine groups in click chemistry reactions. In particular embodiments, TFP esters form amide bonds with amine groups on the nanoparticle. In particular embodiments, TFP esters bind to amine groups of a polymer (e.g., a positively charged polymer, such as PEI). In particular examples, the linker includes a maleimide group that can attach to a thiol on the targeting moiety, for instance, via a maleimide reaction. Thiol groups can be exposed on the targeting moiety (e.g., an antibody) by, in some examples, breaking disulfide bonds (e.g., reduction). In particular embodiments, unbound linkers are quenched (e.g., by adding excess azide) after attachment of the linker and / or the targeting moiety to avoid hydrophobic and / or crosslinking aggregation and retain nanoparticle functionality.

[0163] In particular embodiments, the linker includes a PEG spacer between two functional groups. The PEG spacer may include 1 to 24 ethylene glycol units. In particular embodiments, the PEG spacer includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 ethylene glycol units. In particular embodiments, the PEG spacer includes 4, 12, or 24 ethylene glycol units. PEG spacers with up to 4 ethylene glycol units may be referred to as OEG spacers. In particular embodiments, the PEG spacer has a length of 1 to 8 nanometers (nm). In particular embodiments, the PEG spacer has a length of 2 to 5 nm. In particular embodiments, the PEG spacer has a length of 2, 4, 5, 6, 12, or 24 nm.

[0164] In particular embodiments, the linker includes a DBCO group, a PEG spacer, and a TFP ester. In particular embodiments, the linker includes a DBCO group linked to a TFP ester by aF053-6005PCT / 24-085-WO-PCT PEG spacer with 4 to 12 ethylene glycol units (e.g., a 4-12 PEG spacer). In particular embodiments, the linker includes a DBCO group linked to a TFP ester by a PEG spacer with 4 ethylene glycol units (e.g., DBCO-PEG4-TFP).

[0165] In particular embodiments, the AuNP / CRISPR nanoparticle includes 200 to 4000 linkers per nanoparticle. In particular embodiments, the AuNP / CRISPR nanoparticle includes 250 to 3000 linkers per nanoparticle. In particular embodiments, the AuNP / CRISPR nanoparticle includes 500 to 3000 linkers per nanoparticle. In particular embodiments, the AuNP / CRISPR nanoparticle includes 5 to 500 linkers per RNP. In particular embodiments, the AuNP / CRISPR nanoparticle includes 10 to 250 linkers per RNP. The ratio of linkers per nanoparticle can improve nanoparticle uptake by maximizing targeting moiety addition while retaining functionality of other components.

[0166] In particular embodiments, using an optimized two step method of seeding-growth, highly monodisperse AuNPs were synthesized in 3 different size ranges (15 nm, 50nm, 100 nm) and conjugated with Cpf1 gRNA and endonuclease. Because of the strong electrostatic repulsion between the negatively charged surface and negatively charged gRNA it is difficult to attach the gRNA to the surface of AuNPs without, for example, the thiol modification. In particular embodiments, in the second step, after purification of the gRNA conjugated AuNPs, Cpf1 endonuclease is added and incubated with gRNA conjugated AuNPs to facilitate its binding to the 5’ handle of the gRNA (Dong, et al., Nature, 2016.532(7600): p.522-526). The compact structure of the designed nanoparticle containing both gRNA and Cpf1 endonuclease results in a conformation which increases the stability against degrading agents and facilitates the uptake of the AuNP / CRISPR nanoparticle by cells owing to an overall neutral charge (i.e., zeta potential). While special relevance was given to optimizing the disclosed nanoparticle for CRISPR / Cpf1, the same concept may be applied to other CRISPR classes. Also, along with the gRNA and Cpf1 endonuclease, 18 spacer thiol modified single stranded DNA (ssDNA) can be attached to the surface of AuNPs to obtain a novel nanoparticle with the aim of being used in homology directed repair (HDR). In particular embodiments, the concentration of AuNPs in one or more of the steps described herein is at least 1.5 mg / mL. In particular embodiments, the concentration of AuNPs in one or more of the steps described herein is at least 2 mg / mL. In particular embodiments, the concentration of RNPs in one or more of the steps described herein is at least 2 mg / mL.

[0167] In particular embodiments, a spacer-thiol linker can be added to either of the Cpf1 or Cas9 proteins themselves or engineered variants of the foregoing (e.g., as described below), by addition of a cysteine residue on either the N- or C-terminus. The nuclease protein can then be added as a first layer on the gold nanoparticle surface. This spacer-thiol linker can increase theF053-6005PCT / 24-085-WO-PCT stability of the protein and increase cutting efficiency. In particular embodiments, an RNA complex is formed between gRNA and nuclease and then attached to the surface of gold nanoparticles through a spacer-thiol linker.

[0168] As indicated previously, adding gene-editing components of a bacterial origin as a first loading step can provide beneficial shielding of these components following administration to a subject with pre-existing immunity to the component. The shielding can be due to other gene- editing components (e.g., donor templates) and need not rely on a protective polymer shell. In particular embodiments, a polymer shell is excluded. In particular embodiments, the shielding may permit serial in vivo administration.

[0169] In particular embodiments, gRNAs can be added to AuNPs in different AuNP / gRNA w / w ratios (0.25, 0.5, 1, 1.5, 2, 3, 4, 5, 6) and mixed. Citrate buffer with the pH of 3 can be added to the mixture in 10 mM concentration to screen the negative repulsion between negatively charged gRNA and AuNP. After stirring for 5 min, nanoparticles can be centrifuged down and the unbound gRNA can be visualized by agarose gel electrophoresis. After determining the optimal conjugation concentration, 1 µL of 63 µM Cpf1 nuclease can be added to AuNP / gRNA solution and incubated for 20 min.

[0170] Importantly, the use of a citrate buffer provides significant advantages in manufacturing. Previous methods have relied on the use of NaCl to screen the negatively-charged nanoparticle surface and reduce repulsion of similarly negatively-charged DNA. However, NaCl can cause irreversible aggregation of gold nanoparticles, so it must be added gradually over time with incremental changes in concentration. Generally, NaCl must be added over a 48 hour time period to avoid aggregation. When citrate buffer is used with a pH of 3, this binding can happen with higher efficiency in less than 3 minutes. Zhang, et al. (2012). Journal of the American Chemical Society 134(17): 7266-7269 reducing the cost of goods and time in the GMP manufacturing facility.

[0171] In particular embodiments, the linker is attached to the nanoparticle and / or the targeting moiety at a particular pH. For instance, charge repulsion can reduce the efficiency of the attachment of the linker to the nanoparticle and / or the targeting moiety. Controlling the pH during nanoparticle synthesis can stabilize one or more components (e.g., the positively charged polymer, the nuclease, the targeting moiety). In some examples, the pH is 5.0 to 7.8 during the attachment of the linker and / or the targeting moiety. In some examples, the pH is 5.5 to 7.0 during the attachment of the linker and / or the targeting moiety. In some examples, the pH is 6.0 during the attachment of the linker and / or the targeting moiety. In particular embodiments, the pH is above 5.5 during the attachment of the linker and / or the targeting moiety to stabilize the nuclease.F053-6005PCT / 24-085-WO-PCT In particular embodiments, the pH is equal to the isoelectric point for the targeting moiety during the attachment of the linker and / or the targeting moiety. In some examples, the linker is attached to the nanoparticle at a first pH and the targeting moiety is attached to the nanoparticle at a second pH. In particular embodiments, the first pH is higher than the second pH.

[0172] In particular embodiments, a nanoparticle is layered, such as during synthesis to include PEI or other positively charged polymer for increasing surface area and conjugating larger ssDNA or other molecules, such as targeting moieties and / or large donor templates. This nanoparticle can be prepared in a layer-by-layer form and positively charged polymers (such as; PEI in different molecular weights and forms) can be used to coat the negatively charged surface of either gold nanoparticles or CRISPR coated gold nanoparticles to attach either gene editing components and other components (such as antibody binding domains). Layering essentially increases the surface area of the nanoparticle available for conjugating molecules such as large oligonucleotides with or without other proteins.

[0173] In particular embodiments, PEI can be added as a second layer and ssDNA can be added as a third layer. Alternatively, the conjugation steps can be changed by adding ssDNA as a second layer and PEI as a third layer. In particular embodiments, PEI, polymers, and ssDNA are not included as a first layer, as this layer can be reserved for RNP complexes coupled to linkers.

[0174] Particular embodiments can utilize a gold nanoparticle associated with at least four active layers wherein the first layer includes dsDNA, the second layer includes gRNA, the third layer includes a nuclease, and the fourth layer includes the targeting moiety and wherein the first layer is the closest active layer to the surface of the nanoparticle core, the second layer is the second closest active layer to the surface of the nanoparticle core, the third layer is third closest active layer to the nanoparticle core, and the fourth layer is the farthest active layer from the nanoparticle core. Attaching a targeting moiety to the nuclease, directly or through a linker, also provides immune shielding benefits.

[0175] Size and morphology of prepared AuNP / CRISPR nanoparticles can be characterized by imaging under transmission electron microscope (TEM). AuNPs can be added to copper grids and allowed to dry out overnight. Imaging is carried out at 120 kV. In particular embodiments, AuNP concentration is between 0.05 mg / mL and 0.2 mg / mL.

[0176] CRISPR coating can be visualized by negative staining electron microscopy. AuNP / CRISPR nanoparticle can be stained with 0.7% uranyl formate and 2% uranyl acetate, respectively. Stained sample (4 µL) can be added to carbon-coated copper grid and incubated for 1 min and blotted with a piece of filter paper. After three washing cycles with 20 µl stain solution, 4 µl stain solution can be added to the grids and blotted and air dried.F053-6005PCT / 24-085-WO-PCT

[0177] Also, AuNP / CRISPR nanoparticles can be characterized by Nanodrop UV-visible spectrophotometer by analyzing the shifts in localized surface plasmon resonance (LSPR) peak of the AuNPs before and after conjugation with CRISPR components.

[0178] (V) Nanoparticle Compositions and Cell Formulations. Nanoparticles can be formulated alone or in combination into compositions for administration to subjects. The nucleic acids, gene editing components, targeting moiety, and / or particle disclosed herein (individually, collectively, or in grouped combinations referred to as "active ingredients") can be provided as part of compositions formulated for administration to subjects.

[0179] Exemplary carriers for nanoparticle compositions and / or cell formulations include saline, buffered saline, physiological saline, water, HEPES buffer, Hanks' solution, Ringer's solution, Normosol-R (Abbott Labs), Plasma-Lyte A® (Baxter Laboratories, Inc., Morton Grove, IL), glycerol, ethanol, and combinations thereof. In particular embodiments, nanoparticle compositions and / or cell formulations are administered to subjects as soon as reasonably possible following their initial formulation.

[0180] In particular embodiments, carriers can be supplemented with human serum albumin (HSA) or other human serum components or fetal bovine serum or other species serum components. In particular embodiments, a carrier for infusion includes buffered saline with 5% HSA or dextrose. Additional isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0181] Carriers can include buffering agents, such as citrate buffers, succinate buffers, tartrate buffers, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, or trimethylamine salts.

[0182] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which helps to prevent component adherence to container walls. Typical stabilizers can include polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sodium azide; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, alpha-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins such as HSA, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose,F053-6005PCT / 24-085-WO-PCT maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran. Stabilizers are typically present in the range of from 0.1 to 10,000 parts by weight based on therapeutic weight.

[0183] Therapeutically effective amounts of nanoparticles within a composition can include at least 0.1% w / v or w / w particles; at least 1% w / v or w / w particles; at least 10% w / v or w / w particles; at least 20% w / v or w / w particles; at least 30% w / v or w / w particles; at least 40% w / v or w / w particles; at least 50% w / v or w / w particles; at least 60% w / v or w / w particles; at least 70% w / v or w / w particles; at least 80% w / v or w / w particles; at least 90% w / v or w / w particles; at least 95% w / v or w / w particles; or at least 99% w / v or w / w particles.

[0184] Therapeutically effective amounts of cells within cell-based formulations can be greater than 102cells, greater than 103cells, greater than 104cells, greater than 105cells, greater than 106cells, greater than 107cells, greater than 108cells, greater than 109cells, greater than 1010cells, or greater than 1011cells.

[0185] In particular embodiments, the compositions include nanoparticles of at least 0.1% w / v or w / w of the composition; at least 1% w / v or w / w of composition; at least 10% w / v or w / w of composition; at least 20% w / v or w / w of composition; at least 30% w / v or w / w of composition; at least 40% w / v or w / w of composition; at least 50% w / v or w / w of composition; at least 60% w / v or w / w of composition; at least 70% w / v or w / w of composition; at least 80% w / v or w / w of composition; at least 90% w / v or w / w of composition; at least 95% w / v or w / w of composition; or at least 99% w / v or w / w of composition.

[0186] Compositions disclosed herein can be formulated for administration by, for example, injection, infusion, perfusion, or lavage. The compositions disclosed herein can further be formulated for intraosseous, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral and / or subcutaneous administration and more particularly by intraosseous, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intratumoral, intramuscular, intravesicular, or subcutaneous injection.

[0187] In particular embodiments, it can be necessary or beneficial to freeze dry a nanoparticle composition and / or to cryopreserve a cell-based formulation. Such techniques are well known to those of ordinary skill in the art.

[0188] (VI) Methods of Use. Methods disclosed herein include treating subjects (humans, non- human primates, veterinary animals (dogs, cats, reptiles, birds, etc.) livestock (horses, cattle, goats, pigs, chickens, etc.) and research animals (monkeys, rats, mice, fish, etc.)) withF053-6005PCT / 24-085-WO-PCT formulations disclosed herein. Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and / or therapeutic treatments.

[0189] An "effective amount" is the amount of a formulation necessary to result in a desired physiological change in the subject. For example, an effective amount can provide a therapeutic effect in a genetic, infectious, or malignant disease. Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause a statistically significant effect in an animal model or in vitro assay relevant to the assessment of a genetic, infectious, or malignant disease’s development or progression.

[0190] A "prophylactic treatment" includes a treatment administered to a subject who does not display signs or symptoms of a genetic, infectious, or malignant disease or displays only early signs or symptoms of a genetic, infectious, or malignant disease such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the disease further. Thus, a prophylactic treatment functions as a preventative treatment against a disease.

[0191] A "therapeutic treatment" includes a treatment administered to a subject who displays symptoms or signs of a genetic, infectious, or malignant disease and is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms of the disease. The therapeutic treatment can reduce, control, or eliminate the presence or activity of the genetic, infectious, or malignant disease and / or reduce control or eliminate side effects of the disease.

[0192] Function as an effective amount, prophylactic treatment or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.

[0193] In particular embodiments, therapeutically effective amounts provide anti-disease effects. Anti-disease effects include a decrease in the number of diseased cells, an increase in life expectancy, inhibited diseased cell proliferation, decrease in disease-related symptoms, reduced disease-associated pain, and / or reduced relapse or re-occurrence of disease following treatment.

[0194] Examples of diseases that can be treated using the nanoparticle compositions of the disclosure include a monogenetic blood disorder, hemophilia, Grave's Disease, rheumatoid arthritis, pernicious anemia, Multiple Sclerosis (MS), inflammatory bowel disease, systemic lupus erythematosus (SLE), Wiskott-Aldrich syndrome (WAS), chronic granulomatous disease (CGD), Battens disease, adrenoleukodystrophy (ALD) or metachromatic leukodystrophy (MLD), muscular dystrophy, pulmonary aveolar proteinosis (PAP), pyruvate kinase deficiency, Shwachmann- Diamond-Blackfan anemia, dyskeratosis congenita, cystic fibrosis, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (Lou Gehrig's disease), acute lymphoblasticF053-6005PCT / 24-085-WO-PCT leukemia (ALL), acute myelogenous leukemia (AML), agnogenic myeloid metaplasia, amegakaryocytosis / congenital thrombocytopenia, ataxia telangiectasia, β-thalassemia major, CLL, chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia, common variable immune deficiency (CVID), complement disorders, congenital (X-linked) agammaglobulinemia, familial erythrophagocytic lymphohistiocytosis, Hodgkin's lymphoma, Hurler's syndrome, hyper IgM, IgG subclass deficiency, juvenile myelomonocytic leukemia, mucopolysaccharidoses, multiple myeloma, myelodysplasia, non-Hodgkin's lymphoma, paroxysmal nocturnal hemoglobinuria (PNH), primary immunodeficiency diseases with antibody deficiency, pure red cell aplasia, refractory anemia, selective IgA deficiency, severe aplastic anemia, SCD, and / or specific antibody deficiency.

[0195] Particular embodiments include treatment of bacterial and / or parasitic infections. One exemplary parasite includes malaria-causing Plasmodium.

[0196] For administration, therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and / or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest. The actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of disease, stage of disease, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.

[0197] Therapeutically effective amounts of nanoparticles can range from 0.1 to 5 µg / kg or from 0.5 to 1 µg / kg. In other examples, a dose can include 1 µg / kg, 30 µg / kg, 90 µg / kg, 150 µg / kg, 500 µg / kg, 750 µg / kg, 0.1 to 5 mg / kg or from 0.5 to 1 mg / kg. In other examples, a dose can include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg, or more.

[0198] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or yearly). In particular embodiments, the treatment protocol may be dictated by a clinical trial protocol or an FDA- approved treatment protocol.

[0199] Therapeutically effective amounts can be administered by, e.g., injection, infusion, perfusion, or lavage. Routes of administration can include intraosseous, intravenous, intradermal,F053-6005PCT / 24-085-WO-PCT intraarterial, intraparenteral, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, and / or subcutaneous administration.

[0200] Where necessary or beneficial, nanoparticle compositions and / or cell formulations can include a local anesthetic such as lidocaine to ease pain at a site of injection.

[0201] Particular embodiments include nanoparticles that can be targeted to specific selected cells and accomplish delivery of all required genetic-engineering components by mixing the nanoparticles with cells in culture ex vivo or by administering the nanoparticles to a subject. In particular embodiments, the nanoparticles include: (1) a targeting moiety; and (2) gene-editing components.

[0202] When the disclosed nanoparticles are added to a heterogeneous mixture of cells (e.g., an ex vivo blood product that has undergone minimal manipulation), the engineered nanoparticles bind to selected cell populations and are internalized into the target cell. This process provides entry for the genetic engineering components the nanoparticles carry, and consequently the selected cells become genetically modified. Provision of all components required for genetic engineering on a single particle ensures that a cell that takes up the particle receives all necessary components rather than a subset thereof.

[0203] The nanoparticles can be used for therapies where a loss-of-function mutation is needed, but importantly, can also provide all components needed for gene addition or correction of a specific mutation. The described approaches are safe (i.e., no off-target toxicity), reliable, scalable, easy to manufacture, synthetic, plug-and-play (i.e., the same basic platform can be used to deliver different therapeutic nucleic acids), and compatible with ex vivo manufacturing and in vivo administration.

[0204] In particular embodiments, compositions are administered to a subject. In particular embodiments, cells can be obtained from a subject or donor and genetically modified at a target site before administration to the subject to treat a condition. Common sources of appropriate cells include mobilized peripheral blood samples, bone marrow samples, and / or umbilical cord blood.

[0205] Hematopoietic stem cells (HSC) are stem cells that can give rise to all blood cell types such as the white blood cells of the immune system (e.g., virus-fighting T cells and antibody- producing B cells) and red blood cells. The therapeutic administration of HSC can be used to treat a variety of adverse conditions including immune deficiency diseases, blood disorders, malignant cancers, infections, and radiation exposure (e.g., cancer treatment, accidental, or attack-based). As examples, more than 80 primary immune deficiency diseases are recognized by the World Health Organization. These diseases are characterized by an intrinsic defect in the immuneF053-6005PCT / 24-085-WO-PCT system in which, in some cases, the body is unable to produce any or enough antibodies against infection. In other cases, cellular defenses to fight infection fail to work properly. Typically, primary immune deficiencies are inherited disorders.

[0206] In particular embodiments, a therapeutically effective treatment induces or increases production of hemoglobin; induces or increases production of beta-globin, or alpha-globin; and / or increases the availability of oxygen to cells in the body.

[0207] In particular embodiments, a therapeutically effective treatment increases blood cell counts, improves blood cell function, and / or increases oxygenation of cells.

[0208] In particular embodiments, a therapeutically effective treatment increases the production of coagulation / clotting factor VIII or coagulation / clotting factor IX, causes the production of normal versions of coagulation factor VIII or coagulation factor IX, reduces the production of antibodies to coagulation / clotting factor VIII or coagulation / clotting factor IX, and / or causes the proper formation of blood clots.

[0209] In particular embodiments, a therapeutically effective treatment causes the degradation of mucopolysaccharides in lysosomes, reduces, eliminates, prevents, or delays the swelling in various organs, including the head (exp. Macrosephaly), the liver, spleen, tongue, or vocal cords; reduces fluid in the brain; reduces heart valve abnormalities; prevents or dilates narrowing airways, reduces or prevent upper respiratory conditions like infections and sleep apnea; and / or reduces, eliminates, prevents, or delays the destruction of neurons and / or the symptoms associated with the destruction of neurons.

[0210] In particular embodiments, therapeutically effective amounts may provide function to immune and other blood cells, reduce or eliminate an immune-mediated condition; and / or reduce or eliminate a symptom of the immune-mediated condition.

[0211] In particular embodiments, particular methods of use include in the treatment of conditions where corrected cells have a selective advantage over non-corrected cells. For example, in FA and SCID, corrected cells have an advantage and only transducing the therapeutic gene into a “few” HSPCs is sufficient for therapeutic efficacy.

[0212] Additional methods of treatment can be found in International Patent Application PCT / US2016 / 014378, filed 1 / 21 / 2016 and US Provisional Application Nos. 62 / 351,761, filed 6 / 17 / 2016 and 62 / 428,994, filed 12 / 1 / 2016, each of which is specifically incorporated herein in their entirety.

[0213] Ex Vivo Methods of Use. The nanoparticles disclosed herein can be used in ex vivo cell manufacturing. For example, the methods can include obtaining cells from a subject. Cells can, e.g., be obtained from a subject using any procedure generally known in the art.F053-6005PCT / 24-085-WO-PCT

[0214] Sources of HSPC or other lymphocytes include umbilical cord blood, placental blood, bone marrow and peripheral blood. Methods regarding collection, anti-coagulation and processing, etc. of blood samples are known. See, for example, Alsever et al., 1941, N.Y. St. J. Med.41:126; De Gowin, et al., 1940, J. Am. Med. Ass.114:850; Smith, et al., 1959, J. Thorac. Cardiovasc. Surg. 38:573; Rous and Turner, 1916, J. Exp. Med. 23:219; and Hum, 1968, Storage of Blood, Academic Press, New York, pp. 26-160. Sources of HSPC or lymphocytes also include bone marrow (see Kodo et al., 1984, J. Clin Invest. 73:1377-1384), embryonic cells, aortal-gonadal- mesonephros derived cells, lymph, liver, thymus, and spleen from age-appropriate donors. All collected samples can be screened for undesirable components and discarded, treated, or used according to accepted current standards at the time.

[0215] In particular embodiments, HSC in peripheral blood are mobilized prior to collection. Peripheral blood HSC can be mobilized by any method. Peripheral blood HSC can be mobilized by treating the subject with any agent(s), described herein or known in the art, that increase the number of HSC circulating in the peripheral blood of the subject. For example, in particular embodiments, peripheral blood is mobilized by treating the subject with one or more cytokines or growth factors (e.g., G-CSF, kit ligand (KL), IL-l, IL-7, IL-8, IL-11, Flt3 ligand, SCF, thrombopoietin, or GM-CSF (such as sargramostim)). Different types of G-CSF that can be used in the methods for mobilization of peripheral blood include filgrastim and longer acting G-CSF-pegfilgrastim. In particular embodiments, peripheral blood is mobilized by treating the subject with one or more chemokines (e.g., macrophage inflammatory protein-1α (MIP1α / CCL3)), chemokine receptor ligands (e.g., chemokine receptor 2 ligands GROβ and GROβΔ4), chemokine receptor analogs (e.g., stromal cell derived factor-1α (SDF-1α) protein analogs such as CTCE-0021, CTCE-0214, or SDF-1α such as Met-SDF-lβ), or chemokine receptor antagonists (e.g., chemokine (C-X-C motif) receptor 4 (CXCR4) antagonists such as AMD3100).

[0216] In particular embodiments, peripheral blood is mobilized by treating the subject with one or more anti-integrin signaling agents (e.g., function blocking anti-very late antigen 4 (VLA-4) antibody, or anti-vascular cell adhesion molecule 1 (VCAM-1)).

[0217] In particular embodiments, peripheral blood is mobilized by treating the subject with one or more cytotoxic drugs such as cyclophosphamide, etoposide or paclitaxel.

[0218] In particular embodiments, peripheral blood can be mobilized by administering to a subject one or more of the agents listed above for a certain period of time. For example, the subject can be treated with one or more agents (e.g., G-CSF) via injection (e.g., subcutaneous, intravenous or intraperitoneal), once daily or twice daily, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days prior to collection of HSC. In specific embodiments, HSC are collected within 1, 2, 3, 4, 5, 6, 7, 8,F053-6005PCT / 24-085-WO-PCT 12, 14, 16, 18, 20 or 24 hours after the last dose of an agent used for mobilization of HSC into peripheral blood. In particular embodiments, HSC are mobilized by treating the subject with two or more different types of agents described above or known in the art, such as a growth factor (e.g., G-CSF) and a chemokine receptor antagonist (e.g., CXCR4 receptor antagonist such as AMD3100), or a growth factor (e.g., G-CSF or KL) and an anti-integrin agent (e.g., function blocking VLA-4 antibody). In particular embodiments, different types of mobilizing agents are administered concurrently or sequentially. For additional information regarding methods of mobilization of peripheral blood see, e.g., Craddock et al., 1997, Blood 90(12):4779-4788; Jin et al., 2008, Journal of Translational Medicine 6:39; Pelus, 2008, Curr. Opin. Hematol.15(4):285- 292; Papayannopoulou et al., 1998, Blood 91(7):2231-2239; Tricot et al., 2008, Haematologica 93(11):1739-1742; and Weaver et al., 2001, Bone Marrow Transplantation 27(2):S23-S29).

[0219] In particular embodiments, nanoparticles can be administered to a subject (e.g., intravenously) following mobilization while HSC remain in circulation.

[0220] HSC from peripheral blood can be collected from the blood through a syringe or catheter inserted into a subject's vein. For example, in particular embodiments, the peripheral blood can be collected using an apheresis machine. Blood flows from the vein through the catheter into an apheresis machine, which separates the white blood cells, including HSC from the rest of the blood and then returns the remainder of the blood to the subject's body. Apheresis can be performed for several days (e.g., 1 to 5 days) until enough HSC have been collected.

[0221] In particular embodiments, no further collection or isolation of HSPC or lymphocytes is needed before exposing the acquired sample to nanoparticles disclosed herein because the nanoparticles selectively target selected cell types within a heterogeneous cell population. In particular embodiments, the acquired sample has undergone minimal manipulation.

[0222] In particular embodiments, it may be beneficial to engage in some limited further cell collection and isolation before exposure to nanoparticles disclosed herein. In particular embodiments, HSPC or lymphocytes can be collected and isolated from a sample using any appropriate technique. Appropriate collection and isolation procedures include magnetic separation; fluorescence activated cell sorting (FACS; Williams et al., 1985, J. Immunol. 135:1004; Lu et al., 1986, Blood 68(1):126-133); affinity chromatography; agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody; "panning" with antibody attached to a solid matrix (Broxmeyer et al., 1984, J. Clin. Invest. 73:939-953); selective agglutination using a lectin such as soybean (Reisner et al., 1980, Proc. Natl. Acad. Sci. U.S.A. 77:1164); etc. Particular embodiments can utilize limited isolation. Limited isolation refers to crude cell enrichment, for example, by removal of red blood cells and / or adherent phagocytes.F053-6005PCT / 24-085-WO-PCT

[0223] In particular embodiments, a subject sample (e.g., a blood sample) can be processed to select / enrich for a particular cellular profile. The sample can be processed by using, for example, CD34+ HSPC using anti-CD8 or anti-CD34 antibodies directly or indirectly conjugated to magnetic particles in connection with a magnetic cell separator, for example, the CliniMACS® Cell Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany). In particular embodiments, cells can be enriched for and / or isolated based on CD34 alone; CD133+ alone; CD90+ alone; CD164+ alone; CD46+ alone; or LH+ alone. In particular embodiments, cells can be enriched for and / or isolated based on one or more of CD34; CD133+; CD90+; CD164+; CD46+; or LH+ in various combinations. In particular embodiments, LH+ means that a cell expresses the LH receptor.

[0224] Particular HSC populations include HSC1 (Lin-CD34+CD38-CD45RA-CD90+CD49f+) and HSC2 (CD34+CD38-CD45RA-CD90- CD49f+). For example, in particular embodiments, human HSC1 can be identified by the following profile: CD34+ / CD38- / CD45RA- / CD90+ or CD34+ / CD45RA- / CD90+ and mouse LT-HSC can be identified by Lin-Sca1+ckit+CD150+CD48- Flt3-CD34- (where Lin represents the absence of expression of any marker of mature cells including CD3, Cd4, CD8, CD11b, CD11c, NK1.1, Gr1, and TER119). Thus, HSC1 can include the marker profile: LHR+ / CD34+ / CD38- / CD45RA- / CD90+. In addition to expression of LHR, in particular embodiments, HSC1 can be identified by the following profile: Lin- / CD34+ / CD38- / CD45RA- / CD90+ / CD49f+. Thus, HSC1 can include the marker profile: LHR+ / Lin- / CD34+ / CD38- / CD45RA- / CD90+ / CD49f+. In addition to expression of LHR, in particular embodiments, HSC2 can be identified by the following profile: CD34+ / CD38- / CD45RA- / CD90- / CD49f+. Thus, HSC2 can include the marker profile: LHR+ / CD34+ / CD38- / CD45RA- / CD90- / CD49f+. Based on the foregoing profiles, expression of LHR can be combined with presence or absence of the following one or more markers to prepare HSC1 and / or HSC2 cell populations: Lin / CD34 / CD38 / CD45RA / CD90 / CD49f as well as CD133. Various other combinations may also be used so long as the marker combination reliably isolates HSC1 or HSC2. In particular embodiments, HSC are identified by a CD133+ profile. In particular embodiments, HSC are identified by a CD34+ / CD133+ profile. In particular embodiments, HSC are identified by a CD164+ profile. In particular embodiments, HSC are identified by a CD34+ / CD164+ profile. In particular embodiments, HSC are identified by a CD34+ / CD38- profile.

[0225] In particular embodiments, cells are modified ex vivo with minimal manipulation using a targeted nanoparticle disclosed herein. In particular embodiments, minimal manipulation means that a sample containing the selected cell type is washed before administration of nanoparticles disclosed herein. Following administration of the nanoparticles, the biological sample can be re-F053-6005PCT / 24-085-WO-PCT infused into a patient. In particular embodiments, minimal manipulation means that a sample containing the selected cell type is washed before administration of nanoparticles disclosed herein. Following administration of the nanoparticles, the biological sample can be cryo-preserved before thawing and re-infusion into a patient. In particular embodiments, minimal manipulation means that a sample containing the selected cell type is cryopreserved, followed by thawing and administration of nanoparticles, cryopreserved, thawed, and re-infused into a patient.

[0226] In particular embodiments, minimal manipulation means that no cell selection or purification processes are required before the genetic modification. In particular embodiments, minimal manipulation means that no cell selection or purification processes are used before the genetic modification. In particular embodiments, minimal manipulation means that a patient cell product is only washed to remove platelets, but that no further cell selection or purification steps are performed. In particular embodiments, minimal manipulation means that no electroporation or viral vector delivery is required to deliver gene editing components. In particular embodiments, minimal manipulation means that no electroporation or viral vector delivery is used to deliver gene editing components.

[0227] In particular embodiments, nanoparticles will be combined with cells before or at the early stages of an expansion procedure. This approach will allow modification of a smaller number of cells, with the modification being propagated throughout the cell population as it expands. In particular embodiments, following exposure to nanoparticles disclosed herein that provide a targeted genetic modification, expansion of the modified cells can occur.

[0228] Expansion can occur in the presence of one more growth factors, such as: angiopoietin- like proteins (Angptls, e.g., Angptl2, Angptl3, Angptl7, Angpt15, and Mfap4); erythropoietin; fibroblast growth factor-1 (FGF-1); Flt-3 ligand (Flt-3L); granulocyte colony stimulating factor (G- CSF); granulocyte-macrophage colony stimulating factor (GM-CSF); insulin growth factor-2 (IFG- 2); interleukin-3 (IL-3); interleukin-6 (IL-6); interleukin-7 (IL-7); interleukin-11 (IL-11); stem cell factor (SCF; also known as the c-kit ligand or mast cell growth factor); thrombopoietin (TPO); and analogs thereof (wherein the analogs include any structural variants of the growth factors having the biological activity of the naturally occurring growth factor; see, e.g., WO 2007 / 1145227 and U.S. Patent Publication No.2010 / 0183564).

[0229] In particular embodiments, the amount or concentration of growth factors suitable for expanding cells is the amount or concentration effective to promote proliferation. Cell populations are preferably expanded until a sufficient number of cells are obtained to provide for at least one infusion into a human subject, typically around 104 cells / kg to 109 cells / kg.

[0230] The amount or concentration of growth factors suitable for expanding cells depends onF053-6005PCT / 24-085-WO-PCT the activity of the growth factor preparation, and the species correspondence between the growth factors and cell population, etc. Generally, when the growth factor(s) and cell population are of the same species, the total amount of growth factor in the culture medium ranges from 1 ng / ml to 5 µg / ml, from 5 ng / ml to 1 µg / ml, or from 5 ng / ml to 250 ng / ml. In particular embodiments, the amount of growth factors can be in the range of 5-1000 or 50-100 ng / ml.

[0231] In particular embodiments, growth factors are present in an expansion culture condition at the following concentrations: 25-300 ng / ml SCF, 25-300 ng / ml Flt-3L, 25-100 ng / ml TPO, 25-100 ng / ml IL-6 and 10 ng / ml IL-3. In particular embodiments, 50, 100, or 200 ng / ml SCF; 50, 100, or 200 ng / ml of Flt-3L; 50 or 100 ng / ml TPO; 50 or 100 ng / ml IL-6; and 10 ng / ml IL-3 can be used.

[0232] The cell population (e.g., HSPC) can be expanded in a tissue culture dish onto which an extracellular matrix protein such as fibronectin (FN), or a fragment thereof (e.g., CH-296 (Dao et. al., 1998, Blood 92(12):4612-21)) or RetroNectin® (a recombinant human fibronectin fragment; (Clontech Laboratories, Inc., Madison, WI) is bound.

[0233] Notch agonists can be particularly useful for expanding HSC. In particular embodiments, HSC can be expanded by exposing the HSC to an immobilized Notch agonist, and 50 ng / ml or 100 ng / ml SCF; to an immobilized Notch agonist, and 50 ng / ml or 100 ng / ml of each of Flt-3L, IL- 6, TPO, and SCF; or an immobilized Notch agonist, and 50 ng / ml or 100 ng / ml of each of Flt-3L, IL-6, TPO, and SCF, and 10 ng / ml of IL-11 or IL-3.

[0234] For additional general information regarding appropriate culturing and / or expansion conditions, see U.S. Patent No.7,399,633; U.S. Patent Publication No.2010 / 0183564; Freshney Culture of Animal Cells, Wiley-Liss, Inc., New York, NY (1994)); Vamum-Finney et al., 1993, Blood 101:1784-1789; Ohishi et al., 2002, J. Clin. Invest.110:1165-1174; Delaney et al., 2010, Nature Med. 16(2): 232-236; WO 2006 / 047569A2; WO 2007 / 095594A2; U.S. Patent 5,004,681; WO 2011 / 127470 A1; WO 2011 / 127472A1; and See Chapter 2 of Regenerative Medicine, Department of Health and Human Services, August 2006, and the references cited therein.

[0235] (VII) Reference Levels Derived from Control Populations. Obtained values for parameters associated with a therapy described herein can be compared to a reference level derived from a control population, and this comparison can indicate whether a therapy described herein is effective for a subject in need thereof. Reference levels can be obtained from one or more relevant datasets from a control population. A "dataset" as used herein is a set of numerical values resulting from evaluation of a sample (or population of samples) under a desired condition. The values of the dataset can be obtained, for example, by experimentally obtaining measures from a sample and constructing a dataset from these measurements. As is understood by one of ordinary skill in the art, the reference level can be based on e.g., any mathematical or statisticalF053-6005PCT / 24-085-WO-PCT formula useful and known in the art for arriving at a meaningful aggregate reference level from a collection of individual data points; e.g., mean, median, median of the mean, etc. Alternatively, a reference level or dataset to create a reference level can be obtained from a service provider such as a laboratory, or from a database or a server on which the dataset has been stored.

[0236] A reference level from a dataset can be derived from previous measures derived from a control population. A "control population" is any grouping of subjects or samples of like specified characteristics. The grouping could be according to, for example, clinical parameters, clinical assessments, therapeutic regimens, disease status, severity of condition, etc. In particular embodiments, the grouping is based on age range (e.g., 0-2 years) and non- immunocompromised status. In particular embodiments, a normal control population includes individuals that are age-matched to a test subject and non-immune compromised. In particular embodiments, age-matched includes, e.g., 0-6 months old; 0-1 year old; 0-2 years old; 0-3 years old; 10-15 years old, as is clinically relevant under the circumstances. In particular embodiments, a control population can include those that have an immune deficiency and have not been administered a therapeutically effective amount.

[0237] In particular embodiments, the relevant reference level for values of a particular parameter associated with a therapy described herein is obtained based on the value of a particular corresponding parameter associated with a therapy in a control population to determine whether a therapy disclosed herein has been therapeutically effective for a subject in need thereof.

[0238] In particular embodiments, conclusions are drawn based on whether a sample value is statistically significantly different or not statistically significantly different from a reference level. A measure is not statistically significantly different if the difference is within a level that would be expected to occur based on chance alone. In contrast, a statistically significant difference or increase is one that is greater than what would be expected to occur by chance alone. Statistical significance or lack thereof can be determined by any of various methods well-known in the art. An example of a commonly used measure of statistical significance is the p-value. The p-value represents the probability of obtaining a given result equivalent to a particular data point, where the data point is the result of random chance alone. A result is often considered significant (not random chance) at a p-value less than or equal to 0.05. In particular embodiments, a sample value is “comparable to” a reference level derived from a normal control population if the sample value and the reference level are not statistically significantly different.

[0239] (VIII) Kits. The disclosure also provides kits containing any one or more of the elements disclosed in the methods and compositions herein. In particular embodiments, a kit can include a targeting moiety, a nanoparticle, gene-editing components, targeting elements, cutting elements,F053-6005PCT / 24-085-WO-PCT gRNA, a nuclease capable of cutting a target sequence in a cell, HDR template, homology arms, and / or therapeutic nucleic acid sequences. Elements may be provided individually or in combinations, and may be provided in any suitable container, such as a vial, a bottle, a bag or a tube. In some embodiments, the kit includes instructions in one or more languages, for example in more than one language.

[0240] In particular embodiments, a kit includes one or more reagents for use in a process utilizing one or more of the elements described herein. Reagents may be provided in any suitable container. For example, a kit may provide one or more reaction or storage buffers. Reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g., in concentrate or lyophilized form). A buffer can be any buffer, including a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH from 7 to 10. In some embodiments, the kit includes a homologous recombination template polynucleotide.

[0241] Particular embodiments can include TFP ester, PEG, OEG, or DBCO. Particular embodiments can include linkers that include TFP ester, DBCO, and a PEG spacer. Particular embodiments can include quenchers, such as free azide and / or or free thiol. Particular embodiments can include enzymes, such as N-acetylgalactosaminyltransferase (GalNAc transferase). Particular embodiments can include reducing agents, such as dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), 2-Mercaptoethanol, or glutathione (GSH).

[0242] The Exemplary Embodiments and Examples below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0243] (IX) Exemplary Embodiments. 1. A method of selectively genetically modifying cells of a selected cell type, wherein the method includes: contacting a population of cells including the selected cell type with a nanoparticle, wherein the nanoparticle includes: i) up to 1000 targeting moieties linked to a nanoparticle via 500-3000 linkers attached to the nanoparticle, wherein the targeting moieties selectively bind an antigen expressed by the selected cell type and result in internalization of the bound nanoparticle by the selected cell type; and ii) gene-editing components including a targeting element and a cutting element. 2. The method of embodiment 1, wherein the linkers include heterofunctional linkers.F053-6005PCT / 24-085-WO-PCT 3. The method of embodiment 2, wherein the heterofunctional linkers are attached to the nanoparticle via click chemistry. 4. The method of embodiment 2 or 3, wherein the targeting moieties are attached to the linkers via click chemistry. 5. The method of embodiment 3 or 4, wherein the click chemistry includes strain promoted azide- alkyne click (SPAAC) chemistry or copper-catalyzed Azide-alkyne click (CuAAC) chemistry. 6. The method of any of embodiments 3-5, wherein the linker and / or the targeting moiety include a primary amine or a sulfhydryl. 7. The method of any of embodiments 3-6, wherein the heterofunctional linkers include a primary amine and the targeting moiety includes a sulfhydryl. 8. The method of any of embodiments 1-7, wherein the linkers include a dibenzocyclooctyne (DBCO) group. 9. The method of any of embodiments 1-8, wherein the linkers include a trifluoromethylphenyl (TFP) ester. 10. The method of any of embodiments 1-9, wherein the linker attaches to an azide, an amine, or a thiol on the targeting moiety. 11. The method of embodiment 10, wherein the targeting moiety is modified to include the azide, the amine, or the thiol. 12. The method of embodiment 10 or 11, wherein the linker attaches to the azide on the targeting moiety by azide-alkyne cycloaddition. 13. The method of any of embodiments 1-12, wherein the linkers include a 4-24 unit PEG spacer. 14. The method of any of embodiments 1-13, wherein the linkers include a 4, 12, or 24 unit PEG spacer. 15. The method of any of embodiments 1-14, wherein the targeting moieties includes a protein, an aptamer, or a small molecule. 16. The method of embodiment 15, wherein the protein includes an antibody, a peptide, or a ligand. 17. The method of embodiment 16, wherein the antibody binds CD133, CD117, or CD90. 18. The method of embodiment 16 or 17, wherein the antibody includes an anti-CD133 antibody, an anti-CD117 antibody, or an anti-CD90 antibody. 19. The method of any of embodiments 16-18, wherein the antibody includes αCD133:7, αCD117:104D2 or αCD90:5E10. 20. The method of any of embodiments 16-19, wherein the ligand includes stem cell factor (SCF). 21. The method of any of embodiments 15-20, wherein the protein includes human luteinizingF053-6005PCT / 24-085-WO-PCT hormone. 22. The method of any of embodiments 15-21, wherein the aptamer binds CD133. 23. The method of any of embodiments 15-22, wherein the aptamer includes A15 or B19. 24. The method of any of embodiments 15-23, wherein the small molecule includes degarelix acetate. 25. The method of any of embodiments 1-24, wherein the targeting moieties are attached to the gene-editing components by the linker. 26. The method of any of embodiments 1-25, wherein the cutting element includes a nuclease. 27. The method of embodiment 26, wherein the nuclease includes Cpf1 or Cas9. 28. The method of any of embodiments 1-27, wherein the targeting element includes a guide RNA (gRNA). 29. The method of any of embodiments 1-28, wherein the targeting element includes a single gRNA. 30. The method of embodiment 29, wherein the gRNA includes: UAAUUUCUACUCUUGUAGAUUUCGGACCCGUGCUACAACUU (SEQ ID NO: 2); UAAUUUCUACUCUUGUAGAUAUAGAAUAGCCUCAUAUUUUA (SEQ ID NO: 3); UAAUUUCUACUCUUGUAGAUGAGCUGUUGGCAUCAUGUUCCUG (SEQ ID NO: 4); UAAUUUCUACUCUUGUAGAUUCCAAACCUCCUAAAUGAUAC (SEQ ID NO: 5); or UAAUUUCUACUCUUGUAGAUCACCCGAUCCACUGGGGAGCA (SEQ ID NO: 6). 31. The method of embodiment 29 or 30, wherein the gRNA targets a sequence including: TTTGTGTCCCCGTTTTGGTTGGTAAAC (SEQ ID NO: 7); TTTAAAAATCAATACCGATAATAATGA (SEQ ID NO: 8); TTTCTTAATATGAATATTAATATCGGT (SEQ ID NO: 9); TTTCCGTATCTGGAAGGGGCATCTTGG (SEQ ID NO: 10); TTTCCTTAGGACCGGAAGGATTACAGC (SEQ ID NO: 11); TTTGCCTAAAAGGCACTATGTCAAATG (SEQ ID NO: 12); TTTGGAGCTGTTGGCATCATGTTCCTG (SEQ ID NO: 13); TTTGATTCTTTTCTATCTCAGGACAGA (SEQ ID NO: 14); TTTATAGACATCCCACACTGTAGTTCT (SEQ ID NO: 15); TTTATTAATTTGAGAACCAACATAAGG (SEQ ID NO: 16); TTTATTTTCTTTTTGGTAAGAAGGAAC (SEQ ID NO: 17); TTTCACACACACACACACACACACACA (SEQ ID NO: 18); TTTGGAGCTGTTGGCATCATGTTCCTG (SEQ ID NO: 19); TTTATCCAAACCTCCTAAATGATAC (SEQ ID NO: 20);F053-6005PCT / 24-085-WO-PCT TTTACACCCGATCCACTGGGGAGCA (SEQ ID NO: 21); or TTTTTGATTCTTTTCTATCTCAGGACA (SEQ ID NO: 22). 32. The method of any of embodiments 1-31, wherein the targeting moieties are linked to the cutting element by the linker. 33. The method of embodiment 32, wherein the linker binds to an amine group of the cutting element. 34. The method of any of embodiments 1-33, wherein the nanoparticle includes 10 to 250 linkers per cutting element. 35. The method of any of embodiments 1-34, wherein the nanoparticle further includes an endosomal polymer shell. 36. The method of embodiment 35, wherein the targeting moiety is linked to the endosomal polymer shell by the linker. 37. The method of any of embodiments 1-36, wherein the nanoparticle is a gold nanoparticle. 38. The method of embodiment 37, wherein the gold nanoparticle is associated with a polyethylenimine (PEI) layer. 39. The method of embodiment 38, wherein the PEI layer is closest to the surface of the gold nanoparticle. 40. The method of any of embodiments 1-39, wherein the gene-editing components further include a donor template. 41. The method of embodiment 40, wherein the donor template includes double-stranded DNA (dsDNA). 42. The method of embodiment 40 or 41, wherein the donor template includes single-stranded DNA (ssDNA). 43. The method of any of embodiments 40-42, wherein the donor template includes a therapeutic gene. 44. The method of embodiment 43, wherein the therapeutic gene includes or encodes skeletal protein 4.1, glycophorin, p55, the Duffy allele, globin family genes, WAS, phox, dystrophin, pyruvate kinase, CLN3, ABCD1, arylsulfatase A, SFTPB, SFTPC, NLX2.1, ABCA3, GATA1, ribosomal protein genes, TERT, TERC, DKC1, TINF2, CFTR, LRRK2, PARK2, PARK7, PINK1, SNCA, PSEN1, PSEN2, APP, SOD1, TDP43, FUS, ubiquilin 2, C9ORF72, α2β1, αvβ3, αvβ5, αvβ63, BOB / GPR15, Bonzo / STRL-33 / TYMSTR, CCR2, CD55, CXCR4, aminopeptidase-N, HHV-7, ICAM, ICAM-1,LDLR / α2MR / LRP, PVR; PRR1 / HveC, laminin receptor, 101F6, 123F2, 53BP2, abl, ABLI, ADP, aFGF, APC, ApoAI, ApoAIV, ApoE, ATM, BAI-1, BDNF, Beta*(BLU), bFGF, BLC1, BLC6,F053-6005PCT / 24-085-WO-PCT BRCA1, BRCA2, CBFA1, CBL, C-CAM, CFTR, CNTF, COX-1, CSFIR, CTS-1, cytosine deaminase, DBCCR-1, DCC, Dp, DPC-4, E1A, E2F, EBRB2, erb, ERBA, ERBB, ETS1, ETS2, ETV6, Fab, FancA, FancB, FancC, FancD1, FancD2, FancE, FancF, FancG, FancI, FancJ, FancL, FancM, FancN, FancO, FancP, FancQ, FancR, FancS, FancT, FancU, FancV, and FancW, FCC, FGF, FGR, FHIT, fms, FOX, FUS 1, FUS1, FYN, G-CSF, GDAIF, Gene 21, Gene 26, GM-CSF, GMF, gsp, HCR, HIC-1, HRAS, hst, IGF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, ING1, interferon α, interferon β, interferon γ, IRF-1, JUN, KRAS, LCK, LUCA-1, LUCA-2, LYN, MADH4, MADR2, MCC, mda7, MDM2, MEN-I, MEN-II, MLL, MMAC1, MYB, MYC, MYCL1, MYCN, neu, NF-1, NF-2, NGF, NOEY1, NOEY2, NRAS, NT3, NT5, OVCA1, p16, p21, p27, p53, p57, p73, p300, PGS, PIM1, PL6, PML, PTEN, raf, Rap1A, ras, Rb, RB1, RET, rks-3, ScFv, scFV ras, SEM A3, SRC, TAL1, TCL3, TFPI, thrombospondin, thymidine kinase, TNF, TP53, trk, T-VEC, VEGF, VHL, WT1, WT-1, YES, zac1, iduronidase, IDS, GNS, HGSNAT, SGSH, NAGLU, GUSB, GALNS, GLB1, ARSB, HYAL1, F8, F9, HBB, CYB5R3, γC, JAK3, IL7RA, RAG1, RAG2, DCLRE1C, PRKDC, LIG4, NHEJ1, CD3D, CD3E, CD3Z, CD3G, PTPRC, ZAP70, LCK, AK2, ADA, PNP, WHN, CHD7, ORAI1, STIM1, CORO1A, CIITA, RFXANK, RFX5, RFXAP, RMRP, DKC1, TERT, TINF2, DCLRE1B, or SLC46A1. 45. The method of any of embodiments 40-44, wherein the donor template includes homology arms. 46. The method of embodiment 45, wherein the homology arms are 10 nucleotides– 5000 nucleotides in length. 47. The method of any of embodiments 1-46, wherein the nanoparticle is a gold nanoparticle (AuNP) associated with at least three layers wherein: a first layer includes a gRNA with a 3’ end and a 5’ end, wherein the 3’ end is conjugated to a spacer with a thiol modification, and the 5’ end is conjugated to Cpf1, and wherein the thiol modification is covalently linked to the surface of the AuNP; a second layer includes a donor template including a therapeutic gene and homology-arms; and a third layer includes the targeting moieties, wherein the third layer is the farthest layer from the surface of the AuNP. 48. The method of embodiment 47, wherein the AuNP is associated with at least four layers wherein a first layer includes a donor template, a second layer includes gRNA, a third layer includes a nuclease, and a fourth layer includes the targeting moieties and wherein the first layer is the closest active layer to the surface of an AuNP core, the second layer is the second closest active layer to the surface of the AuNP core, the third layer is third closest active layer to the AuNP core, and the fourth layer is the farthest active layer from the AuNP core.F053-6005PCT / 24-085-WO-PCT 49. The method of any of embodiments 1-48, wherein the selected cell type includes hematopoietic stem cells (HSC), hematopoietic progenitor cells (HPC), hematopoietic stem and progenitor cells (HSPC), T cells, natural killer (NK) cells, B cells, a macrophages, monocytes, mesenchymal stem cells (MSC), white blood cells (WBC), mononuclear cells (MNC), endothelial cells (EC), stromal cells, or a bone marrow fibroblasts. 50. The method of any of embodiments 1-49, wherein the selected cell type includes HSC. 51. The method of any of embodiments 1-50, wherein the selected cell type includes HSPC. 52. The method of any of embodiments 1-51, wherein the selected cell type includes CD34+ / CD38- HSC. 53. A method including: attaching a targeting moiety to a nanoparticle including gene-editing components by a linker including a 4-24 PEG spacer. 54. The method of embodiment 53, wherein the targeting moiety includes an azide. 55. The method of embodiment 54, further including: modifying the targeting moiety to display the azide via an enzymatic reaction. 56. The method of embodiment 55, wherein the enzymatic reaction includes adding the azide to the targeting moiety using a N-acetylgalactosaminyltransferase (GalNAc transferase). 57. The method of any of embodiments 53-56, wherein the targeting moiety includes a thiol. 58. The method of embodiment 57, further including: modifying the targeting moiety to display the thiol via a reduction of disulfide bonds of the targeting moiety. 59. The method of any of embodiments 53-58, wherein the attaching is at a pH of 5.5 - 7.8. 60. The method of any of embodiments 53-59, wherein the attaching is at a pH of 6.0 - 7.5. 61. The method of any of embodiments 53-60, wherein the attaching includes exposing the nanoparticle to the linker, a concentration of the linker being 250X-3000X higher than a concentration of the nanoparticle. 62. The method of any of embodiments 53-61, wherein the attaching includes exposing the nanoparticle to the linker, a concentration of the linker being 500X-1000X higher than a concentration of the nanoparticle. 63. The method of any of embodiments 53-62, wherein the attaching includes exposing the nanoparticle to the linker, a concentration of the linker being 1000X higher than a concentration of the nanoparticle. 64. The method of any of embodiments 52-63, wherein the attaching includes exposing the nanoparticle to the targeting moiety at a concentration of 50-500 targeting moieties per nanoparticle. 65. The method of any of embodiments 52-64, wherein the linker is a heterofunctional linker.F053-6005PCT / 24-085-WO-PCT 66. The method of any of embodiments 52-65, wherein the linker includes a DBCO group. 67. The method of any of embodiments 52-66, wherein the linker includes a TFP ester. 68. The method of any of embodiments 52-67, wherein the targeting moiety includes a protein, an aptamer, or a small molecule. 69. The method of embodiment 68, wherein the protein includes an antibody, a peptide, or a ligand. 70. The method of embodiment 69, wherein the antibody binds CD133, CD117, or CD90. 71. The method of embodiment 69 or 70, wherein the antibody includes an anti-CD133 antibody, anti-CD117 antibody, or anti-CD90 antibody. 72. The method of any of embodiments 69-71, wherein the antibody includes αCD133:7, αCD117:104D2 or αCD90:5E10. 73. The method of any of embodiments 53-72, further including: quenching free linkers by adding quencher after attaching the targeting moiety to the nanoparticle. 74. The method of embodiment 73, wherein the quencher includes free azides or free thiols. 75. The method of embodiment 73 or 74, wherein a concentration of the quencher is 100X higher than a concentration of the linker. 76. The method of any of embodiments 53-75, wherein the gene-editing components include a targeting element and a cutting element. 77. The method of embodiment 76, wherein the attaching includes exposing the nanoparticle to the linker, a concentration of the linker being 10X-250X higher than a concentration of the cutting element. 78. A nanoparticle including: i) up to 1000 targeting moieties linked to the nanoparticle via 500-3000 linkers attached to the nanoparticle, wherein the targeting moieties selectively bind an antigen expressed by a selected cell type and result in internalization of the bound nanoparticle by the selected cell type; and ii) gene-editing components including a targeting element and a cutting element. 79. The nanoparticle of embodiment 78, wherein the linkers include heterofunctional linkers. 80. The nanoparticle of embodiment 79, wherein the heterofunctional linkers are attached to the nanoparticle via click chemistry. 81. The nanoparticle of embodiment 79 or 80, wherein the targeting moieties are attached to the linkers via click chemistry. 82. The nanoparticle of embodiment 81, wherein the click chemistry includes strain promoted azide-alkyne click (SPAAC) chemistry or copper-catalyzed Azide-alkyne click (CuAAC) chemistry. 83. The nanoparticle of embodiment 81 or 82, wherein the linker and / or the targeting moietyF053-6005PCT / 24-085-WO-PCT include a primary amine or a sulfhydryl. 84. The nanoparticle of any of embodiments 81-83, wherein the linker includes a primary amine and the targeting moiety includes a sulfhydryl. 85. The nanoparticle of any of embodiments 79-84, wherein the linkers include a dibenzocyclooctyne (DBCO) group. 86. The nanoparticle of any of embodiments 79-85, wherein the linker includes a trifluoromethylphenyl (TFP) ester. 87. The nanoparticle of any of embodiments 79-86, wherein the linker attaches to an azide, an amine, or a thiol on the targeting moiety. 88. The nanoparticle of embodiment 87, wherein the targeting moiety is modified to include the azide, the amine, or the thiol. 89. The nanoparticle of embodiment 87 or 88, wherein the linker attaches to the azide on the targeting moiety by azide-alkyne cycloaddition. 90. The nanoparticle of any of embodiments 78-89, wherein the linkers include a 4-24 unit PEG spacer. 91. The nanoparticle of any of embodiments 78-90, wherein the linkers include a 4, 12, or 24 unit PEG spacer. 92. The nanoparticle of any of embodiments 78-91, wherein the targeting moiety includes a protein, an aptamer, or a small molecule. 93. The nanoparticle of embodiment 92, wherein the protein includes an antibody, a peptide, or a ligand. 94. The nanoparticle of embodiment 93, wherein the antibody binds CD133, CD117, or CD90. 95. The nanoparticle of embodiment 93 or 94, wherein the antibody includes an anti-CD133 antibody, anti-CD117 antibody, or anti-CD90 antibody. 96. The nanoparticle of any of embodiments 93-95, wherein the antibody includes αCD133:7, αCD117:104D2 or αCD90:5E10. 97. The nanoparticle of any of embodiments 93-96, wherein the ligand includes stem cell factor (SCF). 98. The nanoparticle of any of embodiments 92-97, wherein the aptamer binds CD133. 99. The nanoparticle of any of embodiments 92-98, wherein the aptamer includes A15 or B19. 100. The nanoparticle of any of embodiments 92-99, wherein the protein includes human luteinizing hormone. 101. The nanoparticle of any of embodiments 92-100, wherein the small molecule includes degarelix acetate.F053-6005PCT / 24-085-WO-PCT 102. The nanoparticle of any of embodiments 78-101, wherein the gene-editing components include a cutting element and a targeting element. 103. The nanoparticle of embodiment 102, wherein the cutting element includes a nuclease. 104. The nanoparticle of embodiment 103, wherein the nuclease includes Cpf1 or Cas9. 105. The nanoparticle of any of embodiments 102-104, wherein the targeting moiety is linked to the cutting element. 106. The nanoparticle of embodiment 105, wherein the targeting moiety is linked to the cutting element with a linker. 107. The nanoparticle of embodiment 105 or 106, wherein the targeting moiety is linked to the cutting element with polyethylene glycol. 108. The nanoparticle of any of embodiments 102-107, wherein the targeting element includes guide RNA (gRNA). 109. The nanoparticle of embodiment 108, wherein the gRNA includes: UAAUUUCUACUCUUGUAGAUUUCGGACCCGUGCUACAACUU (SEQ ID NO: 2); UAAUUUCUACUCUUGUAGAUAUAGAAUAGCCUCAUAUUUUA (SEQ ID NO: 3); UAAUUUCUACUCUUGUAGAUGAGCUGUUGGCAUCAUGUUCCUG (SEQ ID NO: 4); UAAUUUCUACUCUUGUAGAUUCCAAACCUCCUAAAUGAUAC (SEQ ID NO: 5); or UAAUUUCUACUCUUGUAGAUCACCCGAUCCACUGGGGAGCA (SEQ ID NO: 6). 110. The nanoparticle of embodiment 108 or 109, wherein the gRNA targets a sequence including: TTTGTGTCCCCGTTTTGGTTGGTAAAC (SEQ ID NO: 7); TTTAAAAATCAATACCGATAATAATGA (SEQ ID NO: 8); TTTCTTAATATGAATATTAATATCGGT (SEQ ID NO: 9); TTTCCGTATCTGGAAGGGGCATCTTGG (SEQ ID NO: 10); TTTCCTTAGGACCGGAAGGATTACAGC (SEQ ID NO: 11); TTTGCCTAAAAGGCACTATGTCAAATG (SEQ ID NO: 12); TTTGGAGCTGTTGGCATCATGTTCCTG (SEQ ID NO: 13); TTTGATTCTTTTCTATCTCAGGACAGA (SEQ ID NO: 14); TTTATAGACATCCCACACTGTAGTTCT (SEQ ID NO: 15); TTTATTAATTTGAGAACCAACATAAGG (SEQ ID NO: 16); TTTATTTTCTTTTTGGTAAGAAGGAAC (SEQ ID NO: 17); TTTCACACACACACACACACACACACA (SEQ ID NO: 18); TTTGGAGCTGTTGGCATCATGTTCCTG (SEQ ID NO: 19); TTTATCCAAACCTCCTAAATGATAC (SEQ ID NO: 20);F053-6005PCT / 24-085-WO-PCT TTTACACCCGATCCACTGGGGAGCA (SEQ ID NO: 21); or TTTTTGATTCTTTTCTATCTCAGGACA (SEQ ID NO: 22). 111. The nanoparticle of any of embodiments 78-110, wherein the gene-editing components further include a donor template. 112. The nanoparticle of embodiment 111, wherein the donor template includes dsDNA. 113. The nanoparticle of embodiment 111 or 112, wherein the donor template includes ssDNA. 114. The nanoparticle of any of embodiments 111-113, wherein the donor template provides a therapeutic gene. 115. The nanoparticle of embodiment 114, wherein the therapeutic gene includes or encodes skeletal protein 4.1, glycophorin, p55, the Duffy allele, globin family genes, WAS, phox, dystrophin, pyruvate kinase, CLN3, ABCD1, arylsulfatase A, SFTPB, SFTPC, NLX2.1, ABCA3, GATA1, ribosomal protein genes, TERT, TERC, DKC1, TINF2, CFTR, LRRK2, PARK2, PARK7, PINK1, SNCA, PSEN1, PSEN2, APP, SOD1, TDP43, FUS, ubiquilin 2, C9ORF72, α2β1, αvβ3, αvβ5, αvβ63, BOB / GPR15, Bonzo / STRL-33 / TYMSTR, CCR2, CD55, CXCR4, aminopeptidase-N, HHV-7, ICAM, ICAM-1,LDLR / α2MR / LRP, PVR; PRR1 / HveC, laminin receptor, 101F6, 123F2, 53BP2, abl, ABLI, ADP, aFGF, APC, ApoAI, ApoAIV, ApoE, ATM, BAI-1, BDNF, Beta*(BLU), bFGF, BLC1, BLC6, BRCA1, BRCA2, CBFA1, CBL, C-CAM, CFTR, CNTF, COX-1, CSFIR, CTS-1, cytosine deaminase, DBCCR-1, DCC, Dp, DPC-4, E1A, E2F, EBRB2, erb, ERBA, ERBB, ETS1, ETS2, ETV6, Fab, FancA, FancB, FancC, FancD1, FancD2, FancE, FancF, FancG, FancI, FancJ, FancL, FancM, FancN, FancO, FancP, FancQ, FancR, FancS, FancT, FancU, FancV, and FancW, FCC, FGF, FGR, FHIT, fms, FOX, FUS 1, FUS1, FYN, G-CSF, GDAIF, Gene 21, Gene 26, GM-CSF, GMF, gsp, HCR, HIC-1, HRAS, hst, IGF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, ING1, interferon α, interferon β, interferon γ, IRF-1, JUN, KRAS, LCK, LUCA-1, LUCA-2, LYN, MADH4, MADR2, MCC, mda7, MDM2, MEN-I, MEN-II, MLL, MMAC1, MYB, MYC, MYCL1, MYCN, neu, NF-1, NF-2, NGF, NOEY1, NOEY2, NRAS, NT3, NT5, OVCA1, p16, p21, p27, p53, p57, p73, p300, PGS, PIM1, PL6, PML, PTEN, raf, Rap1A, ras, Rb, RB1, RET, rks-3, ScFv, scFV ras, SEM A3, SRC, TAL1, TCL3, TFPI, thrombospondin, thymidine kinase, TNF, TP53, trk, T-VEC, VEGF, VHL, WT1, WT-1, YES, zac1, iduronidase, IDS, GNS, HGSNAT, SGSH, NAGLU, GUSB, GALNS, GLB1, ARSB, HYAL1, F8, F9, HBB, CYB5R3, γC, JAK3, IL7RA, RAG1, RAG2, DCLRE1C, PRKDC, LIG4, NHEJ1, CD3D, CD3E, CD3Z, CD3G, PTPRC, ZAP70, LCK, AK2, ADA, PNP, WHN, CHD7, ORAI1, STIM1, CORO1A, CIITA, RFXANK, RFX5, RFXAP, RMRP, DKC1, TERT, TINF2, DCLRE1B, or SLC46A1. 116. The nanoparticle of any of embodiments 111-115, wherein the donor template includesF053-6005PCT / 24-085-WO-PCT homology arms. 117. The nanoparticle of embodiment 116, wherein the homology arms are 10 nucleotides – 5000 nucleotides. 118. The nanoparticle of any of embodiments 78-117, wherein the nanoparticle is a gold nanoparticle. 119. The nanoparticle of any of embodiments 78-118, wherein the targeting moiety is linked to the nanoparticle through electrostatic anchoring. 120. The nanoparticle of any of embodiments 78-119, wherein the nanoparticle further includes an endosomal polymer shell. 121. The nanoparticle of embodiment 120, wherein the targeting moiety is linked to the endosomal polymer shell via a linker. 122. The nanoparticle of embodiment 121, wherein the linker includes polyethylene glycol (PEG) or oligoethylene glycol (OEG). 123. The nanoparticle of any of embodiments 78-122, wherein the nanoparticle is associated with a polyethylene glycol layer or a polyethylenimine layer. 124. The nanoparticle of embodiment 123, wherein the polyethylenimine layer is closest to the surface of the nanoparticle. 125. The nanoparticle of any of embodiments 78-124, wherein the nanoparticle includes four layers, wherein a first layer includes ssDNA, a second layer includes gRNA, a third layer includes a nuclease, and a fourth layer includes the targeting moiety; wherein the first layer is the closest layer to the surface of the nanoparticle, the second layer is the second closest layer to the surface of the nanoparticle, the third layer is third closest layer to the nanoparticle, and the fourth layer is the furthest layer from the nanoparticle. 126. A composition including the nanoparticle of any of embodiments 78-125 and a pharmaceutically acceptable carrier. 127. The composition of embodiment 126, wherein the composition is formulated for intraosseous delivery. 128. A cell genetically-modifed by the method of any of embodiments 1-77. 129. The cell of embodiment 128, wherein the cell is a hematopoietic stem cell (HSC), a hematopoietic progenitor cell (HPC), a hematopoietic stem and progenitor cell (HSPC), a T cell, a natural killer (NK) cell, a B cell, a macrophage, a monocyte, a mesenchymal stem cell (MSC),F053-6005PCT / 24-085-WO-PCT a white blood cell (WBC), a mononuclear cell (MNC), a endothelial cell (EC), a stromal cell, and / or a bone marrow fibroblast. 130. The cell of embodiment 128, wherein the cell includes an HSC. 131. The cell of embodiment 128, wherein the cell includes an HSPC. 132. The cell of embodiment 128, wherein the cell includes a CD34+ / CD38- HSC. 133. The cell of any of embodiments 128-132, wherein the cell is genetically modified in vivo. 134. The cell of any of embodiments 128-132, wherein the cell is genetically modified ex vivo. 135. A formulation including the cell of any of embodiments 128-134. 136. A method of treating a subject in need thereof including administering a composition of embodiments 126 or 127 or a formulation of embodiment 135 to the subject. 137. The method of embodiment 136, wherein the administering includes intraosseous delivery. 138. A kit including: nanoparticles; gene-editing components; heterofunctional linkers including 4-12 PEG spacers; and targeting moieties. 139. The kit of embodiment 138, wherein the heterofunctional linkers include a DBCO group. 140. The kit of embodiment 138 or 139, wherein the heterofunctional linkers include a TFP ester. 141. The kit of any of embodiments 138-140, further including a quencher. 142. The kit of embodiment 141, wherein the quencher includes free azide and / or free thiol. 143. The kit of any of embodiments 138-142, further including an enzyme configured to attach an azide to the targeting moieties. 144. The kit of embodiment 143, wherein the enzyme includes GalNAc transferase. 145. The kit of any of embodiments 138-144, further including a reducing agent. 146. The kit of embodiment 145, wherein the reducing agent includes at least one of dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), 2-Mercaptoethanol, or glutathione (GSH). 147. The kit of any of embodiments 138-146, wherein a concentration of the heterofunctional linkers is 250X-3000X higher than a concentration of the nanoparticles. 148. The kit of any of embodiments 138-147, wherein a concentration of the targeting moieties is 50X-500X higher than a concentration of the nanoparticles.F053-6005PCT / 24-085-WO-PCT 149. The kit of any of embodiments 138-148, wherein the gene-editing components include a targeting element and a cutting element. 150. The kit of embodiment 149, wherein a concentration of the heterofunctional linkers is 10X-250X higher than a concentration of the cutting element. 151. The kit of embodiment 149 or 150, wherein the cutting element includes a nuclease. 152. The kit of embodiment 151, wherein the nuclease includes Cpf1 or Cas9. 153. The kit of any of embodiments 149-152, wherein the targeting element includes a guide RNA (gRNA). 154. The kit of embodiment 153, wherein the targeting element includes a single gRNA. 155. The kit of embodiment 153 or 154, wherein the gRNA includes: UAAUUUCUACUCUUGUAGAUUUCGGACCCGUGCUACAACUU (SEQ ID NO: 2); UAAUUUCUACUCUUGUAGAUAUAGAAUAGCCUCAUAUUUUA (SEQ ID NO: 3); UAAUUUCUACUCUUGUAGAUGAGCUGUUGGCAUCAUGUUCCUG (SEQ ID NO: 4); UAAUUUCUACUCUUGUAGAUUCCAAACCUCCUAAAUGAUAC (SEQ ID NO: 5); or UAAUUUCUACUCUUGUAGAUCACCCGAUCCACUGGGGAGCA (SEQ ID NO: 6). 156. The kit of any of embodiments 153-155, wherein the gRNA targets a sequence including: TTTGTGTCCCCGTTTTGGTTGGTAAAC (SEQ ID NO: 7); TTTAAAAATCAATACCGATAATAATGA (SEQ ID NO: 8); TTTCTTAATATGAATATTAATATCGGT (SEQ ID NO: 9); TTTCCGTATCTGGAAGGGGCATCTTGG (SEQ ID NO: 10); TTTCCTTAGGACCGGAAGGATTACAGC (SEQ ID NO: 11); TTTGCCTAAAAGGCACTATGTCAAATG (SEQ ID NO: 12); TTTGGAGCTGTTGGCATCATGTTCCTG (SEQ ID NO: 13); TTTGATTCTTTTCTATCTCAGGACAGA (SEQ ID NO: 14); TTTATAGACATCCCACACTGTAGTTCT (SEQ ID NO: 15); TTTATTAATTTGAGAACCAACATAAGG (SEQ ID NO: 16); TTTATTTTCTTTTTGGTAAGAAGGAAC (SEQ ID NO: 17); TTTCACACACACACACACACACACACA (SEQ ID NO: 18); TTTGGAGCTGTTGGCATCATGTTCCTG (SEQ ID NO: 19); TTTATCCAAACCTCCTAAATGATAC (SEQ ID NO: 20); TTTACACCCGATCCACTGGGGAGCA (SEQ ID NO: 21); or TTTTTGATTCTTTTCTATCTCAGGACA (SEQ ID NO: 22). 157. The kit of any of embodiments 138-156, wherein the targeting moieties include at least one of a protein, an aptamer, or a small molecule.F053-6005PCT / 24-085-WO-PCT 158. The kit of embodiment 157, wherein the protein includes an antibody, a peptide, or a ligand. 159. The kit of embodiment 158, wherein the antibody binds CD133, CD117, or CD90. 160. The kit of embodiment 158, wherein the antibody includes an anti-CD133 antibody, anti- CD117 antibody, or anti-CD90 antibody. 161. The kit of embodiment 158, wherein the antibody includes αCD133:7, αCD117:104D2 or αCD90:5E10. 162. The kit of any of embodiments 158-162, wherein the ligand includes stem cell factor (SCF). 163. The kit of any of embodiments 157-162, wherein the aptamer binds CD133. 164. The kit of any of embodiments 157-163, wherein the aptamer includes A15 or B19. 165. The kit of any of embodiments 157-164, wherein the protein includes human luteinizing hormone. 166. The kit of any of embodiments 157-165, wherein the small molecule includes degarelix acetate.

[0244] (X) Experimental Examples. Example 1. Hematopoietic Stem and Progenitor Cell Specific Antibodies and Intraosseous Administration Enhance Delivery of a CRISPR Nanoformulation.

[0245] Abstract: Hematopoietic stem cells (HSC) have been a target for CRISPR gene editing to treat multiple diseases. Current state of the art for HSC gene editing occurs ex vivo, however, these methods remain challenging for global dissemination. In vivo delivery of CRISPR gene editing could facilitate global access but is inhibited by the rare and diffuse composition of HSC throughout the bone marrow niche. A layered gold nanoformulation to deliver CRISPR into HSC in vitro without electroporation (CRISPR-AuNP) has previously been described. Here, the goal was to test this CRISPR-AuNP in vivo in a humanized murine model of hematopoiesis. One method to access HSC in vivo is to administer mobilizing agents such as G-CSF and AMD3100, which reversibly increase HSC concentrations in peripheral blood. Initial experiments in humanized mice (n=3) showed no toxicity associated with a single intravenous injection of CRISPR-AuNP under mobilization, but only transient gene editing (5.0 ± 2.5%) in peripheral blood leukocytes at 2 weeks after administration. To improve HSC-specific editing, actively targeted CRISPR-AuNPs were designed, including a conjugated targeting moiety in the outermost layer. Optimal HSC targeting moieties were selected based on observed internalization by confocal microscopy within 3 hours of addition to primary human CD34+ HSPCs. Three antibodies (αCD133:clone7, αCD117:104D2 and αCD90:5E10), two aptamers (A15, B19), and one ligand (SCF) were selected for further testing. Whole bone marrow from six biological replicates were analyzed by flow cytometry to determine targeting moiety specificity. αCD133 and αCD117 showed the highest specificity of 56.9 ± 7 and 66.4 ± 1% of CD34+ / CD38- cells, respectively, vs.F053-6005PCT / 24-085-WO-PCT 6.87 ± 0.25 and 1.17 ± 0.18% of total CD34- cells (SCF had the lowest specificity at 85.6 ± 6.07% CD34- binding) and were then loaded onto CRISPR-AuNP via electrostatic binding at neutral pH (FIG.11). The resulting particle had a hydrodynamic diameter of 114.3 nm with a polydispersity index of 0.147 by dynamic light scattering. These particles were injected intraosseously into one femur of humanized mice (n=12), to increase local concentration within the bone marrow. Mice did not show signs of adverse events or toxicity by weight measurements or behavior by daily visual inspection. Gold tracing by inductively-coupled plasma mass spectrometry at necropsy showed 14.2 ± 0.81% of the Au dose within the injected femur (p<0.001 over bare AuNP control), with enhanced retention of targeted CRISPR-AuNP in the distal femurs (FIG. 1B). This data demonstrates that CRISPR-AuNP are well-tolerated in vivo, that active targeting with antibodies and intraosseous injection can increase bone marrow retention, and these combined methods are associated with persistent editing of peripheral blood leukocytes up to 12 weeks after administration in humanized mice.

[0246] Example 2. Comparison of Electrostatic Modification Efficiency of Antibodies to CIRSPR- AuNP Relative to Click Chemistry Anchoring.

[0247] Abstract: Previous targeted CRISPR AuNP formulations (Example 1) relied on electrostatic adhesion for retention of targeting moieties. Initial studies in mice show increased tissue retention by targeted particles. However, further investigation revealed a loss of the targeting group under repeated purification / shear force (FIG.14C), an expected processes during prolonged in vivo residence time. Here, direct covalent modification using two step modification, TFP ester amide formation, and subsequent strain promoted azide-alkyne cycloaddition chemistry (SPAAC) to strengthen the antibody binding was investigated (FIG. 19). Antibody- Azide4 active groups were added using SiteClick Azido modification kit which produces a repeatable 4 azide modifications per antibody on the inactive stem region of the heavy chain (HC). Tetrafluorophenyl esters (FIG.20) were used for amide linkage to primary amines present on the Cas9 or PEI under mild reaction conditions. Due to reduced diffusion to nanoparticle surfaces, TFP was selected as its reduced hydrolysis rate improves modification efficiency over longer reactions times relative to industry standard N-hydroxy succinimide (NHS) esters. Using a range of linker mol ratios to free RNP, reactions were carried out at pH 7.4 in 10 mM HEPES (<10% DMSO v / v), 2 mg / mL RNP concentration, and incubation for 12 hrs. The resulting amide formation was found to be directly proportional to decreased in vitro DNA cutting activity of the Cas9 nuclease, with heavy inhibition of activity ≥50 molar ratio (FIG.17). The subsequent click reaction was carried out under the same conditions, using Cyanine 3-Azide (Cy3-Az) as a linkage reporter. Running SDS-PAGE, it is clear a loss of RNP solubility under heavy modification, likely due toF053-6005PCT / 24-085-WO-PCT hydrophobic effects, is the major cause for the loss of DNA lysis (FIG.14A). Initial AuNP studies were carried out on CRISPR AuNP using the same conjugation conditions, testing a range from 0-100 linkers per AuNP, the click reporter being antibody-alexa647-Azide, added at 1:2 molar ratio to the highest linker. However, initial results showed no modification up to the max 100 linker / Au core. Confirmation of the linker on AuNP activity was conducted using an RNP only coated AuNP (AuRNP) and 1000 linker / Au core, with Cy3-Az as the reporter. This resulted in the 31.4 ± 5.6 dye / Au core, suggesting the linkers were active on the Au surface but the antibody binding was restricted. As the linker could react with the small molecule dye, it was hypothesized that a combination of steric interference, competition with PEI and / or charge repulsion were lowering the AuNP binding. To reduce PEI competition, AuRNP was used for further testing. Three differing PEG spacer lengths were selected (PEG4, 12 and 24, or 2, 5 and 12 nm length) to reduce steric hindrance and were tested alongside pH charge reduction (between pH 5.5, where the Cas9 RNP becomes unstable (FIG. 22), to physiological pH of 7.4). Analyzed separately, the linker length had no substantial impact on particle stability by DLS, with equal modification ratios weakly decreasing particle size / PDI (FIG.11 Bottom). Decreasing pH resulted in slightly increased size trend without the linkers present, however, when combined with longer linker lengths, resulted in particle aggregation below pH 5.5 (FIG.12). This low pH linker instability was confirmed by fluorescent tracing of ATTO550-trRNA (FIG.23), showing statistical loss of the critical RNA component only at pH 5. This is possibly due to increased molecular strain / reduced lysine positive charge lowering the trRNA binding energy. The RNA loss was also correlated with aggregation of the gold cores / insolubility. SDS-PAGE allowed quantification of click binding, shown as fraction of heavy chain modified to RNP as a ratio against the unconjugated light chain as a loading control. This analysis shows no advantage to larger linkers at pH 6 (FIG.13B) so DBCO-PEG4-TFP was selected. Utilizing this modification method, antibody binds to AuRNP at 10.1 / Au core and results in a particle size of 110.5 nm. Using the knowledge here, antibody conjugation was carried out on the CRISPR AuNP, which contains a modified PEI coating which is directly anchored to the gold surface and positive charge (charge of AuNP controlled by PEI addition level, FIG. 24). The resulting modification contained 15.6 antibodies conjugated to the CRISPR AuNP (FIG. 25). Combined the protocol detailed here elucidates the materials and methods required for targeting modification of CRISPR AuNP and its derivatives. Using the detailed antibodies specific to HSPCs the combination of targeting formulations have great potential for effective editing in blood products without prior purification of the blood stem and progenitor cells. This would greatly simplify manufacturing by permitting minimal manipulation genetic editing ex vivo and / or targeting of the desired cells in vivo either in blood circulation or inF053-6005PCT / 24-085-WO-PCT the bone marrow.

[0248] (XI) Closing Paragraphs. The nucleic acid and amino acid sequences provided herein are shown using letter abbreviations for nucleotide bases and amino acid residues, as defined in 37 C.F.R. §1.831-1.835 and set forth in WIPO Standard ST.26 (implemented on July 1, 2022). Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included in embodiments where it would be appropriate.

[0249] Variants of the sequences disclosed and referenced herein are also included. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTAR™ (Madison, Wisconsin) software. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.

[0250] In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p.224). Naturally occurring amino acids are generally divided into conservative substitution families as follows: Group 1: Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (acidic): Aspartic acid (Asp), and Glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), Glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (large aliphatic, nonpolar residues): Isoleucine (Ile), Leucine (Leu), Methionine (Met), Valine (Val) and Cysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gln, Cys, Ser, and Thr; Group 8 (large aromatic residues): Phenylalanine (Phe), Tryptophan (Trp), and Tyr; Group 9 (non- polar): Proline (Pro), Ala, Val, Leu, Ile, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Val, Leu, and Ile; Group 10 (small aliphatic, nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0251] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol.157(1), 105-32).F053-6005PCT / 24-085-WO-PCT Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (−0.4); Thr (−0.7); Ser (−0.8); Trp (−0.9); Tyr (−1.3); Pro (−1.6); His (−3.2); Glutamate (−3.5); Gln (−3.5); aspartate (−3.5); Asn (−3.5); Lys (−3.9); and Arg (−4.5).

[0252] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity.

[0253] As detailed in US 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Thr (−0.4); Pro (−0.5±1); Ala (−0.5); His (−0.5); Cys (−1.0); Met (−1.3); Val (−1.5); Leu (−1.8); Ile (−1.8); Tyr (−2.3); Phe (−2.5); Trp (−3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0254] As outlined above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. As indicated elsewhere, variants of gene sequences can include codon optimized variants, sequence polymorphisms, splice variants, and / or mutations that do not affect the function of an encoded product to a statistically-significant degree.

[0255] Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.

[0256] “% sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences as determined by the matchF053-6005PCT / 24-085-WO-PCT between strings of such sequences. “Identity” (often referred to as “similarity”) can be readily calculated by known methods, including those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol.215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111- 20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y.). Within the context of this disclosure it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. As used herein "default values" will mean any set of values or parameters, which originally load with the software when first initialized.

[0257] Variants also include nucleic acid molecules that hybridize under stringent hybridization conditions to a sequence disclosed herein and provide the same function as the reference sequence. Exemplary stringent hybridization conditions include an overnight incubation at 42 °C in a solution including 50% formamide, 5XSSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5XDenhardt's solution, 10% dextran sulfate, and 20 µg / ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1XSSC at 50 °C. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, moderately high stringency conditions include an overnight incubation at 37°C in a solution including 6XSSPE (20XSSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 µg / ml salmon spermF053-6005PCT / 24-085-WO-PCT blocking DNA; followed by washes at 50 °C with 1XSSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g., 5XSSC). Variations in the above conditions may be accomplished through the inclusion and / or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

[0258] "Specifically binds" refers to an association of a binding domain (of, for example, a targeting moiety and select cell) to its cognate binding molecule with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1 / M) equal to or greater than 105 M-1, while not significantly associating with any other molecules or components in a relevant environment sample. Binding domains may be classified as "high affinity" or "low affinity". In particular embodiments, "high affinity" binding domains refer to those binding domains with a Ka of at least 107 M-1, at least 108 M-1, at least 109 M-1, at least 1010 M-1, at least 1011 M-1, at least 1012 M-1, or at least 1013 M-1. In particular embodiments, "low affinity" binding domains refer to those binding domains with a Ka of up to 107 M-1, up to 106 M-1, up to 105 M- 1. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10-5 M to 10-13 M). In certain embodiments, a binding domain may have "enhanced affinity," which refers to a selected or engineered binding domains with stronger binding to a cognate binding molecule than a wild type (or parent) binding domain. For example, enhanced affinity may be due to a Ka (equilibrium association constant) for the cognate binding molecule that is higher than the reference binding domain or due to a Kd (dissociation constant) for the cognate binding molecule that is less than that of the reference binding domain, or due to an off-rate (Koff) for the cognate binding molecule that is less than that of the reference binding domain. A variety of assays are known for detecting binding domains that specifically bind a particular cognate binding molecule as well as determining binding affinities, such as Western blot, ELISA, and BIACORE® analysis (see also, e.g., Scatchard, et al., 1949, Ann. N.Y. Acad. Sci.51:660; and U.S. Patent Nos.5,283,173, 5,468,614, or the equivalent).

[0259] Unless otherwise indicated, the practice of the present disclosure can employ conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4nd Edition (2012); F. M. Ausubel, et al. eds., Current Protocols in Molecular Biology, (2003); the series Methods In EnzymologyF053-6005PCT / 24-085-WO-PCT (Academic Press, Inc.); Behlke, et al., Polymerase Chain Reaction: Theory and Technology (2019); Greenfield, ed. Antibodies, A Laboratory Manual, Second Edition (2014); and Capes- Davis and R. I. Freshney, eds. Freshney's Culture of Animal Cells 8th Edition (2021).

[0260] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant reduction in gene editing of selected cell types as described herein.

[0261] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0262] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testingF053-6005PCT / 24-085-WO-PCT measurements.

[0263] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0264] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0265] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0266] Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching.

[0267] In closing, it is to be understood that the embodiments of the invention disclosed hereinF053-6005PCT / 24-085-WO-PCT are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

[0268] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and / or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0269] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

F053-6005PCT / 24-085-WO-PCT CLAIMS What is claimed is:

1. A method of selectively genetically modifying hematopoietic stem and progenitor cells (HSPCs) within a subject, wherein the method comprises: intraosseously administering a nanoparticle comprising: i) up to 1000 targeting moieties linked to a gold nanoparticle via 500-3000 dibenzocyclooctyne (DBCO)-polyethylene glycol (PEG)4- trifluoromethylphenyl (TFP) ester linkers attached to the nanoparticle, the targeting moieties comprising an antibody that binds CD133, CD117, or CD90 and results in internalization of the bound nanoparticle by the HSPCs, ii) Cas9, and iii) single guide RNA (gRNA).

2. The method of claim 1, wherein the antibody comprises clone7, 104D2 or 5E10.

3. A method of selectively genetically modifying cells of a selected cell type, wherein the method comprises: contacting a population of cells comprising the selected cell type with a nanoparticle, wherein the nanoparticle comprises: i) up to 1000 targeting moieties linked to a nanoparticle via 500-3000 linkers attached to the nanoparticle, wherein the targeting moieties selectively bind an antigen expressed by the selected cell type and result in internalization of the bound nanoparticle by the selected cell type; and ii) gene-editing components comprising a targeting element and a cutting element.

4. The method of claim 3, wherein the linkers comprise heterofunctional linkers.

5. The method of claim 4, wherein the heterofunctional linkers are attached to the nanoparticle via click chemistry.

6. The method of claim 4, wherein the targeting moieties are attached to the linkers via click chemistry.

7. The method of claim 5, wherein the click chemistry comprises strain promoted azide-alkyne click (SPAAC) chemistry or copper-catalyzed Azide-alkyne click (CuAAC) chemistry.

8. The method of claim 5, wherein the linker and / or the targeting moiety comprise a primary amine or a sulfhydryl.

9. The method of claim 5, wherein the heterofunctional linkers comprise a primary amine and the targeting moiety comprises a sulfhydryl.

10. The method of claim 3, wherein the linkers comprise a dibenzocyclooctyne (DBCO) group.

11. The method of claim 3, wherein the linkers comprise a trifluoromethylphenyl (TFP) ester.F053-6005PCT / 24-085-WO-PCT 12. The method of claim 3, wherein the linker attaches to an azide, an amine, or a thiol on the targeting moiety.

13. The method of claim 12, wherein the targeting moiety is modified to comprise the azide, the amine, or the thiol.

14. The method of claim 12, wherein the linker attaches to the azide on the targeting moiety by azide-alkyne cycloaddition.

15. The method of claim 3, wherein the linkers comprise a 4-24 unit PEG spacer.

16. The method of claim 3, wherein the linkers comprise a 4, 12, or 24 unit PEG spacer.

17. The method of claim 3, wherein the targeting moieties comprises a protein, an aptamer, or a small molecule.

18. The method of claim 17, wherein the protein comprises an antibody, a peptide, or a ligand.

19. The method of claim 18, wherein the antibody binds CD133, CD117, or CD90.

20. The method of claim 18, wherein the antibody comprises an anti-CD133 antibody, an anti- CD117 antibody, or an anti-CD90 antibody.

21. The method of claim 18, wherein the antibody comprises αCD133:7, αCD117:104D2 or αCD90:5E10.

22. The method of claim 18, wherein the ligand comprises stem cell factor (SCF).

23. The method of claim 17, wherein the protein comprises human luteinizing hormone.

24. The method of claim 17, wherein the aptamer binds CD133.

25. The method of claim 17, wherein the aptamer comprises A15 or B19.

26. The method of claim 17, wherein the small molecule comprises degarelix acetate.

27. The method of claim 3, wherein the targeting moieties are attached to the gene-editing components by the linker.

28. The method of claim 3, wherein the cutting element comprises a nuclease.

29. The method of claim 28, wherein the nuclease comprises Cpf1 or Cas9.

30. The method of claim 3, wherein the targeting element comprises a guide RNA (gRNA).

31. The method of claim 3, wherein the targeting element comprises a single gRNA.

32. The method of claim 31, wherein the gRNA comprises: UAAUUUCUACUCUUGUAGAUUUCGGACCCGUGCUACAACUU (SEQ ID NO: 2); UAAUUUCUACUCUUGUAGAUAUAGAAUAGCCUCAUAUUUUA (SEQ ID NO: 3); UAAUUUCUACUCUUGUAGAUGAGCUGUUGGCAUCAUGUUCCUG (SEQ ID NO: 4); UAAUUUCUACUCUUGUAGAUUCCAAACCUCCUAAAUGAUAC (SEQ ID NO: 5); or UAAUUUCUACUCUUGUAGAUCACCCGAUCCACUGGGGAGCA (SEQ ID NO: 6).

33. The method of claim 31, wherein the gRNA targets a sequence comprising:F053-6005PCT / 24-085-WO-PCT TTTGTGTCCCCGTTTTGGTTGGTAAAC (SEQ ID NO: 7); TTTAAAAATCAATACCGATAATAATGA (SEQ ID NO: 8); TTTCTTAATATGAATATTAATATCGGT (SEQ ID NO: 9); TTTCCGTATCTGGAAGGGGCATCTTGG (SEQ ID NO: 10); TTTCCTTAGGACCGGAAGGATTACAGC (SEQ ID NO: 11); TTTGCCTAAAAGGCACTATGTCAAATG (SEQ ID NO: 12); TTTGGAGCTGTTGGCATCATGTTCCTG (SEQ ID NO: 13); TTTGATTCTTTTCTATCTCAGGACAGA (SEQ ID NO: 14); TTTATAGACATCCCACACTGTAGTTCT (SEQ ID NO: 15); TTTATTAATTTGAGAACCAACATAAGG (SEQ ID NO: 16); TTTATTTTCTTTTTGGTAAGAAGGAAC (SEQ ID NO: 17); TTTCACACACACACACACACACACACA (SEQ ID NO: 18); TTTGGAGCTGTTGGCATCATGTTCCTG (SEQ ID NO: 19); TTTATCCAAACCTCCTAAATGATAC (SEQ ID NO: 20); TTTACACCCGATCCACTGGGGAGCA (SEQ ID NO: 21); or TTTTTGATTCTTTTCTATCTCAGGACA (SEQ ID NO: 22).

34. The method of claim 3, wherein the targeting moieties are linked to the cutting element by the linker.

35. The method of claim 34, wherein the linker binds to an amine group of the cutting element.

36. The method of claim 3, wherein the nanoparticle comprises 10 to 250 linkers per cutting element.

37. The method of claim 3, wherein the nanoparticle further comprises an endosomal polymer shell.

38. The method of claim 37, wherein the targeting moiety is linked to the endosomal polymer shell by the linker.

39. The method of claim 3, wherein the nanoparticle is a gold nanoparticle.

40. The method of claim 39, wherein the gold nanoparticle is associated with a polyethylenimine (PEI) layer.

41. The method of claim 40, wherein the PEI layer is closest to the surface of the gold nanoparticle.

42. The method of claim 3, wherein the gene-editing components further comprise a donor template.

43. The method of claim 42, wherein the donor template comprises double-stranded DNA (dsDNA).F053-6005PCT / 24-085-WO-PCT 44. The method of claim 42, wherein the donor template comprises single-stranded DNA (ssDNA).

45. The method of claim 42, wherein the donor template comprises a therapeutic gene.

46. The method of claim 45, wherein the therapeutic gene comprises or encodes skeletal protein 4.1, glycophorin, p55, the Duffy allele, globin family genes, WAS, phox, dystrophin, pyruvate kinase, CLN3, ABCD1, arylsulfatase A, SFTPB, SFTPC, NLX2.1, ABCA3, GATA1, ribosomal protein genes, TERT, TERC, DKC1, TINF2, CFTR, LRRK2, PARK2, PARK7, PINK1, SNCA, PSEN1, PSEN2, APP, SOD1, TDP43, FUS, ubiquilin 2, C9ORF72, α2β1, αvβ3, αvβ5, αvβ63, BOB / GPR15, Bonzo / STRL-33 / TYMSTR, CCR2, CCR3, CCR5, CCR8, CD4, CD46, CD55, CXCR4, aminopeptidase-N, HHV-7, ICAM, ICAM-1, PRR2 / HveB, HveA, α-dystroglycan, LDLR / α2MR / LRP, PVR; PRR1 / HveC, laminin receptor, 101F6, 123F2, 53BP2, abl, ABLI, ADP, aFGF, APC, ApoAI, ApoAIV, ApoE, ATM, BAI-1, BDNF, Beta*(BLU), bFGF, BLC1, BLC6, BRCA1, BRCA2, CBFA1, CBL, C-CAM, CFTR, CNTF, COX-1, CSFIR, CTS-1, cytosine deaminase, DBCCR-1, DCC, Dp, DPC-4, E1A, E2F, EBRB2, erb, ERBA, ERBB, ETS1, ETS2, ETV6, Fab, FancA, FancB, FancC, FancD1, FancD2, FancE, FancF, FancG, FancI, FancJ, FancL, FancM, FancN, FancO, FancP, FancQ, FancR, FancS, FancT, FancU, FancV, and FancW, FCC, FGF, FGR, FHIT, fms, FOX, FUS 1, FUS1, FYN, G-CSF, GDAIF, Gene 21, Gene 26, GM-CSF, GMF, gsp, HCR, HIC-1, HRAS, hst, IGF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, ING1, interferon α, interferon β, interferon γ, IRF-1, JUN, KRAS, LCK, LUCA-1, LUCA-2, LYN, MADH4, MADR2, MCC, mda7, MDM2, MEN-I, MEN-II, MLL, MMAC1, MYB, MYC, MYCL1, MYCN, neu, NF-1, NF-2, NGF, NOEY1, NOEY2, NRAS, NT3, NT5, OVCA1, p16, p21, p27, p53, p57, p73, p300, PGS, PIM1, PL6, PML, PTEN, raf, Rap1A, ras, Rb, RB1, RET, rks-3, ScFv, scFV ras, SEM A3, SRC, TAL1, TCL3, TFPI, thrombospondin, thymidine kinase, TNF, TP53, trk, T-VEC, VEGF, VHL, WT1, WT-1, YES, zac1, iduronidase, IDS, GNS, HGSNAT, SGSH, NAGLU, GUSB, GALNS, GLB1, ARSB, HYAL1, F8, F9, HBB, CYB5R3, γC, JAK3, IL7RA, RAG1, RAG2, DCLRE1C, PRKDC, LIG4, NHEJ1, CD3D, CD3E, CD3Z, CD3G, PTPRC, ZAP70, LCK, AK2, ADA, PNP, WHN, CHD7, ORAI1, STIM1, CORO1A, CIITA, RFXANK, RFX5, RFXAP, RMRP, DKC1, TERT, TINF2, DCLRE1B, or SLC46A1.

47. The method of claim 42, wherein the donor template comprises homology arms.

48. The method of claim 47, wherein the homology arms are 10 nucleotides– 5000 nucleotides in length.

49. The method of claim 3, wherein the nanoparticle is a gold nanoparticle (AuNP) associated with at least three layers wherein: a first layer comprises a gRNA with a 3’ end and a 5’ end, wherein the 3’ end is conjugatedF053-6005PCT / 24-085-WO-PCT to a spacer with a thiol modification, and the 5’ end is conjugated to Cpf1, and wherein the thiol modification is covalently linked to the surface of the AuNP; a second layer comprises a donor template comprising a therapeutic gene and homology- arms; and a third layer comprises the targeting moieties, wherein the third layer is the farthest layer from the surface of the AuNP.

50. The method of claim 49, wherein the AuNP is associated with at least four layers wherein a first layer comprises a donor template, a second layer comprises gRNA, a third layer comprises a nuclease, and a fourth layer comprises the targeting moieties and wherein the first layer is the closest active layer to the surface of an AuNP core, the second layer is the second closest active layer to the surface of the AuNP core, the third layer is third closest active layer to the AuNP core, and the fourth layer is the farthest active layer from the AuNP core.

51. The method of claim 3, wherein the selected cell type comprises hematopoietic stem cells (HSC), hematopoietic progenitor cells (HPC), hematopoietic stem and progenitor cells (HSPC), T cells, natural killer (NK) cells, B cells, a macrophages, monocytes, mesenchymal stem cells (MSC), white blood cells (WBC), mononuclear cells (MNC), endothelial cells (EC), stromal cells, or a bone marrow fibroblasts.

52. The method of claim 3, wherein the selected cell type comprises HSC.

53. The method of claim 3, wherein the selected cell type comprises HSPC.

54. The method of claim 3, wherein the selected cell type comprises CD34+ / CD38- HSC.

55. A method comprising: attaching a targeting moiety to a nanoparticle comprising gene-editing components by a linker comprising a 4-24 PEG spacer.

56. The method of claim 55, wherein the targeting moiety comprises an azide.

57. The method of claim 56, further comprising: modifying the targeting moiety to display the azide via an enzymatic reaction.

58. The method of claim 57, wherein the enzymatic reaction comprises adding the azide to the targeting moiety using a N-acetylgalactosaminyltransferase (GalNAc transferase).

59. The method of claim 55, wherein the targeting moiety comprises a thiol.

60. The method of claim 59, further comprising: modifying the targeting moiety to display the thiol via a reduction of disulfide bonds of the targeting moiety.

61. The method of claim 55, wherein the attaching is at a pH of 5.5 - 7.

8.

62. The method of claim 55, wherein the attaching is at a pH of 6.0 - 7.

5.

63. The method of claim 55, wherein the attaching comprises exposing the nanoparticle to the linker, a concentration of the linker being 250X-3000X higher than a concentration of theF053-6005PCT / 24-085-WO-PCT nanoparticle.

64. The method of claim 55, wherein the attaching comprises exposing the nanoparticle to the linker, a concentration of the linker being 500X-1000X higher than a concentration of the nanoparticle.

65. The method of claim 55, wherein the attaching comprises exposing the nanoparticle to the linker, a concentration of the linker being 1000X higher than a concentration of the nanoparticle.

66. The method of claim 55, wherein the attaching comprises exposing the nanoparticle to the targeting moiety at a concentration of 50-500 targeting moieties per nanoparticle.

67. The method of claim 55, wherein the linker is a heterofunctional linker.

68. The method of claim 55, wherein the linker comprises a DBCO group.

69. The method of claim 55, wherein the linker comprises a TFP ester.

70. The method of claim 55, wherein the targeting moiety comprises a protein, an aptamer, or a small molecule.

71. The method of claim 70, wherein the protein comprises an antibody, a peptide, or a ligand.

72. The method of claim 71, wherein the antibody binds CD133, CD117, or CD90.

73. The method of claim 71, wherein the antibody comprises an anti-CD133 antibody, anti-CD117 antibody, or anti-CD90 antibody.

74. The method of claim 71, wherein the antibody comprises αCD133:7, αCD117:104D2 or αCD90:5E10.

75. The method of claim 55, further comprising: quenching free linkers by adding quencher after attaching the targeting moiety to the nanoparticle.

76. The method of claim 75, wherein the quencher comprises free azides or free thiols.

77. The method of claim 75, wherein a concentration of the quencher is 100X higher than a concentration of the linker.

78. The method of claim 55, wherein the gene-editing components comprise a targeting element and a cutting element.

79. The method of claim 78, wherein the attaching comprises exposing the nanoparticle to the linker, a concentration of the linker being 10X-250X higher than a concentration of the cutting element.

80. A nanoparticle comprising: i) up to 1000 targeting moieties linked to the nanoparticle via 500-3000 linkers attached to the nanoparticle, wherein the targeting moieties selectively bind an antigen expressed by a selected cell type and result in internalization of the bound nanoparticle by the selected cell type; and ii) gene-editing components comprising a targeting element and a cutting element.F053-6005PCT / 24-085-WO-PCT 81. The nanoparticle of claim 80, wherein the linkers comprise heterofunctional linkers.

82. The nanoparticle of claim 81, wherein the heterofunctional linkers are attached to the nanoparticle via click chemistry.

83. The nanoparticle of claim 81, wherein the targeting moieties are attached to the linkers via click chemistry.

84. The nanoparticle of claim 83, wherein the click chemistry comprises strain promoted azide- alkyne click (SPAAC) chemistry or copper-catalyzed Azide-alkyne click (CuAAC) chemistry.

85. The nanoparticle of claim 83, wherein the linker and / or the targeting moiety comprise a primary amine or a sulfhydryl.

86. The nanoparticle of claim 83, wherein the linker comprises a primary amine and the targeting moiety comprises a sulfhydryl.

87. The nanoparticle of claim 81, wherein the linkers comprise a dibenzocyclooctyne (DBCO) group.

88. The nanoparticle of claim 81, wherein the linker comprises a trifluoromethylphenyl (TFP) ester.

89. The nanoparticle of claim 81, wherein the linker attaches to an azide, an amine, or a thiol on the targeting moiety.

90. The nanoparticle of claim 89, wherein the targeting moiety is modified to comprise the azide, the amine, or the thiol.

91. The nanoparticle of claim 89, wherein the linker attaches to the azide on the targeting moiety by azide-alkyne cycloaddition.

92. The nanoparticle of claim 80, wherein the linkers comprise a 4-24 unit PEG spacer.

93. The nanoparticle of claim 80, wherein the linkers comprise a 4, 12, or 24 unit PEG spacer.

94. The nanoparticle of claim 80, wherein the targeting moiety comprises a protein, an aptamer, or a small molecule.

95. The nanoparticle of claim 94, wherein the protein comprises an antibody, a peptide, or a ligand.

96. The nanoparticle of claim 95, wherein the antibody binds CD133, CD117, or CD90.

97. The nanoparticle of claim 95, wherein the antibody comprises an anti-CD133 antibody, anti- CD117 antibody, or anti-CD90 antibody.

98. The nanoparticle of claim 95, wherein the antibody comprises αCD133:7, αCD117:104D2 or αCD90:5E10.

99. The nanoparticle of claim 95, wherein the ligand comprises stem cell factor (SCF).

100. The nanoparticle of claim 94, wherein the aptamer binds CD133.

101. The nanoparticle of claim 94, wherein the aptamer comprises A15 or B19.F053-6005PCT / 24-085-WO-PCT 102. The nanoparticle of claim 94, wherein the protein comprises human luteinizing hormone.

103. The nanoparticle of claim 94, wherein the small molecule comprises degarelix acetate.

104. The nanoparticle of claim 80, wherein the gene-editing components comprise a cutting element and a targeting element.

105. The nanoparticle of claim 104, wherein the cutting element comprises a nuclease.

106. The nanoparticle of claim 105, wherein the nuclease comprises Cpf1 or Cas9.

107. The nanoparticle of claim 104, wherein the targeting moiety is linked to the cutting element.

108. The nanoparticle of claim 107, wherein the targeting moiety is linked to the cutting element with a linker.

109. The nanoparticle of claim 107, wherein the targeting moiety is linked to the cutting element with polyethylene glycol.

110. The nanoparticle of claim 104, wherein the targeting element comprises guide RNA (gRNA).

111. The nanoparticle of claim 110, wherein the gRNA comprises: UAAUUUCUACUCUUGUAGAUUUCGGACCCGUGCUACAACUU (SEQ ID NO: 2); UAAUUUCUACUCUUGUAGAUAUAGAAUAGCCUCAUAUUUUA (SEQ ID NO: 3); UAAUUUCUACUCUUGUAGAUGAGCUGUUGGCAUCAUGUUCCUG (SEQ ID NO: 4); UAAUUUCUACUCUUGUAGAUUCCAAACCUCCUAAAUGAUAC (SEQ ID NO: 5); or UAAUUUCUACUCUUGUAGAUCACCCGAUCCACUGGGGAGCA (SEQ ID NO: 6).

112. The nanoparticle of claim 110, wherein the gRNA targets a sequence comprising: TTTGTGTCCCCGTTTTGGTTGGTAAAC (SEQ ID NO: 7); TTTAAAAATCAATACCGATAATAATGA (SEQ ID NO: 8); TTTCTTAATATGAATATTAATATCGGT (SEQ ID NO: 9); TTTCCGTATCTGGAAGGGGCATCTTGG (SEQ ID NO: 10); TTTCCTTAGGACCGGAAGGATTACAGC (SEQ ID NO: 11); TTTGCCTAAAAGGCACTATGTCAAATG (SEQ ID NO: 12); TTTGGAGCTGTTGGCATCATGTTCCTG (SEQ ID NO: 13); TTTGATTCTTTTCTATCTCAGGACAGA (SEQ ID NO: 14); TTTATAGACATCCCACACTGTAGTTCT (SEQ ID NO: 15); TTTATTAATTTGAGAACCAACATAAGG (SEQ ID NO: 16); TTTATTTTCTTTTTGGTAAGAAGGAAC (SEQ ID NO: 17); TTTCACACACACACACACACACACACA (SEQ ID NO: 18); TTTGGAGCTGTTGGCATCATGTTCCTG (SEQ ID NO: 19); TTTATCCAAACCTCCTAAATGATAC (SEQ ID NO: 20);F053-6005PCT / 24-085-WO-PCT TTTACACCCGATCCACTGGGGAGCA (SEQ ID NO: 21); or TTTTTGATTCTTTTCTATCTCAGGACA (SEQ ID NO: 22).

113. The nanoparticle of claim 80, wherein the gene-editing components further comprise a donor template.

114. The nanoparticle of claim 113, wherein the donor template comprises dsDNA.

115. The nanoparticle of claim 113, wherein the donor template comprises ssDNA.

116. The nanoparticle of claim 113, wherein the donor template provides a therapeutic gene.

117. The nanoparticle of claim 116, wherein the therapeutic gene comprises or encodes skeletal protein 4.1, glycophorin, p55, the Duffy allele, globin family genes, WAS, phox, dystrophin, pyruvate kinase, CLN3, ABCD1, arylsulfatase A, SFTPB, SFTPC, NLX2.1, ABCA3, GATA1, ribosomal protein genes, TERT, TERC, DKC1, TINF2, CFTR, LRRK2, PARK2, PARK7, PINK1, SNCA, PSEN1, PSEN2, APP, SOD1, TDP43, FUS, ubiquilin 2, C9ORF72, α2β1, αvβ3, αvβ5, αvβ63, BOB / GPR15, Bonzo / STRL-33 / TYMSTR, CCR2, CCR3, CCR5, CCR8, CD4, CD46, CD55, CXCR4, aminopeptidase-N, HHV-7, ICAM, ICAM-1, PRR2 / HveB, HveA, α-dystroglycan, LDLR / α2MR / LRP, PVR; PRR1 / HveC, laminin receptor, 101F6, 123F2, 53BP2, abl, ABLI, ADP, aFGF, APC, ApoAI, ApoAIV, ApoE, ATM, BAI-1, BDNF, Beta*(BLU), bFGF, BLC1, BLC6, BRCA1, BRCA2, CBFA1, CBL, C-CAM, CFTR, CNTF, COX-1, CSFIR, CTS-1, cytosine deaminase, DBCCR-1, DCC, Dp, DPC-4, E1A, E2F, EBRB2, erb, ERBA, ERBB, ETS1, ETS2, ETV6, Fab, FancA, FancB, FancC, FancD1, FancD2, FancE, FancF, FancG, FancI, FancJ, FancL, FancM, FancN, FancO, FancP, FancQ, FancR, FancS, FancT, FancU, FancV, and FancW, FCC, FGF, FGR, FHIT, fms, FOX, FUS 1, FUS1, FYN, G-CSF, GDAIF, Gene 21, Gene 26, GM-CSF, GMF, gsp, HCR, HIC-1, HRAS, hst, IGF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, ING1, interferon α, interferon β, interferon γ, IRF-1, JUN, KRAS, LCK, LUCA-1, LUCA-2, LYN, MADH4, MADR2, MCC, mda7, MDM2, MEN-I, MEN-II, MLL, MMAC1, MYB, MYC, MYCL1, MYCN, neu, NF-1, NF-2, NGF, NOEY1, NOEY2, NRAS, NT3, NT5, OVCA1, p16, p21, p27, p53, p57, p73, p300, PGS, PIM1, PL6, PML, PTEN, raf, Rap1A, ras, Rb, RB1, RET, rks-3, ScFv, scFV ras, SEM A3, SRC, TAL1, TCL3, TFPI, thrombospondin, thymidine kinase, TNF, TP53, trk, T-VEC, VEGF, VHL, WT1, WT-1, YES, zac1, iduronidase, IDS, GNS, HGSNAT, SGSH, NAGLU, GUSB, GALNS, GLB1, ARSB, HYAL1, F8, F9, HBB, CYB5R3, γC, JAK3, IL7RA, RAG1, RAG2, DCLRE1C, PRKDC, LIG4, NHEJ1, CD3D, CD3E, CD3Z, CD3G, PTPRC, ZAP70, LCK, AK2, ADA, PNP, WHN, CHD7, ORAI1, STIM1, CORO1A, CIITA, RFXANK, RFX5, RFXAP, RMRP, DKC1, TERT, TINF2, DCLRE1B, or SLC46A1.

118. The nanoparticle of claim 113, wherein the donor template comprises homology arms.

119. The nanoparticle of claim 118, wherein the homology arms are 10 nucleotides – 5000F053-6005PCT / 24-085-WO-PCT nucleotides.

120. The nanoparticle of claim 80, wherein the nanoparticle is a gold nanoparticle.

121. The nanoparticle of claim 80, wherein the targeting moiety is linked to the nanoparticle through electrostatic anchoring.

122. The nanoparticle of claim 80, wherein the nanoparticle further comprises an endosomal polymer shell.

123. The nanoparticle of claim 122, wherein the targeting moiety is linked to the endosomal polymer shell via a linker.

124. The nanoparticle of claim 123, wherein the linker comprises polyethylene glycol (PEG) or oligoethylene glycol (OEG).

125. The nanoparticle of claim 80, wherein the nanoparticle is associated with a polyethylene glycol layer or a polyethylenimine layer.

126. The nanoparticle of claim 125, wherein the polyethylenimine layer is closest to the surface of the nanoparticle.

127. The nanoparticle of claim 80, wherein the nanoparticle comprises four layers, wherein a first layer comprises ssDNA, a second layer comprises gRNA, a third layer comprises a nuclease, and a fourth layer comprises the targeting moiety; wherein the first layer is the closest layer to the surface of the nanoparticle, the second layer is the second closest layer to the surface of the nanoparticle, the third layer is third closest layer to the nanoparticle, and the fourth layer is the furthest layer from the nanoparticle.

128. A composition comprising the nanoparticle of claim 80 and a pharmaceutically acceptable carrier.

129. The composition of claim 128, wherein the composition is formulated for intraosseous delivery.

130. A cell genetically-modifed by the method of claim 3.

131. The cell of claim 130, wherein the cell is a hematopoietic stem cell (HSC), a hematopoietic progenitor cell (HPC), a hematopoietic stem and progenitor cell (HSPC), a T cell, a natural killer (NK) cell, a B cell, a macrophage, a monocyte, a mesenchymal stem cell (MSC), a white blood cell (WBC), a mononuclear cell (MNC), a endothelial cell (EC), a stromal cell, and / or a bone marrow fibroblast.

132. The cell of claim 130, wherein the cell comprises an HSC.

133. The cell of claim 130, wherein the cell comprises an HSPC.F053-6005PCT / 24-085-WO-PCT 134. The cell of claim 130, wherein the cell comprises a CD34+ / CD38- HSC.

135. The cell of claim 130, wherein the cell is genetically modified in vivo.

136. The cell of claim 130, wherein the cell is genetically modified ex vivo.

137. A formulation comprising the cell of claim 130.

138. A method of treating a subject in need thereof comprising administering a composition of claim 128 or a formulation of claim 137 to the subject.

139. The method of claim 138, wherein the administering comprises intraosseous delivery.

140. A kit comprising: nanoparticles; gene-editing components; heterofunctional linkers comprising 4-12 PEG spacers; and targeting moieties.

141. The kit of claim 140, wherein the heterofunctional linkers comprise a DBCO group.

142. The kit of claim 140, wherein the heterofunctional linkers comprise a TFP ester.

143. The kit of claim 140, further comprising a quencher.

144. The kit of claim 143, wherein the quencher comprises free azide and / or free thiol.

145. The kit of claim 140, further comprising an enzyme configured to attach an azide to the targeting moieties.

146. The kit of claim 145, wherein the enzyme comprises GalNAc transferase.

147. The kit of claim 140, further comprising a reducing agent.

148. The kit of claim 147, wherein the reducing agent comprises at least one of dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), 2-Mercaptoethanol, or glutathione (GSH).

149. The kit of claim 140, wherein a concentration of the heterofunctional linkers is 250X-3000X higher than a concentration of the nanoparticles.

150. The kit of claim 140, wherein a concentration of the targeting moieties is 50X-500X higher than a concentration of the nanoparticles.

151. The kit of claim 140, wherein the gene-editing components comprise a targeting element and a cutting element.

152. The kit of claim 151, wherein a concentration of the heterofunctional linkers is 10X-250X higher than a concentration of the cutting element.

153. The kit of claim 151, wherein the cutting element comprises a nuclease.

154. The kit of claim 153, wherein the nuclease comprises Cpf1 or Cas9.

155. The kit of claim 151, wherein the targeting element comprises a guide RNA (gRNA).

156. The kit of claim 155, wherein the targeting element comprises a single gRNA.F053-6005PCT / 24-085-WO-PCT 157. The kit of claim 155, wherein the gRNA comprises: UAAUUUCUACUCUUGUAGAUUUCGGACCCGUGCUACAACUU (SEQ ID NO: 2); UAAUUUCUACUCUUGUAGAUAUAGAAUAGCCUCAUAUUUUA (SEQ ID NO: 3); UAAUUUCUACUCUUGUAGAUGAGCUGUUGGCAUCAUGUUCCUG (SEQ ID NO: 4); UAAUUUCUACUCUUGUAGAUUCCAAACCUCCUAAAUGAUAC (SEQ ID NO: 5); or UAAUUUCUACUCUUGUAGAUCACCCGAUCCACUGGGGAGCA (SEQ ID NO: 6).

158. The kit of claim 155, wherein the gRNA targets a sequence comprising: TTTGTGTCCCCGTTTTGGTTGGTAAAC (SEQ ID NO: 7); TTTAAAAATCAATACCGATAATAATGA (SEQ ID NO: 8); TTTCTTAATATGAATATTAATATCGGT (SEQ ID NO: 9); TTTCCGTATCTGGAAGGGGCATCTTGG (SEQ ID NO: 10); TTTCCTTAGGACCGGAAGGATTACAGC (SEQ ID NO: 11); TTTGCCTAAAAGGCACTATGTCAAATG (SEQ ID NO: 12); TTTGGAGCTGTTGGCATCATGTTCCTG (SEQ ID NO: 13); TTTGATTCTTTTCTATCTCAGGACAGA (SEQ ID NO: 14); TTTATAGACATCCCACACTGTAGTTCT (SEQ ID NO: 15); TTTATTAATTTGAGAACCAACATAAGG (SEQ ID NO: 16); TTTATTTTCTTTTTGGTAAGAAGGAAC (SEQ ID NO: 17); TTTCACACACACACACACACACACACA (SEQ ID NO: 18); TTTGGAGCTGTTGGCATCATGTTCCTG (SEQ ID NO: 19); TTTATCCAAACCTCCTAAATGATAC (SEQ ID NO: 20); TTTACACCCGATCCACTGGGGAGCA (SEQ ID NO: 21); or TTTTTGATTCTTTTCTATCTCAGGACA (SEQ ID NO: 22).

159. The kit of claim 140, wherein the targeting moieties comprises a protein, an aptamer, or a small molecule.

160. The kit of claim 159, wherein the protein comprises an antibody, a peptide, or a ligand.

161. The kit of claim 160, wherein the antibody binds CD133, CD117, or CD90.

162. The kit of claim 160, wherein the antibody comprises an anti-CD133 antibody, anti-CD117 antibody, or anti-CD90 antibody.

163. The kit of claim 160, wherein the antibody comprises αCD133:7, αCD117:104D2 or αCD90:5E10.

164. The kit of claim 160, wherein the ligand comprises stem cell factor (SCF).

165. The kit of claim 159, wherein the aptamer binds CD133.

166. The kit of claim 159, wherein the aptamer comprises A15 or B19.F053-6005PCT / 24-085-WO-PCT 167. The kit of claim 159, wherein the protein comprises human luteinizing hormone.

168. The kit of claim 159, wherein the small molecule comprises degarelix acetate.

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