Strategies for developing genome-editing spherical nucleic acids (SNAs)

Abstract

Spherical nucleic acids (SNAs) are an attractive platform for therapeutic delivery due to their chemically tunable structure, biocompatibility, and ability to rapidly enter cells without transfection reagents. This disclosure provides SNAs and strategies for delivering gene editing proteins to cells. The delivered gene editing proteins remain enzymatically active and rapidly enter mammalian cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 154,530, filed February 26, 2021, U.S. Provisional Patent Application No. 63 / 273,086, filed October 28, 2021, and U.S. Provisional Patent Application No. 63 / 290,522, filed December 16, 2021, which are incorporated by reference in their entireties herein.

[0002] Government Interest Statement This invention was made with Government support under Grant No. DJF-15-1200-K-0001730 awarded by the Federal Bureau of Investigation (FBI). The Government has certain rights in this invention.

[0003] Incorporation by Reference of Electronically Submitted Materials This application contains a sequence listing in computer readable form, which is incorporated by reference in its entirety as a separate part of this disclosure and identified as follows: Filename: 2021-043R_Seqlisting.txt; Size: 61,129 bytes; Creation Date: February 25, 2022. [Background technology]

[0004] Genome editing refers to the removal or insertion of specific DNA sequences. Among the members of genome editing proteins, due to its specificity and versatility, CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) protein is utilized as an efficient genome editing tool to edit and regulate genome for clinical translation [P.Horvath,R.Barrangou,Science 2010,327,167-170]. Although considerable progress has been made with the Cas9 enzyme, reduced off-target effects and efficient and direct transduction of Cas9 single guide RNA (sgRNA) complexes remain highly desirable [LY Chou, K. Ming, WC Chan, Chem. Soc. Rev. 2011, 40, 233-245; V. Biju, Chem. Soc. Rev. 2014, 43, 744-764; Y. Lu, AAA Imetti, R. Langer, Z. Gu, Nat. Rev. Mater. 2017, 2, 16075].

[0005] Rapidly programmable nucleases such as clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) proteins and transcription activator-like effector nucleases (TALENs) have the potential to treat a wide range of genetic diseases [Gupta et al., J Clin Invest. 124(10):4154-4161(2014); Hsu et al., Cell 157(6):1262-1278(2014)], but efficient delivery into mammalian cells remains a challenge. Summary of the Invention

[0006] To address current limitations with genome editing, including off-target effects of gene editing proteins and efficient transduction, various non-viral delivery systems, such as cationic liposomes, cationic polymers, and inorganic nanoparticles, have been designed and used to stabilize and enhance the delivery of Cas9-sgRNA complexes [Y. Fu, J. A. Foden, C. Khayter, M. L. Maeder, D. Reyon, J. K. Joung, J. D. Sander, Nat. Biotechnol. 2013, 31, 822-830]. 826, JG Doench, N. Fusi, M. Sullender, M. Hegde, EW Vaimberg, KF Donovan, I. Smith, Z. Tothova, C. Wilen, R. Orchard, HW Virgin, J. Listgarten, DE Root, Nat. Biotechnol. 2016, 34, 184-191, BPK Leinstiver, V. Pattanayak, MS Prew, SQ Tsai, NT N Guyen, Z. Zheng, JK Joung, Nature 2016, 529, 490-495, IMS Laymaker, L. Gao, B. Zetsche, DA Scott, WX Yan, F. Zhang, Science 2016, 351, 84-88]. However, the complex design, release efficiency, and potential toxic and immunogenic side effects of these carriers hinder their rapid clinical adoption. Viral systems are used as the primary means for transducing cells in vivo. These systems suffer from problems related to packaging constraints, immunogenicity, and the longevity of Cas expression that favors off-target events. Therefore, viral vectors are not the best choice for direct in vivo delivery of CRISPR / Cas. The present disclosure relates to spherical nucleic acids that comprise a shell of oligonucleotides bound to a nanoparticle core, and their use in the delivery of gene editing proteins.

[0007] Thus, in some aspects, the disclosure provides a protein-core globular nucleic acid (ProSNA) comprising: (a) a protein core comprising a gene editing protein; and (b) a shell of oligonucleotides bound to the protein core. In some embodiments, each oligonucleotide in the shell of oligonucleotides is covalently bound to the protein core. In some embodiments, each oligonucleotide in the shell of oligonucleotides is bound to the protein core via a linker. In further embodiments, the linker is a cleavable linker, a non-cleavable linker, a traceless linker, or a combination thereof. In still further embodiments, the linker is a carbamate alkylene dithiolate linker. In some embodiments, at least one oligonucleotide in the shell of oligonucleotides is a protein-core-NH-C(O)-OC 2-5 Alkylene-SSC 2-7 Alkylene-oligonucleotide or protein-core-NH-C(O)-O-CH 2 -Ar-SSC 2-7 In some embodiments, at least one oligonucleotide in the shell of oligonucleotides is a protein-core-NH-C(O)-OC(ZA)(ZB)C 1-4 Alkylene-C(XA)(XB)-SSC(YA)(YB)C 1-6 In some embodiments, at least one oligonucleotide within the shell of oligonucleotides comprises the following structure: alkylene-oligonucleotides, wherein ZA, ZB, XA, XB, YA, and YB are each independently H, Me, Et, or iPr. In some embodiments, at least one oligonucleotide within the shell of oligonucleotides comprises the following structure: protein-core-NH-C(O)-OC(XA)(XB)-Ar-SSC(YA)(YB)C 2-6In some embodiments, the linker is an amide alkylene dithiolate linker. In further embodiments, at least one oligonucleotide within the shell of oligonucleotides comprises a protein-core-NH-C(O)-C 2-5 Alkylene-SSC 2-7 In some embodiments, at least one oligonucleotide in the shell of oligonucleotides comprises a protein-core-NH-C(O)-C 1-4 Alkylene-C(XA)(XB)-SSC(YA)(YB)C 1-6 In some embodiments, the linker is an amide alkylene thioether linker. In further embodiments, at least one oligonucleotide in the shell of oligonucleotides comprises a protein-core-NH-C(O)-C 2-4 Alkylene-N-succinimidyl-SC 2-6 Includes alkylene-oligonucleotides.

[0008] In some aspects, the present disclosure provides a spherical nucleic acid (SNA) comprising (a) a nanoparticle core, (b) a shell of oligonucleotides bound to the outer surface of the nanoparticle core, and (c) a gene-editing protein. In some embodiments, the nanoparticle core is a liposome core or a lipid nanoparticle core. In further embodiments, the lipid nanoparticle core comprises an ionizable lipid, a phospholipid, a sterol, and a lipid-polyethylene glycol (lipid-PEG) conjugate. In some embodiments, each oligonucleotide in the shell of oligonucleotides is covalently attached to the exterior of the lipid nanoparticle core via a lipid-PEG conjugate. In some embodiments, the gene-editing protein is encapsulated in the lipid nanoparticle core. In some embodiments, a ProSNA of the present disclosure is encapsulated in the lipid nanoparticle core. In some embodiments, a ribonucleoprotein (RNP) complex is encapsulated in the lipid nanoparticle core, the RNP comprising the gene-editing protein, a clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA), and a transactivating crRNA (tracrRNA). In some embodiments, the liposome core comprises a plurality of lipid groups. In some embodiments, the gene editing protein is encapsulated in the liposome core. In some embodiments, the ProSNA of the present disclosure is encapsulated in the liposome nanoparticle core. In some embodiments, a ribonucleoprotein (RNP) complex is encapsulated in the lipid nanoparticle core, the RNP comprising the gene editing protein, CRISPR RNA (crRNA), and transactivating crRNA (tracrRNA). In some embodiments, the plurality of lipid groups comprises a lipid selected from the group consisting of the phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine families of lipids.In some embodiments, the lipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG), 1,2-dioleo ... The lipid anchor group is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE). In some embodiments, at least one oligonucleotide in the shell of oligonucleotides is attached to the exterior of the liposome or lipid nanoparticle core via a lipid anchor group. In some embodiments, the lipid anchor group is attached to the 5' terminus or 3' terminus of at least one oligonucleotide. In further embodiments, the lipid anchor group is tocopherol or cholesterol. In some embodiments, the gene editing protein is a CRISPR-associated protein (Cas). In further embodiments, Cas is Cas9, Cas12, Cas13, or a combination thereof. In some embodiments, at least one oligonucleotide in the oligonucleotide shell is modified at its 5' terminus and / or 3' terminus with dibenzocyclooctyl (DBCO). In some embodiments, the oligonucleotide shell comprises single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, or a combination thereof. In some embodiments, at least one oligonucleotide in the oligonucleotide shell is a modified oligonucleotide. In some embodiments, the oligonucleotide shell comprises about 2 to about 100 oligonucleotides. In further embodiments, the oligonucleotide shell comprises about 10 to about 80 oligonucleotides.In some embodiments, the oligonucleotide shell comprises from about 5 to about 50 oligonucleotides. In further embodiments, the oligonucleotide shell comprises from about 5 to about 20 oligonucleotides. In yet further embodiments, the oligonucleotide shell comprises about 14 oligonucleotides. In some embodiments, the oligonucleotide shell comprises about 15 oligonucleotides. In some embodiments, each oligonucleotide in the oligonucleotide shell is from about 5 to about 100 nucleotides in length. In further embodiments, each oligonucleotide in the oligonucleotide shell is from about 10 to about 50 nucleotides in length. In some embodiments, one or more oligonucleotides in the oligonucleotide shell are selected from the group consisting of (GGX). n In some embodiments, the nucleic acid sequence is (GGX), where n is 2 to 20 and X is a nucleobase (A, C, T, G, or U). n The nucleotide sequence is at the 5' end of one or more oligonucleotides. In some embodiments, (GGX) n The nucleotide sequence is at the 3' end of one or more of the oligonucleotides. In some embodiments, one or more of the oligonucleotides within the shell of oligonucleotides has the sequence (GGT) n In some embodiments, the sequence includes a nucleotide sequence, wherein n is 2 to 20. In some embodiments, the sequence includes a nucleotide sequence, wherein n is 2 to 20. n The nucleotide sequence is at the 5' end of one or more of the oligonucleotides. In some embodiments, the nucleotide sequence is (GGT) nThe nucleotide sequence is at the 3' end of one or more oligonucleotides. In some embodiments, the diameter of the ProSNA or SNA is about 1 nanometer (nm) to about 500 nm. In some embodiments, the diameter of the SNA is about 50 nanometers or less. In some embodiments, at least one oligonucleotide in the shell of the oligonucleotide is a targeting oligonucleotide. In some embodiments, the shell of the oligonucleotide comprises an inhibitory oligonucleotide, an immunostimulatory oligonucleotide, a gene editor substrate DNA or RNA, or a combination thereof. In further embodiments, the inhibitory oligonucleotide is an antisense oligonucleotide, a small interfering RNA (siRNA), an aptamer, a short hairpin RNA (shRNA), a DNAzyme, or an aptazyme. In some embodiments, the immunostimulatory oligonucleotide is a CpG motif-containing oligonucleotide, a double-stranded DNA oligonucleotide, or a single-stranded RNA oligonucleotide. In further embodiments, each of the immunostimulatory oligonucleotides is a toll-like receptor (TLR) agonist. In still further embodiments, the TLR is selected from the group consisting of toll-like receptor 1 (TLR1), toll-like receptor 2 (TLR2), toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), toll-like receptor 5 (TLR5), toll-like receptor 6 (TLR6), toll-like receptor 7 (TLR7), toll-like receptor 8 (TLR8), toll-like receptor 9 (TLR9), toll-like receptor 10 (TLR10), toll-like receptor 11 (TLR11), toll-like receptor 12 (TLR12), and toll-like receptor 13 (TLR13).

[0009] In some aspects, the present disclosure provides a composition comprising a plurality of protein-core globular nucleic acids (ProSNAs) as described herein. In some embodiments, the composition further comprises a guide RNA. In some embodiments, at least two ProSNAs comprise different protein cores.

[0010] In some aspects, the present disclosure provides a composition comprising a plurality of spherical nucleic acids (SNAs) of the present disclosure. In some embodiments, at least two of the SNAs comprise different nanoparticle cores.

[0011] In some aspects, the present disclosure provides a method of delivering a gene editing protein to a cell, the method comprising contacting the cell with a ProSNA of the present disclosure.

[0012] In some aspects, the disclosure provides a method of delivering a gene editing protein to a cell, the method comprising contacting the cell with a composition of the disclosure.

[0013] In some aspects, the present disclosure provides a method of delivering a gene editing protein to a cell, comprising contacting the cell with an SNA of the present disclosure.

[0014] In some aspects, the disclosure provides a method of delivering a gene editing protein to a cell, the method comprising contacting the cell with a composition of the disclosure.

[0015] In some aspects, the present disclosure provides a method of treating, ameliorating, and / or preventing a disorder in a subject, comprising administering to the subject an effective amount of (i) a ProSNA of the present disclosure, (ii) a SNA of the present disclosure, (iii) a composition of the present disclosure, or (iv) a combination thereof. In some embodiments, the disorder is cancer, an infectious disease, an autoimmune disease, a neurodegenerative disease, a genetic disease, a cardiovascular disease, or a combination thereof.

[0016] In some aspects, the disclosure provides a fusion protein comprising the following arranged from N-terminus to C-terminus: (i) one or more GALA peptides, (ii) a gene editing protein, and (iii) a nuclear localization signal (NLS) as follows: In some embodiments, the one or more GALA peptides comprise three consecutive GALA peptides. In various embodiments, each of the one or more GALA peptides comprises or consists of an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:22. In some embodiments, the one or more GALA peptides comprises or consists of an amino acid sequence set forth in SEQ ID NO:26. In some embodiments, the gene editing protein is a CRISPR-associated protein (Cas). In further embodiments, the Cas is Cas9, Cas12, Cas13, or a combination thereof. In some embodiments, the Cas9 comprises or consists of an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:25. In some embodiments, the Cas12 comprises or consists of an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO:27. In some embodiments, the Cas13 comprises or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 29. In various embodiments, the NLS comprises or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 28.

[0017] In some aspects, the present disclosure provides a composition comprising a fusion protein of the present disclosure and a physiologically acceptable carrier.

[0018] In a further aspect, the present disclosure provides a ProSNA as described herein, wherein the gene editing protein is a fusion protein of the present disclosure.

[0019] In some aspects, the present disclosure provides an SNA as described herein, wherein the gene editing protein is a fusion protein of the present disclosure.

[0020] In a further aspect, the disclosure provides a method of delivering a gene editing protein to a cell, the method comprising contacting the cell with a fusion protein described herein.

[0021] In some aspects, the disclosure provides a method of delivering a gene editing protein to a cell, the method comprising contacting the cell with a composition of the disclosure comprising a fusion protein.

[0022] In a further aspect, the present disclosure provides a method of treating, ameliorating, and / or preventing a disorder in a subject, comprising administering to the subject an effective amount of (i) a fusion protein of the present disclosure, (ii) a composition of the present disclosure comprising the fusion protein, or (iii) a combination thereof. In some embodiments, the disorder is cancer, an infectious disease, an autoimmune disease, a neurodegenerative disease, a genetic disease, a cardiovascular disease, or a combination thereof. [Brief description of the drawings]

[0023] [Figure 1] Schematic diagram of the synthesis of CRISPR-SNA: concentrated Cas9 RNP is encapsulated in liposomes, most of the unencapsulated RNP is removed via SEC, liposomes are extruded to reduce polydispersity, DBCO-DNA is added to functionalize the liposomes with DNA, liposomes are incubated with proteinase K to digest remaining unencapsulated Cas9, and finally digested Cas9 is removed via SEC. [Diagram 2]Shown are: (A) DLS of CRISPR SNA after DNA functionalization and cleaning. (B) Standard curve of Cy3-DNA fluorescence with SNA samples (diluted in half). (C) ICP-OES quantification of phosphorus (and therefore phospholipid) concentration in CRISPR SNA samples, including the standard curve (blue), SNA samples (red), and SNA samples after correction for DNA concentration obtained in B. SNA concentrations are calculated using Equation 1. (D) Standard curve of Alexa647-RNP fluorescence with SNA samples (blue) plotted with a linear fit. [Diagram 3] It is shown that the RNPs maintain activity throughout the SNA synthesis procedure. (A) Schematic of in vitro Cas9 activity testing. (B) Activity testing of Cas9 RNPs that were freshly prepared (B1), modified with Alexa dye (B2), then concentrated on an Amicon 10K filter (B3), then subjected to 7 cycles of freeze / thaw / sonication (B4), then passed through a Sepharose 6b SEC column (B5), and then extruded three times through 0.2 μM and 0.1 μM PES membranes (B6). [Figure 4]Demonstrating that CRISPR-SNAs protect active RNPs from proteinases and indicate encapsulation. (A) Size-exclusion fractions collected from a Superdex 200 column after incubation of proteinase K with empty SNAs and Alexa-RNPs (top) or a mixture of CRISPR SNAs and encapsulated Alexa-RNPs (bottom). Cy3 (DNA) fluorescence is shown in red, Alexa647 (Cas9) fluorescence is shown in blue, and colocalization of Cy3 and Cas9 fluorescence is shown in pink. (B) In vitro Cas9 activity tests were performed with no Cas9 (1); fresh Cas9 without (2) and with (3) proteinase K; Alexa modified Cas9 without (4) and with (5) proteinase K; CRISPR liposomes without (6), with (7), and with added proteinase K after disruption of liposomes with Tween 20 (8); and finally CRISPR SNA without (9), with (10), and with added proteinase K after disruption of liposomes with Tween 20 (11). [Diagram 5] We show that CRISPR-SNAs are actively taken up into mammalian cells. Five picomole equivalents of Alexa RNPs from each sample were incubated with C166-GFP cells for 16 hours, after which Alexa 647 fluorescence was measured with allophycocyanin (APC) excitation and emission filters. Histograms of Alexa-RNP fluorescence for untreated cells (red, overlapping with empty liposomal spherical nucleic acid (LSNA), empty Cy3-modified LSNA (light green), RNP encapsulated in liposomes (orange), Alexa-RNP transfected with RNAiMax, and finally CRISPR-SNAs (dark green). [Figure 6]Structural characterization of ProSNA (dashed red trace) Cas9. (A) TEM characterization of Cas9 SNA. (B) and (C) Denaturing gel electrophoresis and zeta potential of unmodified Cas9, Cas9 AF647, Cas9 azide, and Cas9 SNA. (D) UV-vis absorbance spectra used to quantify the functionalization of Cas9 with AlexaFluor647 and DNA. [Figure 7] Results from cell experiments demonstrating biocompatibility and cellular uptake are shown: (a) Cell viability of HaCat, HEK293T, hMSC, or Raw264.7 cells treated with Cas9 SNA for 48 h, (b) cellular uptake of Cas9 (white) and Cas9 SNA (black) as determined by flow cytometry. [Figure 8] Figure 1 shows genome editing of HEK293T / EGFP cells by Cas9 SNA. (a) Surveyor assays for DNase I hypersensitive sites, (b) GRIN2B, and (c) EGFP. d) Flow cytometry of HEK293T / EGFP cells treated with Cas9 SNA. [Figure 9] Schematic design of GeoCas9 engineering fused with GALA endosomal peptide at the N-terminus is shown. [Figure 10] Quantitative molar extinction coefficients of GeoCas9 at (a) 260 nm and (b) 280 nm are shown. The molar extinction coefficients were determined by the Pierce bicinchoninic acid assay and used to quantify the concentrations of GeoCas9 and Cas9 SNA. [Figure 11] FIG. 1 shows the structure of Alexa Fluor™ 647 NHS ester (AF647) used to prepare Cas9-AF647. [Figure 12]Shown is the UV-Vis spectrum of AF-647 fluorophore modified Cas9. Spectroscopy was determined at ambient temperature on a Cary5000 spectrophotometer. Protein and AF647 concentrations were calculated from absorbance at 650 nm and 280 nm, respectively. The AF647 fluorophore was used to calculate protein concentration after DNA modification and to track protein uptake in both flow cytometry and confocal imaging experiments. Inset: details of calculation of fluorophores per Cas9. [Figure 13] FIG. 1 shows the structure of the NHS-PEG4-azide linker used to prepare azide-terminated Cas9 (Cas9-AF647-azide). [Figure 14] MALDI-MS spectra of unmodified Cas9-AF647 (blue) and Cas9-AF647-azide (red) are shown. To calculate the number of NHS-PEG4-azides per protein, MALDI-MS was used to determine the mass difference between the unmodified and azide-modified proteins. Each linker conjugation results in a mass increase of 275 m / z. [Figure 15] Determination of the number of DNA strands on Cas9 ProSNA using UV-Vis spectra. Spectra were determined on a Cary5000 spectrophotometer. Protein and DNA concentrations were calculated from the absorbance at 650 nm and 260 nm, respectively. Inset: Details of the calculation of DNA per Cas9. [Figure 16] (a) FPLC size-exclusion chromatogram (SEC) analysis of Cas9 SNA (b) and Cas9-AF647-azide. The solid line corresponds to absorbance at 650 nm and the dashed line corresponds to absorbance at 260 nm. All samples were run on a SEC650 column (Bio-Rad) at a flow rate of 1 mL / min at 4° C. [Figure 17] Shown is an SDS-PAGE gel biostability analysis of (a) Cas9 and (b) Cas9 ProSNA incubated with trypsin (proteinase), showing that Cas9 was degraded over the course of 1 hour (as evidenced by the disappearance of the Cas9 protein band), while Cas9 ProSNA remained. [Figure 18] Cell viability measurements by live / dead assay of Cas9 ProSNA in HaCat cells. Live cells were stained with calcium AM and dead cells were stained with propidium iodide (PI). No significant cytotoxicity was observed after treatment with Cas9 protein as determined by fluorescence microscopy. Scale bar: 300 μm. [Figure 19] Flow histograms showing cellular uptake of AF647-modified Cas9 ProSNA and native Cas9 in HaCat cells. Flow cytometry was used to measure uptake of Cas9 ProSNA or native protein in HaCat cells after treatment with 20 nM protein for 4 hours. [Figure 20] 1 shows the results of nuclear transport efficiency of HaCat cells treated with Cas9-AF647 and Cas9 ProSNA at different time points, demonstrating enhanced nuclear import of Cas9 ProSNA. [Figure 21] SURVEYOR assay for detection of double-strand break-induced microinsertions and deletions. Schematic of the SURVEYOR assay used to determine Cas9-mediated cleavage efficiency. First, genomic PCR (gPCR) is used to amplify the Cas9 target region from a heterogeneous population of modified and unmodified cells, and the gPCR products are slowly rehybridized to generate heteroduplexes. The reannealed heteroduplexes are cleaved by T7EI nuclease, while homoduplexes are left intact. Cas9-mediated cleavage efficiency (indel %) is calculated based on the percentage of DNA cleaved. [Figure 22] Genome editing analysis: Flow cytometry histogram results of HEK293T / EGFP cells treated with Cas9 protein or Cas9 ProSNA. [Diagram 23] We show that surface-reactive lysine chemistry enables DNA conjugation to Cas9. [Figure 24] Figure 2 shows that the structure of Cas9 was retained after DNA functionalization. [Diagram 25] 14 shows that Cas9 ProSNA demonstrated enhanced stability against proteinase degradation. [Figure 26] We show that cells incubated with Cas9 ProSNAs demonstrate high cell viability in multiple cell types, including HaCaT, HEK293T, hMSC, and RAW264.7 cells. [Figure 27] 1 shows enhanced cellular uptake by cells treated with Cas9 ProSNA as observed by AlexaFluor647 fluorescence. [Figure 28] 1 illustrates the barriers and advantages to cellular delivery of gene editing proteins provided by SNAs comprising proteins (e.g., fusion proteins) of the present disclosure. [Figure 29] We show that Cas9 SNA fused with GALA and NLS demonstrated significant endosomal escape and nuclear transport efficiency. [Diagram 30] We show that Cas9 ProSNA achieved high gene editing efficiency for both insertions and deletions compared to the control Cas9 protein in HaCaT and hMSC cells. [Diagram 31] We demonstrate the editing efficiency of Cas9 ProSNA in macrophage-like RAW264.7 cells. Cas9 ProSNA demonstrated increased gene editing activity compared to control Cas9 protein and commercial transfection agents. [Diagram 32] We demonstrate the gene silencing activity of Cas9 ProSNA in HEK293T cells. Cas9 ProSNA demonstrated increased knockdown of GFP compared to the control Cas9 protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Spherical nucleic acids (SNAs) are a class of nanoparticles functionalized with a dense layer of oligonucleotides surrounding an exchangeable nanoparticle core. This nucleic acid shell confers several functions: the oligonucleotide coating reduces endonuclease activity on the nanoparticle surface and forms a highly concentrated salt cloud that interacts with cell surface proteins, resulting in high cellular uptake in virtually all cell lines. The combination of these unique properties allows SNAs to operate as easily tunable, single-body drugs.

[0025] term Language such as "from," "to," "up to," "at least," "greater than," "less than," and the like all refer to ranges that include the recited numbers and can then be broken down into sub-ranges.

[0026] Ranges include each individual member. Thus, for example, a group having 1 to 3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members.

[0027] As used in this specification and the appended claims, the articles "a" and "an" refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.

[0028] "About" and "approximately" are generally intended to mean an acceptable degree of error for the quantity measured given the nature or precision of the measurement. Exemplary degrees of error are within 20-25 percent (%), e.g., within 20 percent, within 10 percent, within 5 percent, within 4 percent, within 3 percent, within 2 percent, or within 1 percent of the stated value or range of values.

[0029] The terms "polynucleotide" and "oligonucleotide" are interchangeable when used herein.

[0030] As used herein, a "linker" is a moiety that connects an oligonucleotide to the protein core of a protein-core globular nucleic acid (ProSNA) as described herein. In any of the aspects or embodiments of the present disclosure, the linker is a cleavable linker, a non-cleavable linker, a traceless linker, or a combination thereof.

[0031] A "subject" is a vertebrate organism. The subject can be a non-human mammal (e.g., a mouse, a rat, or a non-human primate), or the subject can be a human subject.

[0032] As used herein, the terms "administering," "administer," "administration," and the like refer to any mode of transferring, delivering, introducing, or transporting a therapeutic agent to a subject in need of treatment with such agent. Such modes include, but are not limited to, oral, topical, intravenous, intraarterial, intraperitoneal, intramuscular, intratumor, intradermal, intranasal, and subcutaneous administration.

[0033] As used herein, "treating" and "treatment" refer to any reduction in the severity and / or onset of symptoms associated with abnormal scarring. Thus, "treating" and "treatment" include therapeutic and prophylactic measures. One skilled in the art will appreciate that any degree of protection from or amelioration of abnormal scarring will be beneficial to a subject, such as a human patient. The patient's quality of life is improved by any degree of reduction in the severity of symptoms and / or delaying the onset of symptoms in a subject.

[0034] As used herein, a "targeting oligonucleotide" is an oligonucleotide that directs SNA to a specific tissue and / or a specific cell type. In some embodiments, the targeting oligonucleotide is an aptamer. Thus, in some embodiments, the SNA of the present disclosure comprises an aptamer attached to the exterior of the nanoparticle core, and the aptamer is designed to bind to one or more receptors on the surface of a specific cell type.

[0035] As used herein, an "immunostimulatory oligonucleotide" is an oligonucleotide that can stimulate (e.g., induce or enhance) an immune response. Typical examples of immunostimulatory oligonucleotides are CpG motif-containing oligonucleotides, single-stranded RNA oligonucleotides, double-stranded RNA oligonucleotides, and double-stranded DNA oligonucleotides. A "CpG motif" is a cytosine-guanine dinucleotide sequence. Single-stranded RNA sequences can be recognized by toll-like receptors 8 and 9, double-stranded RNA sequences can be recognized by toll-like receptor 3, and double-stranded DNA can be recognized by toll-like receptor 3 and cyclic GMP-AMP synthase (cGAS).

[0036] The term "inhibitory oligonucleotide" refers to an oligonucleotide that reduces the production or expression of a protein, for example, by preventing the translation of mRNA into a protein in a ribosome, or that is sufficiently complementary to either the gene or mRNA encoding one or more of the target proteins to specifically bind (hybridize) to one or more target genes or mRNAs, thereby reducing the expression or biological activity of the target protein. Inhibitory oligonucleotides include, but are not limited to, isolated or synthetic short hairpin RNA (shRNA or DNA), antisense oligonucleotides (e.g., antisense RNA or DNA, chimeric antisense DNA or RNA), miRNA and miRNA mimics, small interfering RNA (siRNA), DNA or RNA inhibitors of innate immune receptors, aptamers, DNAzymes, or aptazymes.

[0037] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which they are cited, and may, in some cases, be incorporated herein in their entirety.

[0038] Gene-editing proteins The SNA of the present disclosure comprises one or more gene editing proteins. The gene editing proteins contemplated by the present disclosure include, but are not limited to, transcription activator-like effector-based nucleases (TALENs), meganucleases, nucleases, zinc finger nucleases (ZFNs), CRISPR-associated proteins, CRISPR / Cas9, Cas9, xCas9, Cas12a (Cpf1), Cas13, Cas13a, Cas14, CasX, CasY, class 1 Cas proteins, class 2 Cas proteins, MAD7, or combinations thereof. In any aspect or embodiment of the present disclosure, genome editing is used to inhibit or reduce the production of a target gene. In certain embodiments, reduction of gene expression and subsequent biologically active protein expression can be achieved by insertion / deletion of nucleotides via non-homologous end joining (NHEJ) or insertion of a suitable donor cassette or insertion of nucleotides via homology directed repair (HDR) leading to a premature stop codon and expression of a non-functional protein.

[0039] As shown in FIG. 28, there are barriers to the entry of gene editing proteins into cells. These barriers include internalization of the gene editing protein (by membrane barriers and large gene editing proteins), ways to achieve nuclear uptake of the gene editing protein, and ways in which the gene editing protein can escape endosomes. Thus, in any of the aspects or embodiments of the present disclosure, the gene editing protein is part of a "fused" protein. The term "fused" in this sense, in various aspects, refers to a protein that comprises or consists of the following elements fused together in N-terminal to C-terminal order: (i) one or more GALA peptides, (ii) a gene editing protein, and (iii) a nuclear localization signal (NLS). In some aspects, the fusion protein comprises or consists of the following elements fused together in N-terminal to C-terminal order: (i) a gene editing protein and (ii) a nuclear localization signal (NLS). The gene editing portion of the fusion protein can be any gene editing protein known in the art and / or described herein, such as, and without limitation, a CRISPR-associated protein (Cas). In various embodiments, the Cas is Cas9, Cas12, Cas13, or a combination thereof. In some embodiments, the Cas9 is as described in Harrington, LB, Paez-Espino, D., Staahl, BT et al. A thermostable Cas9 with increased lifetime in human plasma. Nat Commun 8, 1424 (2017). https: / / doi.org / 10.1038 / s41467-017-01408-4, which is incorporated by reference in its entirety. In some embodiments, the Cas9 comprises or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:25.In some embodiments, Cas12 comprises or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:27 (Strecker J, Jones S, Koopal B, Schmid-Burgk J, Zetsche B, Gao L, Makarova KS, Koonin EV, Zhang F. Nat Commun. 2019 Jan 22;10(1):212. doi:10.1038 / s41467-018-08224-4.10.1038 / s41467-018-08224-4 PubMed 30670702). In some embodiments, Cas13 comprises or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:29 (Smargon AA, Cox DB, Pyzocha NK, Zheng K, Slaymaker IM, Gootenberg JS, Abudayyeh OA, Essletzbichler P, Shmakov S, Makarova KS, Koonin EV, Zhang F. Mol Cell. 2017 Feb 16;65(4):618-630.e7.doi:10.1016 / j.molcel.2016.12.023. Epub 2017 Jan 5.10.1016 / j.molcel.2016.12.023 PubMed 28065598). GALA is known in the art (see, e.g., Schach et al., J. Am. Chem. Soc. 2015, 137, 38, 12199-12202, which is incorporated herein by reference in its entirety) and described herein. The present disclosure contemplates that, in various embodiments, the fusion protein comprises or consists of one, two, three, four, or five GALA peptides in tandem. In some embodiments, the N-terminus of the fusion protein of the present disclosure comprises or consists of three GALA peptides in tandem. In some embodiments, the N-terminus of the fusion protein of the present disclosure comprises or consists of three GALA peptides in tandem, each GALA peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:22.In any of the aspects or embodiments of the present disclosure, the C-terminus of the fusion protein described herein comprises or consists of an NLS sequence. NLS sequences are known in the art (see, for example, Cutrona, G., Carpaneto, E., Ulivi, M. et al. Effects in live cells of a c-myc antigen PNA linked to a nuclear localization signal. Nat Biotechnol 18, 300-303 (2000). https: / / doi.org / 10.1038 / 73745, which is incorporated herein by reference in its entirety). In some embodiments, the NLS sequence is derived from the NLS of the SV40 virus large T antigen and comprises or consists of the amino acid sequence PKKKRKV (SEQ ID NO: 23). In some embodiments, the NLS comprises or consists of the amino acid sequence KRTADGSEFESPKKKRKV (SEQ ID NO: 28). The present disclosure also provides a composition comprising a fusion protein described herein and a pharma- ceutically acceptable carrier. The fusion protein provided by the present disclosure can be used in any of the ProSNA, SNA, compositions, and / or methods described herein.Thus, in some aspects, the ProSNA of the present disclosure comprises (a) a protein core comprising a fusion protein, and (b) a shell of oligonucleotides bound to the protein core.In further aspects, the present disclosure provides an SNA comprising (a) a nanoparticle core, (b) a shell of oligonucleotides bound to the outer surface of the nanoparticle core, and (c) a fusion protein.

[0040] Through in vitro studies using the Streptococcus pyogenes type II CRISPR / Cas system, it has been shown that the only components required for efficient CRISPR / Cas-mediated target DNA or genome modification are a Cas nuclease (e.g., Cas9 nuclease), a CRISPR RNA (crRNA), and a trans-activating crRNA (tracrRNA). The wild-type mechanism of CRISPR / Cas-mediated DNA cleavage occurs through several steps. Transcription of the CRISPR array containing a small fragment (20-30 base pairs) of the encountered (or target) DNA into pre-crRNA, which matures through hybridization with tracrRNA via the direct repeats of the pre-crRNA. Hybridization of the pre-crRNA and tracrRNA, known as the guide RNA (gRNA or sgRNA), associates with the Cas nuclease, which forms a ribonucleoprotein complex that mediates conversion of the pre-crRNA to the mature crRNA. The mature crRNA:tracrRNA duplex directs Cas9 to a DNA target consisting of a protospacer and a required protospacer adjacent motif (CRISPR / cas protospacer adjacent motif; PAM) via heteroduplex formation between the spacer region of the crRNA and the protospacer DNA on the host genome. Cas9 nuclease mediates cleavage of the target DNA upstream of the PAM to generate a double-stranded break within the protospacer or uses a mutated Cas9 nuclease to generate a strand-specific nick, thereby mutating one DNA strand-specific cleavage motif.

[0041] Thus, in various aspects involving gene editing, the SNAs of the disclosure (e.g., ProSNAs, LNP-SNAs, LSNAs) include, in addition to the gene editing protein, a DNA or RNA gene editor substrate (e.g., a guide RNA), which in various embodiments is bound to the surface of the SNA or encapsulated within the SNA. In some embodiments, the SNAs containing the gene editing protein are delivered separately from the DNA or RNA gene editor substrate.

[0042] Other RNA-guided nucleases from related CRISPR systems that have also been adapted for programmable nucleic acid cleavage include Staphylococcus aureus Cas9 (SaCas9), CRISPR from Prevotella or Franciscella I (CpfI), Geobacillus Cas9 (GeoCas9), Campylobacter jejuni Cas9 (CjCas9), metagenomic-derived CRISPR-CasX and CRISPR-CasY, CRISPR-Cas3, and the RNA-cleaving CRISPR-C2c2.

[0043] CRISPR / Cas systems have been modified to perform several functions other than gene knockout and editing, three of which are described below: Catalytically inactivated Cas9 (dCas9) has been fused to transcriptional activation and repression domains, thereby allowing programmable control of gene expression [Gilbert et al., Cell 154, 442-451 (2013); Zalatan et al., Cell 160339-350 (2015)]. dCas9 transcriptional activators in particular allow de novo screening, similar to siRNA or CRISPR knockout libraries, but where genes are overexpressed [Gilbert et al., Cell 159, 647-61 (2014)]. dCas9 fused to fluorescent proteins allows microscopic tracking of specific sites in the genome and the study of sequence-specific nuclear organization [Chen et al., Cell 155, 1479-91 (2013)]. Finally, active Cas9 can be targeted to cut various non-functional genomic regions within the zygote, and the frequency and sequence of mutations in each cell of the mature organism can be used to trace the lineage of cell differentiation during embryonic development [Mckenna et al., Science 42, 237-241 (2016)].

[0044] As used herein, the term TALEN is broad and includes monomeric TALEN that can cut double-stranded DNA without the assistance of another TALEN.The term TALEN is also used to refer to one or both members of a pair of TALENs that are engineered to work together to cut DNA at the same site.The TALENs that work together can be referred to as left TALEN and right TALEN, referring to the handedness of DNA or TALEN pair.

[0045] The term TALEN refers to a protein that contains a transcription activator-like (TAL) effector binding domain and a nuclease domain, including monomeric TALENs that are functional by themselves and others that require dimerization with another monomeric TALEN. Dimerization can result in homodimeric TALENs when both monomeric TALENs are identical, or heterodimeric TALENs when the monomeric TALENs are different. TALENs have been shown to induce genetic modification in immortalized human cells by two major eukaryotic DNA repair pathways, non-homologous end joining (NHEJ) and homology-directed repair. Although TALENs are often used in pairs, monomeric TALENs are known. Cells for treatment with TALENs (and other genetic tools) include cultured cells, immortalized cells, primary cells, primary somatic cells, zygotes, germ cells, primordial germ cells, blastocysts, or stem cells. In some embodiments, TAL effectors can be used to target other protein domains (e.g., non-nuclease protein domains) to specific nucleotide sequences. For example, TAL effectors can be conjugated to protein domains from proteins that interact with or modify other proteins, such as, but not limited to, DNA-interacting enzymes (e.g., methylases, topoisomerases, integrases, transposases, or ligases), transcriptional activators or repressors, or histones. Applications of such TAL effector fusions include, for example, creating or modifying epigenetic regulatory elements, making site-specific insertions, deletions, or repairs in DNA, controlling gene expression, and modifying chromatin structure.

[0046] Thus, in some aspects, the present disclosure provides SNAs (e.g., ProSNAs, LSNAs, LNP-SNAs) for use in delivery of gene editing proteins. In various embodiments, the gene editing proteins are in a ribonucleoprotein (RNP) complex. In various embodiments, the ribonucleoprotein (RNP) complex encapsulated in the SNA comprises CRISPR-associated protein 9 (Cas9) (SEQ ID NO: 1 or SEQ ID NO: 25), CRISPR RNA (crRNA), transactivating crRNA (tracrRNA), and / or transcription activator-like effector nuclease (TALEN). In some embodiments, the Cas9 utilized in the compositions and methods of the present disclosure is EnGen® Cas9 NLS, S. pyogenes (New England Biolabs Catalog No. M0646T). In any of the aspects or embodiments of the disclosure, the nucleotide or amino acid sequence of the disclosure comprises or consists of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a reference or wild-type sequence. In any of the aspects or embodiments of the disclosure, the gene editing protein comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a reference or wild-type sequence. In various embodiments, the gene editing protein is a Cas9 protein that comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a reference or wild-type Cas9 sequence. Thus, in various embodiments, the gene editing protein is a Cas9 protein that comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:1 or SEQ ID NO:25. In further embodiments, the gene editing protein is a Cas12 protein that comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:27.In further embodiments, the gene editing protein is a Cas13 protein comprising or consisting of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:29.

[0047] Spherical nucleic acid (SNAS) As described herein, spherical nucleic acids (SNAs) are a unique class of nanomaterials that comprise a spherical nanoparticle core functionalized with a highly oriented oligonucleotide shell. The oligonucleotide shell comprises one or more oligonucleotides attached to the outer surface of the nanoparticle core. In various embodiments, the oligonucleotide shell comprises inhibitory oligonucleotides, immunostimulatory oligonucleotides, gene editor substrate DNA or RNA, targeting oligonucleotides, or combinations thereof. The nanoparticle core can be either organic (e.g., liposomes), inorganic (e.g., gold, silver, or platinum), polymer-based (e.g., poly(lactic-co-glycolic acid) (PLGA) particles), or hollow (e.g., silica-based). In various embodiments of the present disclosure, the nanoparticle core is a protein (protein-core SNA (ProSNA)), liposome (liposomal SNA (LSNA)), or lipid nanoparticle (LNP-SNA).

[0048] The spherical architecture of the polynucleotide shell confers unique advantages over traditional nucleic acid delivery methods, including entry into nearly all cells independent of transfection agents and resistance to nuclease degradation. Furthermore, SNAs can penetrate biological barriers, including the blood-brain barrier (see, e.g., U.S. Patent Application Publication No. 2015 / 0031745, which is incorporated herein by reference in its entirety) and the blood-tumor barrier, as well as the epidermis (see, e.g., U.S. Patent Application Publication No. 2010 / 0233270, which is incorporated herein by reference in its entirety).

[0049] Protein-core globular nucleic acid (ProSNA) Recently, protein spherical nucleic acids (ProSNAs), which comprise a dense shell of oligonucleotides bound (e.g., covalently attached) to a protein core, have emerged as an exciting new architecture with diverse biological applications in protein delivery, assembly, and intracellular sensing [Brodin, JD; Sprangers, AJ; McMillan, JR; Mirkin, CA DNA-Mediated Cellular Delivery of Functional Enzymes. J. Am. Chem. Soc. 2015, 137(47), 14838-14841; Kusmierz, CD; Bujold, KE; Callmann, CE; Mirkin, CA Defining the Design Parameters for in Vivo Enzyme Delivery Through Protein Spherical Nucleic Acids. ACS Cent. Sci. 2020, 6(5), 815-822]. The dense shell of oligonucleotides promotes cellular uptake, physiological stability, and biocompatibility of proteins relative to their individual components [Giljohann, DA; Seferos, DS; Patel, PC; Millstone, JE; Rosi, NL; Mirkin, CA Oligonucleotide Loading Determines Cellular Uptake of DNA-Modified Gold Nanoparticles. Nano Lett. 2007, 7(12), 3818-3821]. This enhanced cellular internalization of SNAs stems from the three-dimensional architecture of the conjugates and their ability to engage scavenger receptors on the surface of most cells. Importantly, the favorable biological properties of SNAs are independent of their protein cores and therefore can be selected for protein delivery genome editing applications.

[0050] As used herein, "protein-core" includes gene-editing proteins. Thus, in any of the aspects or embodiments of the present disclosure, the gene-editing proteins of the present disclosure generally function as the "core" of a protein-core SNA (SNA). A protein is a molecule that includes one or more polymers of amino acids. In various embodiments of the present disclosure, the protein-core includes or consists of a single protein (i.e., a single polymer of amino acids), a multimeric protein, a peptide (e.g., a polymer of amino acids about 2-50 amino acids in length), or a synthetic fusion protein of two or more proteins. Synthetic fusion proteins include, but are not limited to, expressed fusion proteins (expressed from a single gene) and post-expression fusions in which proteins are chemically conjugated together. In any of the aspects or embodiments of the present disclosure, the protein-core includes or consists of a gene-editing protein. Proteins are understood in the art and can be either naturally occurring or non-naturally occurring.

[0051] Protein-core SNA synthesis. The present disclosure provides compositions and methods in which one or more oligonucleotides are associated and / or attached to the surface of a protein-core SNA via a linker. In various embodiments, the linker can be a cleavable linker, a non-cleavable linker, a traceless linker, or a combination thereof. In some embodiments, the cleavable linker is sensitive to (and cleaved in response to) a reducing agent (e.g., glutathione (GSH), dithiothreitol (DTT)) or a reducing environment (e.g., inside a cell). In various embodiments, the cleavable linker is sensitive to (and cleaved in response to) various chemical stimuli, such as, for example, acidity (e.g., low pH), enzymes (e.g., peptidases), light (e.g., NIR laser), and / or hydrolysis.

[0052] The linker connects the protein-core to the oligonucleotide (i.e., protein-core-LINKER-oligonucleotide) in the disclosed protein-core SNA. In various embodiments, a single oligonucleotide is attached to the linker. In further embodiments, two or more oligonucleotides (e.g., two, three, or more) are attached to a single linker. In general, linkers contemplated by the present disclosure include the following, which may be used alone or in combination in the ProSNAs of the present disclosure: amide, thioether, triazole, oxime, urea, and thiourea. Some specifically contemplated linkers include carbamate alkylene, carbamate alkylene aryl dithiolate linker, amide alkylene dithiolate linker, amide alkylene aryl dithiolate linker, and amide alkylene succinimidyl linker. In some cases, the linker is -NH-C(O)-OC 2-5 Alkylene-SSC 2-7 Alkylene- or -NH-C(O)-C 2-5 Alkylene-SSC 2-7 The carbon alpha to the -SS- moiety can be branched, e.g., -C(XA)(XB)-SS- or -SSC(YA)(YB)-, or combinations thereof, where XA, XB, YA, and YB are independently H, Me, Et, or iPr. The carbon alpha to the protein can be branched, e.g., -C(XA)(XB)-C 2-4 alkylene-SS-, where XA and XB are H, Me, Et, or iPr. In some cases, the linker is -NH-C(O)-O-CH 2 -Ar-SSC 2-7 alkylene- and Ar is a meta or para substituted phenyl. 2-4 Alkylene-N-succinimidyl-SC 2-6 It is alkylene.

[0053] Additional linkers contemplated by the present disclosure include those described in International Patent Publication No. 2018 / 213585, which is incorporated herein by reference in its entirety. In some embodiments, the linker is an SH linker, an SM linker, an SE linker, or an SI linker. The present disclosure contemplates multiple attachment points of the oligonucleotide on the protein-core.

[0054] The oligonucleotides of the present disclosure can be modified at either the 5' or 3' end for attachment to the protein core.

[0055] The oligonucleotides of the disclosure may contain an alkyne moiety, e.g., a DBCO-type moiety for reacting with azides on a protein surface: [ka] where L is a linker to the terminus of the polynucleotide. 2 is C 1-10 Alkylene, -C(O)-C 1-10 Alkylene-Y- and -C(O)-C 1-10 Alkylene-YC 1-10 Alkylene-(OCH 2 CH 2 ) m -Y-, where each Y is independently selected from the group consisting of a bond, C(O), O, NH, C(O)NH, and NHC(O), and m is 0, 1, 2, 3, 4, or 5. For example, the DBCO functional group can be [ka] where the terminal "O" is from the terminal nucleotide on the polynucleotide. The use of this DBCO-type moiety allows for a link between the polynucleotide and the protein when the surface amines are modified. [ka] where L and L 2 are each independently1-10 Alkylene, -C(O)-C 1-10 Alkylene-Y- and -C(O)-C 1-10 Alkylene-YC 1-10 Alkylene-(OCH 2 CH 2 ) m -Y-, where each Y is independently selected from the group consisting of a bond, C(O), O, NH, C(O)NH, and NHC(O), m is 0, 1, 2, 3, 4, or 5, and PN is a polynucleotide. Similar structures in which surface thiols or surface carboxylates of proteins are modified can be made in a similar manner to yield equivalent binding structures.

[0056] Proteins can be modified with surface functional groups (e.g., surface amines, surface carboxylates, surface thiols) using linkers that terminate in an azide functionality: protein-XLN 3 (X is from a surface amino group (e.g., -NH-), a carboxy group (e.g., -C(O)- or -C(O)O-), or a thiol group (e.g., -S-) on the protein; and L is from 1-10 Alkylene, -YC(O)-C 1-10 Alkylene-Y- and -YC(O)-C 1-10 Alkylene-YC 1-10 Alkylene-(OCH 2 CH 2 ) m -Y-, where each Y is independently selected from the group consisting of a bond, C(O), O, NH, C(O)NH, and NHC(O), and m is 0, 1, 2, 3, 4, or 5. 3 The introduction of functional groups can be accomplished using well-known techniques. For example, the surface amines of a protein can be substituted with terminal N 3 to form an amide bond between the amine of the protein and the carboxylate of the activated ester of the linker reagent.

[0057] The oligonucleotide can be modified to include an alkyne functional group at the terminus of the oligonucleotide: oligonucleotide-L. 2 -X-≡-R(L 2 is C 1-10 Alkylene, -C(O)-C 1-10 Alkylene-Y- and -C(O)-C 1-10 Alkylene-YC 1-10 Alkylene-(OCH 2 CH 2 ) m -Y-, each Y is independently selected from the group consisting of a bond, C(O), O, NH, C(O)NH, and NHC(O), m is 0, 1, 2, 3, 4, or 5, X is a bond, and R is H or C 1-10 or X and R together with the carbon to which they are attached form an 8-10 membered carbocyclic or 8-10 membered heterocyclic group). [ka] has.

[0058] Proteins with surface-modified azides and polynucleotides with termini modified to contain alkynes can be reacted together to form a triazole ring in the presence of a copper(II) salt and a reducing agent to generate a copper(I) salt in situ. In some cases, the copper(I) salt is added directly. Reducing agents contemplated include ascorbic acid, ascorbate, sodium borohydride, 2-mercaptoethanol, dithiothreitol (DTT), hydrazine, lithium aluminum hydride, diisobutylaluminum hydride, oxalic acid, Lindlar's catalyst, sulfite compounds, tin compounds, iron compounds, sodium amalgam, tris(2-carboxyethyl)phosphine, hydroquinone, and mixtures thereof.

[0059] The surface functional groups of proteins can be attached to oligonucleotides using other linking chemistries. For example, surface amines can be directly conjugated to carboxylates or activated esters at the termini of oligonucleotides to form amide bonds. Surface carboxylates can be conjugated to amines on the termini of oligonucleotides to form amide bonds. Alternatively, surface carboxylates can be reacted with diamines to form amide bonds at the surface carboxylates and amines at the other termini. This terminal amine can then be modified in a similar manner as for surface amines of proteins. Surface thiols can be conjugated to thiol moieties on polynucleotides to form disulfide bonds. Alternatively, thiols can be conjugated to activated esters on the termini of polynucleotides to form thiocarboxylates. Alternatively, thiols can be conjugated to Michael acceptors (e.g., succinimides) on the termini of polynucleotides to form thioethers.

[0060] In general, a typical procedure for synthesizing protein-core SNA (ProSNA) involves attaching a desired amount of oligonucleotides to the surface of a protein. Attachment is performed by repeating a two-step process: (1) attaching and purifying a linker to the surface of the protein, and (2) attaching and purifying an oligonucleotide (e.g., DNA) to the protein-conjugated linker. These two steps are repeated until a desired amount of oligonucleotides is attached to the protein. It will be understood that the foregoing procedure is exemplary in nature.

[0061] Lipid Nanoparticle Spherical Nucleic Acid (LNP-SNA) Lipid nanoparticle spherical nucleic acid (LNP-SNA) is composed of a lipid nanoparticle core decorated with oligonucleotides. The lipid nanoparticle core contains a gene editing protein, an ionizable lipid, a phospholipid, a sterol, and a lipid-polyethylene glycol (lipid-PEG) conjugate. The oligonucleotide shell contains one or more oligonucleotides attached to the outer surface of the lipid nanoparticle core. The spherical architecture of the oligonucleotide shell confers unique advantages over conventional nucleic acid delivery methods, including entry into almost any cell independent of transfection agents, resistance to nuclease degradation, sequence-based function, targeting, and diagnostics.

[0062] Thus, in various aspects, the present disclosure provides a lipid nanoparticle spherical nucleic acid (LNP-SNA) comprising (a) a lipid nanoparticle core, (b) a shell of oligonucleotides bound to the outer surface of the lipid nanoparticle core, and (c) a gene editing protein. Thus, the LNP-SNA comprises a gene editing protein, an ionizable lipid, a phospholipid, a sterol, and a lipid-polyethylene glycol (lipid-PEG) conjugate. In some embodiments, the ionizable lipid is dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), C12-200, 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), similar lipid / lipidoid structures, or combinations thereof. In some embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dihexadecanoylphosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or a combination thereof.In a further embodiment, the sterol is 3β-hydroxycholest-5-ene (cholesterol), 9,10-secocholesta-5,7,10(19)-trien-3β-ol (vitamin D3), 9,10-secoegosta-5,7,10(19),22-tetraen-3β-ol (vitamin D2), calcipotriol, 24-ethyl-5,22-cholestadien-3β-ol (stigmasterol), 22,23-dihydrostigmasterol (β-sitosterol), 3,28-dihydroxy-lupeol (beta-sitosterol), 5,5-dihydroxy-1,1,2,3,4,4-trimethylol, 5,5-dihydro ... Turin), lupeol, ursolic acid, oleanolic acid, 24α-methylcholesterol (campesterol), 24-ethylcholesta-5,24(28)E-dien-3β-ol (fucosterol), 24-methylcholesta-5,22-dien-3β-ol (brassicasterol), 24-methylcholesta-5,7,22-trien-3β-ol (ergosterol), 9,11-dehydroergosterol, daucosterol, or any of the foregoing sterols modified with one or more amino acids. In some embodiments, the lipid-polyethylene glycol (lipid-PEG) conjugate comprises a 2000 Dalton (Da) polyethylene glycol. In further embodiments, the lipid-polyethylene glycol (lipid-PEG) conjugate is a lipid-PEG-maleimide. In still further embodiments, the lipid-PEG-maleimide is 1,2-dipalmitryl-sn-glycero-3-phosphoethanolamine (DPPE) conjugated to a 2000 Da polyethylene glycol maleimide, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) conjugated to a 2000 Da polyethylene glycol maleimide, or a combination thereof.

[0063] Oligonucleotides contemplated for use according to the present disclosure include those bound to the nanoparticle core through any means (e.g., covalent or non-covalent). In any of the aspects or embodiments of the present disclosure, the oligonucleotides are bound to the exterior of the lipid nanoparticle core via covalent binding to a lipid-polyethylene glycol (lipid-PEG) conjugate of the oligonucleotide. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the oligonucleotides in the oligonucleotide shell are covalently bound to the exterior of the lipid nanoparticle core via a lipid-PEG conjugate. In various embodiments, one or more oligonucleotides in the oligonucleotide shell are bound to the exterior of the lipid nanoparticle core via a lipid anchor group. The lipid anchor group, in various embodiments, is attached to the 5' or 3' terminus of the oligonucleotide. In various embodiments, the lipid anchor group is cholesterol or tocopherol.

[0064] In any of the aspects or embodiments of the present disclosure, the LNP-SNAs are synthesized such that the gene editing protein is encapsulated in the lipid nanoparticle core and a shell of oligonucleotides is attached to the exterior of the lipid nanoparticle core. Generally, and by way of example, lipid nanoparticles (LNPs) may be formulated by diluting lipids and sterols in ethanol.

[0065] Liposomal Spherical Nucleic Acid (LSNA) Liposomes are spherical, self-closed structures of various size ranges that contain one or several hydrophobic lipid bilayers with a hydrophilic core. The diameter of these lipid-based carriers ranges from 0.15 to 1 micrometer, which is significantly larger than the effective therapeutic range of 20 to 100 nanometers. Liposomes called small unilamellar vesicles (SUVs) can be synthesized in the size range of 20 to 50 nanometers, but face challenges such as instability and aggregation that lead to interparticle fusion. This interparticle fusion limits the use of SUVs in therapeutics.

[0066] Liposomal spherical nucleic acid (LSNA) is an attractive platform for therapeutic delivery due to its chemically tunable structure, biocompatibility, and ability to rapidly enter cells without transfection reagents. The present disclosure provides a method for delivering gene editing proteins to cells by encapsulating them in LSNA. The encapsulated gene editing enzymes remain enzymatically active and rapidly enter mammalian cells. These properties make this new form of LSNA a delivery vehicle for gene editing therapeutics.

[0067] Previous SNA-mediated protein delivery strategies require chemical modification of amino acids on proteins, which may inhibit protein function. Proteins encapsulated in LSNAs can be delivered into cells without any chemical modification. Furthermore, cationic lipid-mediated strategies for protein delivery require anionic protein complexes. However, SNA-mediated delivery uses neutral phospholipids and does not require anionic proteins. Therefore, this method is also suitable for the delivery of positively charged proteins such as TALENs.

[0068] Thus, in some aspects, the present disclosure contemplates the use of the LSNAs disclosed herein, including gene editing enzymes (e.g., CRISPR-associated protein 9 (Cas9) (Jinek et al., (2012) Science. 816-821; Zuris et al., Nat Biotechnol. 2015 Jan;33(1):73-80, which are incorporated by reference in their entireties), CRISPR RNA (crRNA), and trans-activating crRNA (tracrRNA), transcription activator-like effector nucleases (TALENs)) and surface-functionalized oligonucleotides in gene editing methods.

[0069] Thus, the present disclosure provides LSNAs for use in methods including, but not limited to, in vitro or in vivo delivery (e.g., to cells) of gene editing proteins. Liposomal particles (e.g., as disclosed in International Patent Application No. PCT / US2014 / 068429, which is incorporated herein by reference in its entirety, particularly with respect to the discussion of liposomal particles) are also contemplated by the present disclosure. The liposomal particles of the present disclosure have at least a substantially spherical shape, an interior and an exterior, and comprise a lipid bilayer. Thus, in various aspects, the present disclosure provides spherical nucleic acids (SNAs) comprising (a) a liposomal core, (b) a shell of oligonucleotides bound to the exterior surface of the liposomal core, and (c) a gene editing protein. The lipid bilayer comprises, in various embodiments, a plurality of lipid groups, including lipids from the phosphocholine family of lipids or the phosphoethanolamine family of lipids. Lipids contemplated by the present disclosure include 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), cardiolipin, lipid A, and combinations thereof. In various embodiments, at least one oligonucleotide within the oligonucleotide shell is attached to the exterior of the liposome core via a lipid anchor group.In further embodiments, the lipid anchor group is attached to the 5' or 3' terminus of at least one oligonucleotide. In yet further embodiments, the lipid anchor group is tocopherol or cholesterol. Thus, in various embodiments, at least one (or all) of the oligonucleotides in the shell of the oligonucleotide is an oligonucleotide-lipid conjugate containing a lipid anchor group, which is adsorbed to the lipid bilayer. The lipid anchor group, in various embodiments, comprises tocopherol, palmitoyl, dipalmitoyl, stearyl, distearyl, or cholesterol. In a further aspect, the present disclosure provides a LSNA comprising a lipid bilayer having a substantially spherical shape and comprising a plurality of lipid groups, a ribonucleoprotein (RNP) complex encapsulated in a liposomal particle, the RNP comprising a gene editing protein (e.g., CRISPR associated protein 9 (Cas9)) and a guide RNA, and one or more oligonucleotides on the surface of the LSNA.

[0070] With respect to the surface density of oligonucleotides on the surface of the LSNAs of the present disclosure, it is contemplated that the LSNAs described herein comprise from about 1 to about 400 oligonucleotides on their surface. In various embodiments, the LSNAs comprise from about 10 to about 100, or from 10 to about 90, or from about 10 to about 80, or from about 10 to about 70, or from about 10 to about 60, or from about 10 to about 50, or from about 10 to about 40, or from about 10 to about 30, or from about 10 to about 20, or from about 50 to about 100, or from about 60 to about 100, or from about 70 to about 100, or from about 80 to about 100, or from about 90 to about 100 oligonucleotides on their surface. In further embodiments, the LSNA comprises or consists of at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, or 400 oligonucleotides on its surface. In some embodiments, the LSNA comprises or consists of 70 oligonucleotides on its surface. Additional surface densities of SNAs are described herein below.

[0071] In some aspects, architectures are disclosed that include tocopherol-modified oligonucleotides. In various embodiments, it is contemplated that the tocopherol is at the 5'-terminus or 3'-terminus of the oligonucleotide or modified form thereof. The tocopherol-modified oligonucleotide includes a lipophilic terminus and a non-lipophilic terminus. The lipophilic terminus includes tocopherol and may be selected from the group consisting of tocopherol derivatives, alpha-tocopherol, beta-tocopherol, gamma-tocopherol, and delta-tocopherol. In further embodiments, the lipophilic terminus includes palmitoyl, dipalmitoyl, stearyl, cholesterol, or distearyl.

[0072] In a further aspect, the present disclosure contemplates that cholesterol or phospholipids are used instead of tocopherol. Cholesterol is attached in solid-phase oligonucleotide synthesis and mixed with the prepared liposomes to form SNA. In some embodiments, liposomes composed of 95% 1,2-dioleoyl-sn-glycero-3 phosphatidylcholine (DOPC) and 5% 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(6-azidohexanoyl) (DPPE-azide) are prepared as described below. DBCO-modified oligonucleotides are then added that react with the azide lipids to functionalize the surface.

[0073] In yet a further embodiment, phospholipid-conjugated oligonucleotides are prepared as follows: First, a phosphatidylethanolamine lipid such as DOPE is reacted with succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB) by mixing 25 mg / mL lipid, 1 equivalent of SMPB, and 1 equivalent of N,N-diisopropylethylamine in chloroform. The mixture is allowed to react overnight. The product is then purified by flash chromatography using a silica column (solvent A: dichloromethane, solvent B: methanol). The thiol-modified oligonucleotide (3' or 5' end modified) is reduced with 0.2 M DTT and 0.1 M phosphate buffer (pH 8) at 40°C for 2 hours. The oligonucleotide is then purified on a size-exclusion column using water. The phosphatidylethanolamine-SMPB lipid is dried over nitrogen gas and dissolved in ethanol in the same volume as the oligonucleotide. The oligonucleotide is then mixed with the lipid such that the reaction is 50:50 water and ethanol. The mixture is reacted overnight, and excess lipid is extracted by washing the reaction mixture with chloroform three times.Then, the aqueous phase and the interface are dried and dissolved in water.It is contemplated that all lipid-conjugated oligonucleotides disclosed herein can be used interchangeably in the preparation of LSNA.The non-lipophilic end of tocopherol-modified oligonucleotide is the oligonucleotide described herein.

[0074] Disclosed herein is a method for making oligonucleotides that contain lipid anchors.For example, first, an oligonucleotide and a phosphoramidite-modified tocopherol are provided.Then, the oligonucleotide is exposed to the phosphoramidite-modified tocopherol to make a tocopherol-modified oligonucleotide.Any chemistry known to those skilled in the art can be used to bind tocopherol (or any lipid anchor), including, but not limited to, amide bond or click chemistry.

[0075] The present disclosure also provides a method for making LSNA. In some embodiments, a phospholipid, a solvent, and a tocopherol-modified oligonucleotide are provided. Then, the phospholipid is added to the solvent to form a first mixture containing liposomes. The size of the liposomes in the first mixture is about 100 nanometers to about 150 nanometers. Next, the liposomes are disrupted to make a second mixture containing liposomes and small unilamellar vesicles (SUVs). The size of the liposomes and SUVs in the second mixture is about 20 nanometers to about 150 nanometers. Next, SUVs having a particle size of about 20 nanometers to about 50 nanometers are isolated from the second mixture. Finally, a tocopherol-modified oligonucleotide is added to the isolated SUVs to make liposomal particles. In various embodiments, the diameter of the LSNAs made by the method of the present disclosure is about 50 nanometers or less. In some embodiments, a plurality of LSNAs are produced, and the particles within the plurality have an average diameter of about 50 nanometers or less (e.g., about 5 nanometers to about 50 nanometers, or about 5 nanometers to about 40 nanometers, or about 5 nanometers to about 30 nanometers, or about 5 nanometers to about 20 nanometers, or about 10 nanometers to about 50 nanometers, or about 10 nanometers to about 40 nanometers, or about 10 nanometers to about 30 nanometers, or about 10 nanometers to about 20 nanometers). In further embodiments, the particles within the plurality of LSNAs produced by the methods of the present disclosure have an average diameter of about 20 nanometers or less, or about 25 nanometers or less, or about 30 nanometers or less, or about 35 nanometers or less, or about 40 nanometers or less, or about 45 nanometers or less.

[0076] In some aspects, the method includes: (1) adding 1×PBS to dry down lipids to a final concentration of 1-25 mg / mL (so that in various embodiments the final concentration is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mg / ml); (2) quick-freezing in liquid nitrogen three times and thawing in a bath sonicator; and (3) extruding through 200, 100, 80, 50, and 30 nm filters. Dual filters are used, typically with 2-10 passes through each filter. In some embodiments, the process is stopped at 50 nm, but if 30 nm structures are desired, an additional 30 nm filter is added. In further aspects, if 30 nm liposomes are desired, one probe is sonicated after step (2). The liposomes are then centrifuged at 21,000 xg for 10 minutes to remove metal chips that may be dislodged by sonication, and the mixture is extruded through a 30 nm filter as described in step (3).

[0077] Thus, in some aspects, the disclosure provides a method of making a LSNA comprising: adding phospholipids to a solvent to form a first mixture, the first mixture comprising a plurality of liposomes; disrupting the plurality of liposomes to create a second mixture, the second mixture comprising liposomes and small unilamellar vesicles (SUVs); isolating the SUVs from the second mixture, the SUVs having a particle size of about 20 nanometers to 50 nanometers; and adding an oligonucleotide or a plurality of oligonucleotides to the isolated SUVs to make the LSNA.

[0078] Oligonucleotides The present disclosure provides spherical nucleic acids (e.g., ProSNAs, LSNAS, LNP-SNAs) comprising a nanoparticle core and a shell of oligonucleotides bound to the exterior of the nanoparticle core. In various embodiments, the shell of oligonucleotides comprises inhibitory oligonucleotides, immunostimulatory oligonucleotides, targeting oligonucleotides, or combinations thereof. As described herein, in some embodiments, the nanoparticle core comprises an encapsulated gene editing protein. In various embodiments, the oligonucleotides of the present disclosure include DNA oligonucleotides, RNA oligonucleotides, modified forms thereof, or combinations thereof. In any aspect or embodiment described herein, the oligonucleotide is single-stranded, double-stranded, or partially double-stranded. In any aspect or embodiment of the present disclosure, the oligonucleotide comprises a detectable marker.

[0079] Modified forms of oligonucleotides, including those with at least one modified internucleotidic linkage as described herein, are also contemplated by the present disclosure. In some embodiments, the oligonucleotide is all or partly a peptide nucleic acid. Other modified internucleoside linkages include at least one phosphorothioate linkage. Still other modified oligonucleotides include those that include one or more universal bases. A "universal base" refers to a molecule that can replace a linkage to any one of A, C, G, T, and U in a nucleic acid by forming a hydrogen bond without significant structural destabilization. Oligonucleotides incorporating universal base analogs can function, for example, as probes in hybridization. Examples of universal bases include, but are not limited to, 5'-nitroindole-2'-deoxyriboside, 3-nitropyrrole, inosine, and hypoxanthine.

[0080] The term "nucleotide" or its plural forms as used herein are interchangeable with the variants discussed herein and otherwise known in the art. The term "nucleobase" or its plural forms as used herein are interchangeable with the variants discussed herein and otherwise known in the art. Nucleotides or nucleobases include the naturally occurring nucleobases A, G, C, T, and U. Non-naturally occurring nucleobases include, for example, but are not limited to, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosine, N',N'-ethano-2,6-diaminopurine, 5-methylcytosine (mC), 5-(C3-C6)-alkynyl-cytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine, and the "non-naturally occurring" nucleobases described in Benner et al., U.S. Pat. No. 5,432,272 and Susan M. Freier and Karl-Heinz Altmann, 1997, Nucleic Acids Research, vol. 25: pp 4429-4443. The term "nucleobase" includes the known purine and pyrimidine heterocycles, as well as their heterocyclic analogs and tautomers.Further naturally occurring and non-naturally occurring nucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan, et al.); Antisense Research and Application, Ed. S.T. Crooke and B. Lebleu, CRC Press, 1993; Chapter 15 by Sanghvi in ​​Englisch et al., 1991, Angewandte Chemie, International Edition, 30:613-722 (see especially pages 622 and 623, and in Concise Encyclopedia of Polymer Science and Engineering, J.I. Kroschwitz Ed., John Wiley & Sons, 1990, pages 858-859; Cook, Anti-Cancer Drug Design 1991, 6, 585-607, each of which is incorporated herein by reference in its entirety). In various embodiments, oligonucleotides also contain one or more "nucleoside bases" or "base units", which are a category of non-naturally occurring nucleotides that contain compounds such as heterocyclic compounds that are not nucleoside bases in the most classical sense, but can function as nucleoside bases, including certain "universal bases".Universal bases include 3-nitropyrrole, optionally substituted indoles (e.g., 5-nitroindoles), and optionally substituted hypoxanthines.Other desirable universal bases include pyrrole, diazole, or triazole derivatives that contain universal bases known in the art.

[0081] Examples of oligonucleotides include those containing modified backbones or non-natural internucleoside linkages. Oligonucleotides with modified backbones include those that have a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are considered to be within the meaning of "oligonucleotide."

[0082] Modified oligonucleotide backbones containing a phosphorus atom include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phototriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs thereof, and those having reverse polarity where one or more internucleotide linkages are 3'-3', 5'-5' or 2'-2' linkages. Also contemplated are oligonucleotides having only one inverted nucleoside residue that contains a single 3'-3' linkage at the 3'-most internucleotide linkage, i.e., that may be abbasic (either no nucleotide or a hydroxyl group in that location). Salts, mixed salts, and free acid forms are also contemplated. Representative United States patents which teach the preparation of the above phosphorus-containing linkages include U.S. Pat. Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,196, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455,233, Nos. 5,466,677, 5,476,925, 5,519,126, 5,536,821, 5,541,306, 5,550,111, 5,563,253, 5,571,799, 5,587,361, 5,194,599, 5,565,555, 5,527,899, 5,721,218, 5,672,697 and 5,625,050, the disclosures of which are incorporated herein by reference.

[0083] Modified oligonucleotide backbones that do not contain phosphorus atoms therein have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include backbones with morpholino linkages; siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and methylenethioformacetyl backbones; riboacetyl backbones; alkene-containing backbones; sulfamic acid backbones; methyleneimino and methylenehydrazino backbones; sulfonic acid and sulfonamide backbones; amide backbones; and N, O, S and CH 2 Other scaffolds exist that have a mixture of component parts, e.g., U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,264,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677, 5,541,307, 5,561,225, 5,59 See Nos. 6,086, 5,602,240, 5,610,289, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, 5,792,608, 5,646,269 and 5,677,439, the disclosures of which are incorporated herein by reference.

[0084] In yet further embodiments, there are oligonucleotide mimetics in which one or more sugars and / or one or more internucleotide bonds of nucleotide units are both replaced with "non-naturally occurring" groups. The bases of the oligonucleotide are maintained for hybridization. In some aspects, this embodiment contemplates peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligonucleotide is replaced with an amide-containing backbone. See, for example, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262, and Nielsen et al., Science, 1991, 254, 1497-1500, the disclosures of which are incorporated herein by reference.

[0085] In still further embodiments, oligonucleotides having phosphorothioate backbones and oligonucleosides having heteroatom backbones are provided, including those having the -CH 2 -NH-O-CH 2 -, -CH 2 -N(CH 3 )-O-CH 2 -, -CH 2 -ON(CH 3 )-CH 2 -, -CH 2 -N(CH 3 )-N(CH 3 )-CH 2 - and -ON(CH 3 )-CH 2 -CH 2 Also contemplated are oligonucleotides having morpholino backbone structures, as described in U.S. Patent No. 5,034,506.

[0086] In various forms, the bond between two consecutive monomers in an oligonucleotide is -CH 2 -, -O-, -S-, -NR H -, >C=O, >C=NR H , >C=S, -Si(R'') 2 -, -SO-, -S(O) 2 -, -P(O) 2-, -PO(BH 3 )-, -P(O,S)-, -P(S) 2 -, -PO(R'')-, -PO(OCH 3 )- and -PO(NHR H ) , wherein R H is hydrogen and C 1-4 -alkyl, and R″ is selected from C 1-6 -alkyl and phenyl. An illustrative example of such a bond is -CH 2 -CH 2 -CH 2 -, -CH 2 -CO-CH 2 -, -CH 2 -CHOH-CH 2 -, -O-CH 2 -O-, -O-CH 2 -CH 2 -, -O-CH 2 -CH=(R when used as a bond to a subsequent monomer) 5 ), -CH 2 -CH 2 -O-, -NR H -CH 2 -CH 2 -, -CH 2 -CH 2 -NR H -, -CH 2 -NR H -CH 2 --, -O-CH 2 -CH 2 -NR H -, -NR H -CO-O-, -NR H -CO-NR H -, -NR H -CS-NR H -, -NR H -C(=NR H )-NR H -, -NR H -CO-CH 2 -NR H -O-CO-O-, -O-CO-CH 2 -O-, -O-CH 2 -CO-O-, -CH 2-CO-NR H -, -O-CO-NR H -, -NR H -CO-CH 2 -, -O-CH 2 -CO-NR H -, -O-CH 2 -CH 2 -NR H -, -CH=NO-, -CH 2 -NR H -O-, -CH 2 -ON=(R when used as a bond to a subsequent monomer) 5 ), -CH 2 -O-NR H -, -CO-NR H -CH 2 -, -CH 2 -NR H -O-, -CH 2 -NR H -CO-, -O-NR H -CH 2 -, -O-NR H , -O-CH 2 -S-, -S-CH 2 -O-, -CH 2 -CH 2 -S-, -O-CH 2 -CH 2 -S-, -S-CH 2 -CH=(R when used as a bond to a subsequent monomer) 5 ), -S-CH 2 -CH 2 -,-S-CH 2 -CH 2 --O-, -S-CH 2 -CH 2 -S-, -CH 2 -S-CH 2 -, -CH 2 -SO-CH 2 -, -CH 2 -SO 2 -CH 2 -, -O-SO-O-, -OS(O) 2 -O-, -OS(O) 2 -CH 2 -, -OS(O) 2 -NR H-,-NR H -S(O) 2 -CH 2 -,-O-S(O) 2 -CH 2 -,-O-P(O) 2 -O-,-O-P(O,S)-O-,-O-P(S) 2 -O-,-S-P(O) 2 -O-,-S-P(O,S)-O-,-S-P(S) 2 -O-,-O-P(O) 2 -S-,-O-P(O,S)-S-,-O-P(S) 2 -S-,-S-P(O) 2 -S-,-S-P(O,S)-S-,-S-P(S) 2 -S-,-O-PO(R’’)-O-,-O-PO(OCH 3 )-O-,-O-PO(OCH 2 CH 3 )-O-,-O-PO(OCH 2 CH 2 S-R)-O-,-O-PO(BH 3 )-O-,-O-PO(NHR N )-O-,-O-P(O) 2 -NR H H-,-NR H -P(O) 2 -O-,-O-P(O,NR H )-O-,-CH 2 -P(O) 2 -O-,-O-P(O) 2 -CH 2 -、and -O-Si(R’’) 2 -O- and among them, -CH 2 -CO-NR H -,-CH 2 -NR H -O-,-S-CH 2 -O-,-O-P(O) 2 -O-O-P(-O,S)-O-,-O-P(S) 2 -O-,-NR H P(O) 2 -O-,-O-P(O,NR H )-O-,-O-PO(R’’)-O-,-O-PO(CH 3)-O- and -O-PO(NHR N )-O-, where R H is hydrogen and C 1-4 -alkyl, and R″ is selected from C 1-6 Further examples are provided by Mesmaeker et.al., Current Opinion in Structural Biology 1995, 5, 343-355 and Susan M. Freier and Karl-Heinz Altmann, Nucleic Acids Research, 1997, vol 25, pp 4429-4443.

[0087] Still other modified forms of oligonucleotides are described in detail in US Patent Application Publication No. 2004 / 0219565, the disclosure of which is incorporated herein by reference in its entirety.

[0088] Modified oligonucleotides may also contain one or more substituted sugar moieties. In certain embodiments, the oligonucleotide comprises one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C 1 ~C 10 Alkyl or C 2 ~C 10 It can be alkenyl and alkynyl. In other embodiments, O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n O.N.H. 2 , and O(CH 2 )n ON[(CH 2 ) n CH 3 ] 2 where n and m are from 1 to about 10. Other oligonucleotides include one of the following at the 2' position: 1 ~C 10 Lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , S.O. 2 CH 3 , O.N.O. 2 , NO 2 , N 3 , NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, or an RNA cleaving group. In one aspect, the modification includes 2'-methoxyethoxy (also known as 2'-O-(2-methoxyethyl) or 2'-MOE, 2'-O-CH 2 CH 2 OCH 3 ) (Martin et al. Helv. Chim. Acta 1995 78486-504), i.e., alkoxyalkoxy groups. Other modifications include O(CH), also known as 2'-dimethylaminooxyethoxy, i.e., 2'-DMAOE. 2 ) 2 ON(CH 3 ) 2 groups, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH 2 -O-CH 2 -N(CH 3 ) 2 Examples include:

[0089] Further modifications include 2'-methoxy (2'-O-CH 3 ), 2'-aminopropoxy (2'-OCH 2 CH 2CH 2 NH 2 ), 2'-allyl (2'-CH 2 -CH=CH 2 ), 2'-O-allyl (2'-O-CH 2 -CH=CH 2 ), and 2'-fluoro (2'-F). The 2' modification can be at the arabino (up) or ribo (down) position. In one aspect, the 2'-arabino modification is a 2'-F. Similar modifications can also be made at other positions on oligonucleotides, such as the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and at the 5' position of the 5' terminal nucleotide. Oligonucleotides can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. See, for example, U.S. Patent Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, 5,567,811, 5,576,427, 5,591,7 22, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, 5,792,747, and 5,700,920, the disclosures of which are incorporated herein by reference in their entireties.

[0090] In some embodiments, sugar modifications include Locked Nucleic Acids (LNAs), in which a 2'-hydroxyl group is attached to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. In certain embodiments, the linkage is a methylene (-CH 2 -) n where n is 1 or 2. LNAs and their preparation are described in WO98 / 39352 and WO99 / 14226.

[0091] Modified nucleotides are described in EP 1072679 and WO 97 / 12896, the disclosures of which are incorporated herein by reference. Modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (succinimide), ... douracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Further modified bases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified bases can also include those in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.Additional nucleobases include those disclosed in U.S. Patent No. 3,687,808, The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons 1990, Englisch et al., 1991 Angewandte Chemie, International Edition, 30:613, and Sanghvi, YS, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., ed., CRC Press, 1993. Some of these bases are useful for increasing binding affinity and include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C, and in certain embodiments are combined with 2'-O-methoxyethyl sugar modifications. See U.S. Patent Nos. 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, and 5,55 See, for example, Nos. 2,540, 5,587,469, 5,594,121, 5,596,091, 5,614,617, 5,645,985, 5,830,653, 5,763,588, 6,005,096, 5,750,692, and 5,681,941, the disclosures of which are incorporated herein by reference.

[0092] Methods for making polynucleotides of a given sequence are well known. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and F. Eckstein (ed.) Oligonucleotides and Analogues, 1st Ed. (Oxford University Press, New York, 1991). Solid-phase synthesis methods are suitable for both polyribonucleotides and polydeoxyribonucleotides (the well-known methods for DNA synthesis are also useful for RNA synthesis). Polyribonucleotides can also be prepared enzymatically. Non-naturally occurring nucleobases can also be incorporated into polynucleotides. See, e.g., U.S. Pat. No. 7,223,833; Katz, J. Am. Chem. Soc., 74:2238 (1951); Yamane, et al., J. Am. Chem. Soc., 83:2599 (1961); Kosturko, et al., Biochemistry, 13:3949 (1974); Thomas, J. Am. Chem. Soc., 76:6032 (1954); Zhang, et al., J. Am. Chem. Soc., 127:74-75 (2005); and Zimmermann, et al., J. Am. Chem. Soc., 124:13684-13685 (2002).

[0093] In various embodiments, the oligonucleotides of the present disclosure or modified forms thereof are generally about 5 to about 100 nucleotides in length. More specifically, the oligonucleotides of the present disclosure may be about 5 to about 90 nucleotides in length, about 5 to about 80 nucleotides in length, about 5 to about 70 nucleotides in length, about 5 to about 60 nucleotides in length, about 5 to about 50 nucleotides in length, about 5 to about 45 nucleotides in length, about 5 to about 40 nucleotides in length, about 5 to about 35 nucleotides in length, about 5 to about 30 nucleotides in length, about 5 to about 25 nucleotides in length, about 5 to about 20 nucleotides in length, about 5 to about 15 nucleotides in length, about 5 to about 10 nucleotides in length, about 10 to about 100 nucleotides in length, about 10 to about 90 nucleotides in length, about 10 to about 80 nucleotides in length, and more particularly, the oligonucleotides of the present disclosure may be about 5 to about 90 nucleotides in length, about 5 to about 80 nucleotides in length, about 5 to about 90 nucleotides in length, about 5 to about 10 ... The oligonucleotide lengths are, for example, about 10 to about 70 nucleotides, about 10 to about 60 nucleotides, about 10 to about 50 nucleotides, about 10 to about 45 nucleotides, about 10 to about 40 nucleotides, about 10 to about 35 nucleotides, about 10 to about 30 nucleotides, about 10 to about 25 nucleotides, about 10 to about 20 nucleotides, about 10 to about 15 nucleotides, about 18 to about 28 nucleotides, about 15 to about 26 nucleotides, and all oligonucleotide lengths intermediate between the specifically disclosed sizes to the extent that the oligonucleotide achieves the desired result. In further embodiments, the oligonucleotides of the disclosure are from about 5 to about 100 nucleotides in length, from about 5 to about 90 nucleotides in length, from about 5 to about 80 nucleotides in length, from about 5 to about 70 nucleotides in length, from about 5 to about 60 nucleotides in length, from about 5 to about 50 nucleotides in length, from about 5 to about 40 nucleotides in length, from about 5 to about 30 nucleotides in length, from about 5 to about 20 nucleotides in length, from about 5 to about 10 nucleotides in length, and any oligonucleotide length intermediate the specifically disclosed sizes such that the oligonucleotide achieves the desired result.Thus, in various embodiments, the oligonucleotides of the present disclosure are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100+ or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides in length. In further embodiments, the oligonucleotides of the disclosure are 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides shorter.In various embodiments, the shell of oligonucleotides bound to the exterior of the nanoparticle core of the SNA comprises a plurality of oligonucleotides all having the same length / sequence, and in some embodiments, the plurality of oligonucleotides comprises one or more oligonucleotides having a different length and / or sequence relative to at least one other oligonucleotide in the plurality. In various embodiments, the nanoparticle core comprises one or more oligonucleotides encapsulated therein.

[0094] In some embodiments, the oligonucleotides in the shell of oligonucleotides are aptamers. Thus, all the features and aspects of oligonucleotides described herein (e.g., length, type (DNA, RNA, modified forms thereof), optional presence of spacers) also apply to aptamers. Aptamers are oligonucleotide sequences that can be evolved to bind to various target analytes of interest. Aptamers can be single-stranded, double-stranded, or partially double-stranded.

[0095] Methods for binding the detectable markers (eg, fluorophores, radiolabels) and therapeutic agents (eg, antibodies) described herein to oligonucleotides are known in the art.

[0096] Spacer. In some aspects and embodiments, one or more oligonucleotides in the shell of the oligonucleotides that are bound to the nanoparticle core of the SNA comprise a spacer. As used herein, "spacer" refers to a moiety that serves to increase the distance between the nanoparticle core and the oligonucleotide, or to increase the distance between individual oligonucleotides when bound to the nanoparticle core in multiple copies, or to improve the synthesis of the SNA. Thus, it is contemplated that the spacer is located between the oligonucleotide and the nanoparticle core.

[0097] In some aspects, the spacer, when present, is an organic moiety. In some aspects, the spacer is a polymer, including but not limited to a water-soluble polymer, a nucleic acid, a polypeptide, an oligosaccharide, a carbohydrate, a lipid, ethyl glycol, or a combination thereof. In any of the aspects or embodiments of the present disclosure, the spacer is an oligo(ethylene glycol)-based spacer. In various embodiments, the oligonucleotide comprises one, two, three, four, five, or more spacer (e.g., spacer-18 (hexaethylene glycol)) moieties. In further embodiments, the spacer is an alkane-based spacer (e.g., C12). In some embodiments, the spacer is an oligonucleotide spacer (e.g., T5). The oligonucleotide spacer can have any sequence that does not interfere with the ability of the oligonucleotide to become bound to the nanoparticle core or the target. In certain aspects, the bases of the oligonucleotide spacer are all adenylic acids, all thymidylic acids, all cytidylic acids, all guanylic acids, all uridylic acids, or all some other modified base.

[0098] In various embodiments, the length of the spacer is or is equal to at least about 2 nucleotides, at least about 3 nucleotides, at least about 4 nucleotides, at least about 5 nucleotides, 5-10 nucleotides, 10 nucleotides, 10-30 nucleotides, or even more than 30 nucleotides.

[0099] SNA surface density. Generally, at least about 2 pmoles / cm 2 An oligonucleotide surface density of at least 15 pmol / cm is suitable for providing a stable SNA. In some embodiments, the surface density of the SNAs of the present disclosure (e.g., ProSNAs, LSNAs, LNP-SNAs) is at least 15 pmol / cm 2 In addition, the oligonucleotide is about 2 pmol / cm 2 ~about 200pmol / cm 2 , or about 10 pmol / cm 2 ~about 100pmol / cm 2Also provided is a method in which the SNA is bound to the nanoparticle core at a surface density of at least about 2 pmol / cm. In a further embodiment, the surface density is at least about 2 pmol / cm. 2 , at least 3 pmol / cm 2 , at least 4 pmol / cm 2 , at least 5 pmol / cm 2 , at least 6 pmol / cm 2 , at least 7 pmol / cm 2 , at least 8 pmol / cm 2 , at least 9 pmol / cm 2 , at least 10 pmol / cm 2 , at least about 15 pmol / cm 2 , at least about 19 pmol / cm 2 , at least about 20 pmol / cm 2 , at least about 25 pmol / cm 2 , at least about 30 pmol / cm 2 , at least about 35 pmol / cm 2 , at least about 40 pmol / cm 2 , at least about 45 pmol / cm 2 , at least about 50 pmol / cm 2 , at least about 55 pmol / cm 2 , at least about 60 pmol / cm 2 , at least about 65 pmol / cm 2 , at least about 70 pmol / cm 2 , at least about 75 pmol / cm 2 , at least about 80 pmol / cm 2 , at least about 85 pmol / cm 2 , at least about 90 pmol / cm 2 , at least about 95 pmol / cm 2 , at least about 100 pmol / cm 2 , at least about 125 pmol / cm 2 , at least about 150 pmol / cm 2 , at least about 175 pmol / cm 2 , at least about 200 pmol / cm 2 , at least about 250 pmol / cm 2, at least about 300 pmol / cm 2 , at least about 350 pmol / cm 2 , at least about 400 pmol / cm 2 , at least about 450 pmol / cm 2 , at least about 500 pmol / cm 2 , at least about 550 pmol / cm 2 , at least about 600 pmol / cm 2 , at least about 650 pmol / cm 2 , at least about 700 pmol / cm 2 , at least about 750 pmol / cm 2 , at least about 800 pmol / cm 2 , at least about 850 pmol / cm 2 , at least about 900 pmol / cm 2 , at least about 950 pmol / cm 2 , at least about 1000 pmol / cm 2 In a further embodiment, the surface density is about 2 pmol / cm 2 Less than 3 pmol / cm 2 Less than 4 pmol / cm 2 Less than 5 pmol / cm 2 Less than 6 pmol / cm 2 Less than 7 pmol / cm 2 Less than 8 pmol / cm 2 Less than 9 pmol / cm 2 Less than 10 pmol / cm 2 Less than 15 pmol / cm 2 Less than 19 pmol / cm 2 Less than 20 pmol / cm 2 Less than about 25 pmol / cm 2 Less than 30 pmol / cm 2 Less than 35 pmol / cm 2 Less than 40 pmol / cm 2 Less than 45 pmol / cm 2 Less than 50 pmol / cm 2 Less than 55 pmol / cm 2 Less than 60 pmol / cm 2 Less than 65 pmol / cm2 Less than 70 pmol / cm 2 Less than about 75 pmol / cm 2 Less than 80 pmol / cm 2 Less than 85 pmol / cm 2 Less than 90 pmol / cm 2 Less than 95 pmol / cm 2 Less than about 100 pmol / cm 2 Less than about 125 pmol / cm 2 Less than 150 pmol / cm 2 Less than about 175 pmol / cm 2 Less than about 200 pmol / cm 2 Less than about 250 pmol / cm 2 Less than 300 pmol / cm 2 Less than about 350 pmol / cm 2 Less than 400 pmol / cm 2 Less than 450 pmol / cm 2 Less than about 500 pmol / cm 2 Less than about 550 pmol / cm 2 Less than 600 pmol / cm 2 Less than 650 pmol / cm 2 Less than 700 pmol / cm 2 Less than about 750 pmol / cm 2 Less than 800 pmol / cm 2 Less than about 850 pmol / cm 2 Less than 900 pmol / cm 2 Less than 950 pmol / cm 2 Less than or about 1000 pmol / cm 2 is less than.

[0100] Alternatively, the density of oligonucleotides bound to the SNA is measured by the number of oligonucleotides bound to the SNA. With respect to the surface density of oligonucleotides bound to the SNA of the present disclosure, it is contemplated that the SNAs described herein comprise from about 1 to about 2,500 or from about 1 to about 500 oligonucleotides on their surface. In various embodiments, the SNA comprises from about 10 to about 500, or from about 10 to about 300, or from about 10 to about 200, or from about 10 to about 190, or from about 10 to about 180, or from about 10 to about 170, or from about 10 to about 160, or from about 10 to about 150, or from about 10 to about 140, or from about 10 to about 130, or from about 10 to about 120, or from about 10 to about 110, or from about 10 to about 100, or from 10 to about 90, or from about 10 to about 80, or from about 10 to about 70, or from about 10 to about 60, or from about 10 to about 50, or from about 10 to about 40, or from about 10 to about 30, or from about 10 to about 20 oligonucleotides within a shell of oligonucleotides bound to the nanoparticle core. In some embodiments, the SNA comprises about 80 to about 140 oligonucleotides within the shell of oligonucleotides bound to the nanoparticle core, in further embodiments, the SNA comprises at least about 5, 10, 20, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 oligonucleotides within the shell of oligonucleotides bound to the nanoparticle core. In further embodiments, the SNA consists of 5, 10, 20, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 oligonucleotides within a shell of oligonucleotides bound to a nanoparticle core.In still further embodiments, the shell of oligonucleotides bound to the nanoparticle core of the SNA comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more oligonucleotides. In some embodiments, the shell of oligonucleotides bound to the nanoparticle core of the SNA consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more oligonucleotides.

[0101] composition The present disclosure also provides a composition comprising the SNA of the present disclosure, or a plurality thereof. In any of the aspects or embodiments of the present disclosure, the composition further comprises a guide RNA. In some embodiments, the composition further comprises a pharma- ceutically acceptable carrier. The term "carrier" refers to a vehicle in which the SNA described herein is administered to a subject. Any conventional medium or agent compatible with the SNA according to the present disclosure can be used. The term carrier includes diluents, excipients, adjuvants, and combinations thereof. Pharmaceutically acceptable carriers are well known in the art (see, for example, Remington's Pharmaceutical Sciences by Martin, 1975, the entire disclosure of which is incorporated herein by reference).

[0102] Exemplary "diluents" include water for injection, saline, buffers such as Tris, acetate, citrate, or phosphate, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents. Exemplary "excipients" include, but are not limited to, stabilizers such as amino acids and amino acid derivatives, polyethylene glycol and polyethylene glycol derivatives, polyols, acids, amines, polysaccharides or polysaccharide derivatives, salts, and surfactants, and pH adjusters. In some embodiments, the SNAs provided herein include an immunostimulatory oligonucleotide (such as, and not limited to, a CpG oligonucleotide) as an adjuvant. Other adjuvants known in the art can also be used in the compositions of the present disclosure. For example, the adjuvant can be aluminum or its salts, mineral oil, Freund's adjuvant, vegetable oil, water-in-oil emulsion, mineral salts, small molecules (e.g., imiquimod, resiquimod), bacterial components (e.g., flagellin, monophosphoryl lipid A), or combinations thereof.

[0103] Use of SNAs in gene regulation In some embodiments of the present disclosure, oligonucleotides associated with SNA (e.g., ProSNA, LNP-SNA, LSNA) inhibit gene expression. Methods of inhibiting expression of gene products include those in which expression of target gene products is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%, compared to expression of the gene product in the absence of SNA. In other words, the methods provided encompass essentially any degree of inhibition of expression of target gene products.

[0104] The degree of inhibition is determined in vivo from bodily fluid or biopsy samples, or by imaging techniques well known in the art, or, alternatively, in cell culture assays as a predictable measure of the degree of inhibition that can generally be expected in vivo resulting from the use of a particular type of SNA and a particular oligonucleotide.

[0105] In some embodiments of the present disclosure, it is contemplated that the SNA serves both a gene inhibition function and a drug delivery function. In such embodiments, a drug (e.g., a therapeutic agent) is associated with the SNA and the particle is additionally functionalized with one or more oligonucleotides designed to effect inhibition of target gene expression.

[0106] In various embodiments, the methods involve the use of oligonucleotides that are 100% complementary, i.e., a perfect match, to the target oligonucleotide, while in other embodiments, the oligonucleotides include the use of oligonucleotides that are at least (meaning greater than or equal to) about 95% complementary to the polynucleotide over the length of the oligonucleotide, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20% complementary to the polynucleotide over the length of the oligonucleotide to the extent that the oligonucleotide is capable of achieving the desired inhibition of the target gene product.

[0107] It is understood in the art that the sequence of an antisense compound does not need to be 100% complementary to the sequence of its target nucleic acid in order to be specifically hybridizable. Furthermore, an oligonucleotide may hybridize on one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., loop or hairpin structures). The percent complementarity is determined over the length of the oligonucleotide. For example, consider an antisense compound in which 18 of the 20 nucleotides of the antisense compound are complementary to a 20 nucleotide region of a 100 nucleotide full length target oligonucleotide, so that the oligonucleotide will be 90 percent complementary. In this example, the remaining non-complementary nucleotides may be clustered or interspersed with complementary nucleotides, and do not need to be adjacent to each other or to complementary nucleobases. The percent complementarity of an antisense compound to a region of a target nucleic acid can be routinely determined using the BLAST (basic local alignment search tool) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0108] The oligonucleotides utilized in such methods are either RNA or DNA. The RNA may be an inhibitory oligonucleotide, such as an inhibitory RNA (RNAi) that performs a regulatory function, and in various embodiments is selected from the group consisting of small inhibitory RNA (siRNA), single-stranded RNA (ssRNA), and ribozymes. Alternatively, the RNA is a microRNA that performs a regulatory function. The DNA, in some embodiments, is antisense-DNA. In some embodiments, the RNA is a piwi-interacting RNA (piRNA).

[0109] Use of SNAs in immune modulation Toll-like receptors (TLRs) are a class of proteins expressed on sentinel cells and play a key role in regulating the innate immune system. The mammalian immune system uses two general strategies to fight infectious diseases. Exposure to pathogens rapidly triggers an innate immune response characterized by the production of immunostimulatory cytokines, chemokines, and polyreactive IgM antibodies. The innate immune system is activated by exposure to pathogen-associated molecular patterns (PAMPs) expressed by a diverse group of infectious microorganisms. Recognition of PAMPs is mediated by members of the Toll-like family of receptors. TLR receptors, such as TLR4, TLR8, and TLR9, which respond to specific oligonucleotides, reside within specialized intracellular compartments called endosomes. Regulatory mechanisms of TLR4, TLR8, and TLR9 receptors are based on, for example, but not limited to, DNA-protein interactions.

[0110] As described herein, synthetic immunostimulatory oligonucleotides containing CpG motifs similar to those found in bacterial DNA stimulate similar responses of TLR receptors. Thus, the CpG oligonucleotides of the present disclosure have the ability to function as TLR agonists. Other TLR agonists contemplated by the present disclosure include, but are not limited to, single-stranded RNA and small molecules (e.g., R848 (resiquimod)). Thus, immunomodulatory (e.g., immunostimulatory) oligonucleotides have a variety of promising therapeutic applications, including the treatment of immune deficiencies and cancer. Thus, in some embodiments, the SNAs of the present disclosure are used in methods to modulate the activity of toll-like receptors (TLRs).

[0111] In some embodiments, the SNAs (e.g., ProSNAs, LSNAs, LNP-SNAs) of the present disclosure comprise oligonucleotides that are TLR antagonists. In some embodiments, the TLR antagonist is single-stranded DNA (ssDNA).

[0112] In some embodiments, the downregulation of the immune system involves knocking down genes involved in the expression of Toll-like receptors. This antisense approach involves the use of the SNA of the present disclosure to inhibit the expression of any toll-like protein.

[0113] Thus, in some embodiments, methods are disclosed that utilize the SNAs described herein to modulate toll-like receptors. The methods use a TLR agonist or TLR antagonist to either upregulate or downregulate toll-like receptor activity, respectively. The methods include contacting a cell having a toll-like receptor with a SNA of the present disclosure, thereby modulating the activity and / or expression of the toll-like receptor. The toll-like receptors that are modulated include one or more of toll-like receptor 1, toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 6, toll-like receptor 7, toll-like receptor 8, toll-like receptor 9, toll-like receptor 10, toll-like receptor 11, toll-like receptor 12, and / or toll-like receptor 13.

[0114] Use of SNAs to treat disorders In some embodiments, the SNAs of the present disclosure (e.g., ProSNA, LSNA, LNP-SNA) are used to treat disorders. Thus, in some aspects, the present disclosure provides a method of treating a disorder, comprising administering an effective amount of the SNAs of the present disclosure to a subject (e.g., a human subject) in need thereof, thereby treating the disorder. In various embodiments, the disorder is cancer, an infectious disease, a pulmonary disease, a gastrointestinal disease, a hematological disease, a viral disease, an inflammatory disease, an autoimmune disease, a neurodegenerative disease, a genetic disease, a cardiovascular disease, or a combination thereof. An "effective amount" of the SNA is, for example, an amount sufficient to affect gene editing and treat the disorder. An effective amount of the SNA is also, for example, an amount that inhibits gene expression, activates an innate immune response, or a combination thereof, to treat the disorder. Thus, a method of activating an innate immune response is also contemplated herein, such a method comprising administering an SNA of the present disclosure to a subject in need thereof in an amount effective to activate an innate immune response in the subject.

[0115] The SNAs of the present disclosure can be administered via any suitable route, such as parenteral administration, intramuscular injection, subcutaneous injection, intradermal administration, and / or mucosal administration, such as oral or intranasal. Additional routes of administration include, but are not limited to, intravenous, intraperitoneal, intranasal, intravaginal, intrarectal, and oral administration. Separate or simultaneous combinations of different routes of administration are also contemplated by the present disclosure.

[0116] Therapeutic Agents The SNAs provided herein optionally further comprise a therapeutic agent or agents. In various embodiments, the therapeutic agent is simply associated with an oligonucleotide in the shell of an oligonucleotide bound to the exterior of the nanoparticle core of the SNA, and / or the therapeutic agent is associated with the nanoparticle core of the SNA, and / or the therapeutic agent is encapsulated in the SNA. In some embodiments, the therapeutic agent is associated with the terminus of an oligonucleotide in the shell of an oligonucleotide that is not bound to a nanoparticle core (e.g., if the oligonucleotide is bound to the nanoparticle core via its 3' terminus, the therapeutic agent is associated with the 5' terminus of the oligonucleotide). Alternatively, in some embodiments, the therapeutic agent is associated with the terminus of an oligonucleotide in the shell of an oligonucleotide that is bound to a nanoparticle core (e.g., if the oligonucleotide is bound to the nanoparticle core via its 3' terminus, the therapeutic agent is associated with the 3' terminus of the oligonucleotide). In some embodiments, the therapeutic agent is covalently associated with an oligonucleotide in the shell of an oligonucleotide that is bound to the exterior of the nanoparticle core of the SNA. In some embodiments, the therapeutic agent is non-covalently associated with an oligonucleotide in the shell of an oligonucleotide that is bound to the exterior of the nanoparticle core of the SNA. However, it should be understood that the present disclosure provides SNAs in which one or more therapeutic agents are both covalently and non-covalently associated with oligonucleotides within the shell of oligonucleotides bound to the exterior of the nanoparticle core of the SNA. It will also be understood that non-covalent associations include hybridization, protein binding, and / or hydrophobic interactions. In some embodiments, the therapeutic agent is administered separately from the SNA of the present disclosure. Thus, in some embodiments, the therapeutic agent is administered before, after, or simultaneously with the SNA of the present disclosure to treat a disorder.

[0117] Therapeutic agents contemplated by the present disclosure include, but are not limited to, a protein (e.g., a therapeutic protein), a growth factor, a hormone, an interferon, an interleukin, an antibody or antibody fragment, a small molecule, a peptide, an antibiotic, an antifungal agent, an antiviral agent, a chemotherapeutic agent, or a combination thereof.

[0118] The term "small molecule" as used herein refers to a chemical compound or drug, or any other low molecular weight organic compound, either natural or synthetic. "Low molecular weight" refers to compounds having a molecular weight of less than 1500 Daltons, typically between 100 and 700 Daltons. EXAMPLES

[0119] With respect to the following examples, references to the use of a "CRISPR-SNA" may refer to the use of a Cas9 protein that does not include any GALA peptide sequence. Also, with respect to the following examples, references to the use of a "Cas9 SNA" may refer to the use of a "fusion" Cas9 protein as described herein that includes, in order from N-terminus to C-terminus, the following structures: (i) one or more GALA peptides, (ii) a gene editing protein, and (iii) a nuclear localization signal (NLS).

[0120] Example 1 Use of LSNAs in gene editing The present disclosure provides a method for delivering gene editing proteins to mammalian cells using spherical nucleic acids. The enzymatically active ribonucleoprotein (RNP) complex of Streptococcus pyogenes Cas9 with tracrRNA and crRNA is synthesized, and then the RNP is encapsulated in liposomes made of 95% 1,2-dioleoyl-sn-glycero-3 phosphatidylcholine (DOPC) and 5% 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(6-azidohexanoyl) (DPPE-azide). The liposomes are then functionalized with 5'DBCO-modified DNA to generate LSNA. These particles contain enzymatically active Cas9 and are efficiently taken up by mammalian cells.

[0121] method Unless otherwise stated, all reagents were purchased from commercial sources and used as received. For oligonucleotide, crRNA, and tracrRNA synthesis, all phosphoramidites and reagents were purchased from Glen Research, Co. (Sterling, VA, USA). All lipids were purchased from Avanti Polar Lipids (Alabaster, AL, USA) in either dry powder form or in chloroform and used without further purification. EnGen® Cas9 NLS (Cas9), proteinase K, and Phusion PCR kit were purchased from New England Biolabs (Ipswich, MA, USA). Alexa Fluor647 NHS ester dye (Alexa647) was purchased from Lumiprobe Corp. (Cockneysville, MD, USA). Plasmids were purchased from AddGene (Cambridge, MA, USA). GelRed dye was purchased from Biotium Inc. (Fremont, CA, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). C166-GFP cells were purchased from ATCC (Manassas, VA, USA) and Opti-MEM was purchased from Life Technologies (Carlsbad, CA).

[0122] Cas9 labeling and quantification To track and quantify Cas9, 2 nmoles of Cas9 was incubated with 10 nmoles of Alexa647 NHS ester in 1×HBS overnight at 4° C. to generate Alexa-Cas9. To remove unreacted dye, Alexa-Cas9 was passed through a NAP5 column equilibrated in 1×HBS and eluted in 1 mL of 1×HBS. 2 nmoles of unmodified Cas9 was exchanged into 1×HBS using the NAP5 column and combined with Alexa Cas9. The concentrations of Cas9 and Alexa dye were calculated using the absorbance at 280 nm and 650 nm, respectively, and the molar ratio of Alexa dye to Cas9 was calculated. Alexa-Cas9 was then diluted to 1 μM. A 20 μL aliquot was set aside for activity and concentration assays.

[0123] Cas9 ribonucleoprotein synthesis and concentration Ten nanomoles of crRNA and tracrRNA were generated by incubating 10 μM crRNA with 10 μM tracrRNA in 1×HBS at 95° C. for 5 min and allowed to cool to room temperature for 10 min. Ten nanomoles of crRNA / tracrRNA complex was then mixed with 4 nmoles of 1 μM Alexa-Cas9 and left at room temperature for 10 min to form Cas9 ribonucleoprotein (RNP). The RNP was then concentrated in an Amicon 10K spin filter for 5 min and then resuspended until the retained liquid volume reached 500 μL or less. Cas9 concentration was again quantified using the absorbance of Alexa647 dye. 20 μL was set aside for activity and concentration measurements.

[0124] Synthesis and purification of SNA To synthesize liposomes encapsulating Cas9 RNP, a dehydrated phospholipid film was generated by lyophilizing a mixture of 3 mg DOPC and 0.15 mg DPPE-azide in chloroform. The lipid film was then rehydrated with 400 μL of Alexa647-labeled ribonucleoprotein complexes (Alexa-RNPs) in 1× HBS at a concentration of 5–8 μM. This solution was then subjected to seven freeze / thaw cycles using liquid nitrogen and a room temperature bath sonicator to generate single unilamellar vesicles (SUVs). SUVs were passed through a column filled with Sepharose 6B and equilibrated in 1× HBS to separate them from unencapsulated RNPs. To reduce polydispersity, SUVs were extruded twice through 200 nm and then 100 nm membrane filters. To remove remaining unencapsulated RNPs, SUVs were incubated with proteinase K (10 U, in 500 μL of 1×NEB buffer 2+1×HBS) at room temperature for 1 hour. SUVs were separated from digested RNPs using a column packed with Superdex 200 and equilibrated in 1×HBS. To generate SNAs, SUVs were then incubated overnight with oligonucleotides functionalized with DBCO at the 5′ end and Cy3 internally (approximately 1 DNA per 20 phospholipids). SNAs were then separated from free oligonucleotides using a column packed with Superdex 200 and equilibrated in 1×HBS. See FIG. 1.

[0125] Quantification of Cas9 and DNA loading To measure SUV concentration, inductively coupled plasma optical emission spectroscopy (ICP-OES) and phosphorus standards were used to calculate the phospholipid concentration. Liposome diameter was measured via dynamic light scattering (DLS) and the number of phospholipids per liposome was calculated using the following Equation 1. SUV concentration was calculated by dividing the phospholipid concentration by the number of phospholipids per SUV. [ka]

[0126] After synthesis of the SNA (Figure 1), the SNA samples were treated with 0.1% Tween 20 detergent (to disrupt liposomes and disperse oligonucleotides) and the concentration of oligonucleotides was measured on a plate reader by comparing Cy3 fluorescence in the SNA samples to a standard curve generated from free DBCO and Cy3-labeled oligonucleotides. The concentration of liposomes was determined by ICP-OES as described above, using the phosphorus concentration corrected based on the concentration of oligonucleotides and the number of phosphorus atoms per oligonucleotide.

[0127] To calculate the concentration of RNP, a standard curve was generated from the set aside Alexa-RNP aliquots. The concentration of RNP was determined by measuring the fluorescence of Alexa647 from the liposome samples, which was then plotted on the linear regression of the Alexa-RNP standard curve in the plate reader.

[0128] A representative synthesis produced 115 nm CRISPR SNAs with approximately 450 DNA strands per particle and encapsulated approximately three RNPs per liposome (Figure 2).

[0129] In vitro Cas9 DNA cleavage assay To measure Cas9 enzyme activity, RNPs targeting the EGFP gene were synthesized and used to generate CRISPR SNAs. Purified plasmid pcDNA3-EGFP was linearized by digestion with the restriction enzyme SmaI. Active RNPs incubated with the linearized plasmid were cut into 2 kb and 4 kb fragments, which were visible on a 1% agarose electrophoresis gel run in TBE buffer for 30 minutes. To verify that RNPs do not degrade or lose activity during the synthesis of CRISPR SNAs, 200 nanograms of linearized plasmids were incubated with 1 pmol and 0.1 pmol of Alexa RNPs immediately after their generation, after freeze / thaw cycles, after size exclusion, and after extrusion. RNPs did not lose activity during these steps (Figure 3).

[0130] RNPs remain active throughout the SNA synthesis procedure - a proteinase stability study To verify that RNPs were encapsulated within the SNA, clean CRISPR SNAs were incubated with proteinase K in NEB's restriction enzyme buffer 2 for 1 hour at room temperature. As a control, Alexa-RNPs were mixed with empty SNAs and incubated with proteinase K. The incubated samples were then eluted in 200 μL fractions through a Superdex 200 size exclusion column equilibrated in 1×HBS. These fractions were then imaged on a fluorescent gel scanner for Cy3 and Alexa Fluor 647 fluorescence. Encapsulated RNPs co-eluted with SNAs after proteinase K digestion, whereas RNPs incubated with empty SNAs were digested and therefore RNP-associated Alexa fluorescence eluted much slower than SNA-associated Cy3 fluorescence.

[0131] To verify that the encapsulated RNPs were still active, an in vitro Cas9 DNA cleavage assay was performed on some samples. Liposomes in the CRISPR SNAs were disrupted with 0.1% Tween 20 detergent either before or after incubating them with proteinase K as described above. In vitro DNA cleavage activity assays were performed after inactivating proteinase K with 1 mM phenylmethylsulfonyl fluoride (PMSF). For control RNPs, Tween had no effect on activity, but proteinase K incubation abolished activity. However, CRISPR SNAs maintained their activity when Tween was added after proteinase K incubation, but showed no activity when Tween was added before proteinase K incubation (Figure 4). This indicated that the RNPs in the CRISPR SNAs were both encapsulated (protected from proteinase digestion) and enzymatically active.

[0132] Cellular uptake studies To determine whether SNAs can deliver RNPs into cells, C166-GFP cells were incubated with CRISPR SNAs, empty SNAs, RNPs encapsulated in naked liposomes, and RNPs complexed with RNAiMAX transfection reagent in Opti-MEM reduced serum medium for 16 hours. The uptake of RNPs labeled with Alexa Fluor647 was then measured via flow cytometry. Cells treated with CRISPR-SNAs had higher median fluorescence and a higher percentage of highly fluorescent (fluorescence >1000AU) cells than cells treated with the RNP / RNAiMAX mixture or RNPs encapsulated in naked liposomes, whereas untreated cells showed little fluorescence (Figure 5). This data indicated that gene editing enzymes encapsulated in liposomal SNAs are actively taken up into mammalian cells.

[0133] Example 2 This example details the synthesis of CRISPR / Cas9 ProSNA as an efficient genome editing delivery platform for Cas9-sgRNA complexes. As described herein, Cas9 serves as the nanoparticle core of ProSNA. Surface lysine amines were reacted at opposite ends with small polyethylene glycol polymers bearing azide and amine-reactive N-hydroxysuccinimide moieties. The covalently attached azide was then reacted with a DNA strand containing a distorted cyclooctyne, dibenzocyclooctyne (DBCO), at the 5' end. The sequence (dGGT) used herein is 10 was selected based on previous studies that showed enhanced cellular uptake of SNAs containing a G-rich shell versus a poly-dT shell. The three-dimensional oligonucleotide shell creates a steric and electrostatic barrier to stabilize the Cas9 proteins and render them functional for cell entry. This strategy allows for the facile generation of genome editing tools with excellent biocompatibility and cellular uptake performance, as well as excellent genome editing activity of approximately 42.5% in human cell lines. Our findings demonstrate that Cas9 ProSNAs have attractive prospects in genome editing and gene silencing.

[0134] material LB broth with agar (cat. no. L2897-250G) and LB broth were purchased from Sigma. Isopropyl β-D-1-thiogalactopyranoside (cat. no. DSI5600) was purchased from dot scientific inc. Phosphate buffered saline (PBS, pH 7.4) was purchased from Gibco Life Technologies. SA MALDI matrix (cat. no. 90032), Alexa Fluor647 (cat. no. A37573), and NHS-PEG4-azide (cat. no. 26130) were purchased from ThermoFisher. T7 RNA polymerase (M0251S) was purchased from NEB. All solutions were prepared using ultrapure water (18.25 MΩ.cm, 25° C.).

[0135] Oligonucleotide design, synthesis, and purification Oligonucleotides were synthesized on solid support using reagents and standard protocols obtained from Glen Research. The products were incubated overnight at room temperature in 30% NH 4 The oligonucleotides were cleaved from the solid support using OH and purified using reverse-phase HPLC with a gradient of 0-75% acetonitrile in triethylammonium acetate buffer over 45 min. After HPLC purification, the final dimethoxytrityl group was removed in 20% acetic acid for 2 h, followed by extraction with ethyl acetate. The mass of the oligonucleotides was confirmed using matrix-assisted laser desorption / ionization mass spectrometry using 3-hydroxypicolinic acid as a matrix. sgRNAs were synthesized using the NEB T7 Transcription Kit according to the manual.

[0136] [Table 1]

[0137] Synthesis and characterization of Cas9 SNAs Expression and purification of Cas9. The Cas9 plasmid (number 87703) was transformed into One Shot® BL21(DE3) Chemically Competent E. coli (Thermo Fisher) by electroshock and the cells were grown overnight on LB agar plates with 100 μg / mL ampicillin. Single colonies were picked and 7 mL cultures were grown overnight in LB broth at 37°C. These cultures were added to 750 mL of 2×YBT broth and 100 μg / mL ampicillin and the cells were grown to an optical density of 0.6-0.9 at 37°C and then induced with 1 mM isopropyl β-D-1-thiogalactopyranoside overnight at 17°C. The cells were pelleted (6000 g, 15 min), resuspended in 100 mL of 1×PBS and then lysed using a high-pressure homogenizer. The cell lysate was clarified by centrifugation at 30000g for 30 min and loaded onto a Bio-Scale™ Mini Profinity™ IMAC cartridge (Bio-Rad). The column was washed with 100 mL of 1×PBS and then eluted with the same buffer containing 250 mM imidazole. The eluted fraction was further purified by dialysis.

[0138] Reaction of surface-accessible cysteines with Alexa Fluor647 (AF647). Cas9 protein was dissolved in 1x phosphate-buffered saline (1x PBS; Thermo Fisher Scientific). Then, 10 equivalents of Alexa Fluor647-C2-maleimide (Thermo Fisher Scientific) dissolved in DMSO was added to approximately 10 μM Cas9 in 1500 μL of 1x PBS, and the reaction was shaken (900 rpm) overnight. Unconjugated Alexa Fluor647 was removed by repeated centrifugation using a 100 kDa filter until the filtrate had no detectable absorbance at 650 nm by UV-Vis. The number of Alexa Fluor647 modifications per protein was calculated based on UV-Vis spectroscopy.

[0139] Reaction of surface-accessible lysines with NHS-PEG4-azide. 50 equivalents of NHS-PEG dissolved in anhydrous DMSO at a concentration of 100 mM. 4 -Azide crosslinker (Thermo Fisher Scientific) was added to approximately 45 μM Cas9-AF647 in 550 μL of 1×PBS. The reaction was shaken (900 rpm) overnight at 25° C. Unconjugated linkers were removed by centrifugation 10 times using a 100 kDa filter. The number of azide modifications was assessed by MALDI-MS using sinapinic acid (Thermo Fisher Scientific) as a matrix on a Bruker AutoFlex-III.

[0140] DNA conjugation. DNA conjugation was performed immediately after purification. 350 equivalents of DBCO-dT-terminated DNA strands were first lyophilized, then 10 μM Cas9-AF647-azide in 450 μL of 1× PBS was added to rehydrate the DNA. The solution was incubated at 25 °C for 72 h with shaking (900 rpm). Unreacted DNA strands were removed by successive centrifugations through a 100 kDa filter until the filtrate had no detectable absorbance at 260 nm. Typically, complete removal of DNA requires 30-40 washing steps. The number of DNA strands per protein was calculated based on UV-Vis spectroscopy and MALDI-MS.

[0141] Binding and cleavage activity of Cas9 SNA-sgRNA complex. To assemble the Cas9 SNA-sgRNA complex, purified Cas9 SNA and sgRNA targeting a non-coding region in the human genome were incubated in 1x NEBuffer 4.1 at concentrations of 30 nM and 60 nM, respectively, for 30 min at 37°C. Cy5-labeled DNA carrying the target sequence was then added to obtain a concentration of 150 nM, and the mixture was further incubated under the same conditions for 30 min. Cleavage activity was examined by mixing 10 μL of the reaction with 2 μL of 6x native loading buffer prior to analysis using a 6% native PAGE gel.

[0142] In vitro investigation of Cas9 SNA. The cell lines HaCaT (human keratinocyte cell line), EGFP-expressing HEK293 (human embryonic kidney cells, HEK293 / EGFP) were purchased from American Type Culture Collection. The cells were incubated at 4°C for 1 h in a humidified room temperature with 5% CO 2 The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in ambient air.

[0143] Cellular uptake in HaCat cells. HaCaT cells were cultured in flow cytometry tubes (0.7 × 10 5 , 0.5 mL) and cultured overnight in DMEM containing 10% FBS. Afterwards, the culture medium was replaced with 450 μL OPTI-MEM, and 50 μL of Cas9 SNA was added and mixed to obtain a final concentration of 20 nM for different time intervals (0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h). For post-treatment, cells were washed with 1×PBS, 300 μL (trypsinized) (Gibco), 300 μL of 1×PBS was added, washed at 300 G for 5 min, and then cells were resuspended in 1 mL PBS. Cells were counted and the density was adjusted to 1×10 in a volume of 1 mL with PBS. 6 The cells were adjusted to 1000 x 100 cells. 1 μL of live / dead dye was added to 1 mL of cell suspension and mixed well. Live / dead staining was performed for 0.5 h and incubated at room temperature for 30 min, protected from light. Cells were washed once with 0.5 mL of PBS and then fixed in 150 μL of 4% paraformaldehyde (Thermo Fisher Scientific) for 15 min. 450 μL of 1x PBS was then added, washed for 3 min, followed by 200 μL of PBS, and then analyzed by flow cytometry using a BD LSRFortessa measuring the fluorescence (excitation 640 nm, emission 655-685 nm) of at least 30,000 single cell events per sample. Raw FCS files were gated based on forward and side scatter intensity and analyzed with FlowJo.

[0144] Cell viability. To assess cell viability, the Standard Cell Counting Kit-8 (CCK-8) assay was utilized. Briefly, cells were plated in 96-well plates (1 × 10 per well). 4 ) and cultured overnight in 200 μL of DMEM medium with 1% FBS. Then, 200 μL of OPTI-MEM medium with 2% FBS containing different concentrations of Cas9 SNA (50 nM, 100 nM, 200 nM, 300 nM, 400 nM, and 500 nM) was added, followed by incubation for 24 h. The medium was then replaced with 200 μL of 10% CCK-8 in PBS. After continuous incubation at 37° C. for 0.5 h, 150 μL of medium was used to measure the absorbance at 450 nm using a microplate reader. Cell viability was also assessed by calcein-AM / PI staining.

[0145] In vitro gene silencing. HEK293 cells that constantly express EGFP (HEK293 / EGFP) were used to evaluate the gene silencing effect of Cas9 SNA. HEK293 / EGFP cells were cultured in 24-well plates (24-well plates, 1.3 × 10 per well). 5 , 0.5 mL) and cultured overnight at 37 °C. The medium was changed to 2% FBS in OPTI-MUM for 5 h. After 6 h of incubation with Cas9 SNA in POTI-MUM to target the coding region of EGFP for 24 h, the cells were replaced with fresh medium and cultured for 5 days. The cells were then digested with trypsin-EDTA solution and resuspended in 0.3 mL PBS for flow cytometry.

[0146] Surveyor assay. HEK290 / EGFP cells were cultured in 24-well plates (5 × 10 cells per well). 4Cells) and cultured overnight at 37 °C. After incubation with assembled Cas9 SNA (100 nM, targeting human DNase I high activity site, human GRIN2B site, and EGFP site for 24 h), cells were replaced with fresh medium and cultured for another 4 days. Cells were then harvested for genomic DNA extraction using a genomic DNA extraction kit. 250 ng of DNA extract was denatured in a total volume of 19 µL in combination with 2 µL of NEBuffer2 (NEB), then reannealed at 95 °C for 5 min, thermal cycling from 95 to 85 °C at 2 °C / s and 85 to 20 °C at 0.2 °C / s. The reannealed DNA was incubated with 1 µL of T7 endonuclease I (10 U / µL, NEB) for 15 min at 37 °C. 10 μL of 50% glycerol was added to the T7 endonuclease reaction, and 12 μL was analyzed on a PAGE gel (Bio-Rad) electrophoresed at 200 V for 30 min, then stained with 1× SYBR Gold (Life Technologies) for 30 min. The Cas9-induced cleaved and uncleaved bands were used to calculate genome editing efficiency using ImageJ. Targeted genome modifications were also detected by Sanger sequencing.

[0147] result Recombinant Cas9 protein was purified from Escherichia coli BL21(DE3) and the binding / cleavage activity of Cas9-sgRNA complex was confirmed in solution. Cas9 ProSNAs were synthesized by a previously developed method. Specifically, Cas9 protein was tagged with Alexa Fluor647 (AF647) to facilitate in vitro tracking and to calculate the concentration of Cas9 SNA. The surface lysine amines were then reacted at the opposite end with a small polyethylene glycol polymer bearing an azide and an amine-reactive N-hydroxysuccinimide moiety. The covalently attached azide was then reacted with a DNA strand containing a distorted cyclooctyne, dibenzocyclooctyne (DBCO), at the 5' end via copper-free click chemistry. The successfully synthesized Cas9 SNA was characterized by transmission electron microscopy (TEM) with an average size of 10 nm (Figure 6a). The purity of the synthesized protein was confirmed using SDS-PAGE gel (Figure 6b). The gel images show clear molecular weight changes after each synthesis step, demonstrating the covalent binding of oligonucleotides rather than nonspecific association with the surface. After functionalization with DNA, the average zeta potential was changed to -15.8 mV (Figure 6c). This enhanced the solution stability with more negative charges. The level of DNA modification was determined by comparing the difference in absorbance at 260 nm between AF Cas9 and ProSNA Cas9 (Figure 6d). These results clearly demonstrated the success of DNA functionalization of Cas9.

[0148] As good biocompatibility is a prerequisite for biological applications, the viability of several cell lines was evaluated. The cytotoxicity of Cas9 SNA was investigated using HaCat, HEK293T / EGFP, hMSC, and Raw264.7 cell lines as models (Figure 7a). Standard CCK-8 was used to determine cell viability. Although the concentration of Cas9 SNA exceeded the typical concentrations used in in vitro experiments, no cytotoxicity was observed. Cytoplasmic delivery of Cas9 SNA was investigated using HaCat cell line as a model. Cells were incubated with 20 nM protein for 0–8 h and their uptake performance was determined by flow cytometry (Figure 7b). Compared to cells incubated with Cas9, Cas9 SNA showed approximately 10-fold increased cellular uptake. The enhanced cellular uptake of Cas9 SNA was attributed to the engagement of cell surface scavenger receptors followed by pit-mediated endocytosis.

[0149] Next, the ability of Cas9 SNA in genome editing was evaluated. Cas9 SNA targeting a DNase I hypersensitive site in the human genome, i.e., a Cas9 SNA that is relatively safe and accessible for genome editing, was delivered to HEK293T / EGFP cells. The Surveyor assay revealed an indel frequency of 39.2% (Figure 8a). Subsequently, a Cas9 SNA targeting a site in a gene associated with a rare neurodevelopmental disorder (i.e., GRIN2B) was also determined, resulting in an indel frequency of 42.5% (Figure 8b). The ability of Cas9 SNA in gene silencing was also evaluated using an sgRNA targeting the coding region of enhanced green fluorescent protein (EGFP). The corresponding indel and EGFP silencing efficiency was 35.5% (Figure 8c). Gene silencing performance was also confirmed by EGFP fluorescence change by flow cytometry, with an efficiency of 17.8%. These results demonstrated that Cas9 SNA achieved very high editing efficiency.

[0150] In conclusion, CRISPR / Cas9 SNA was established for efficient genome editing and gene silencing. Cas9 SNA effectively entered cells by the scavenger receptor pathway. Furthermore, in vitro studies demonstrated that Cas9 SNA led to efficient genome editing and gene silencing with good biocompatibility. This simple and versatile cytoplasmic delivery approach can be extended to biomedical applications of gene therapy, and its excellent biocompatibility opens new avenues in gene therapy and personalized medicine.

[0151] Example 3 This example describes additional experiments using CRISPR / Cas9 ProSNA.

[0152] Design, expression, and purification of Cas9 Construction of Cas9 expression vector. The pET-MBP-NLS-Geo_st expression vector (Addgene Plasmid no. 87703 (Harrington et al., Nat Commun. 2017 Nov 10;8(1):1424. doi:10.1038 / s41467-017-01408-4)) was further engineered by inserting three consecutive GALA peptides (3GALA) at the N-terminus of Geo Cas9 (Figure 9). All sequences used are listed in Table 1. The GALA gene sequence was purchased from Integrated DNA Technologies and cloned using Golden Gate assembly (GG). The pET-MBP-NLS-Geo_st vector was first amplified in a PCR thermocycler (ABI), followed by removal of the original plasmid template by DpnI digestion and gel purification. The 3GALA gene sequence was then subcloned into the amplified vector by GG-assembly. The constructed vector was transformed into One Shot® BL21(DE3) by electroporation and confirmed by conventional Sanger sequencing to obtain the 3GALA Cas9 vector. Note that the C-terminus of Cas9 contained a nuclear localization signal. The amino acid sequence of the fusion protein (SEQ ID NO:24) is shown below.

[0153] [Table 2]

[0154] The bold sequence (WEAALAEALAEALAEHLAEALAEALEALAAWEAALAEALAEALAEHLAEALAEALEALAAWEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID NO: 26)) is a 3GALA peptide. The bold and italicized sequence (PKKKRKV (SEQ ID NO:23)) is the NLS.

[0155] [Table 3]

[0156] Production and purification of Cas9. The recombinant Cas9 overexpression vector with N-terminal 3GALA peptide was transformed into One Shot® BL21(DE3) by electroshock and grown overnight on LB-ampicillin agar plates (100 μg / mL ampicillin). The resulting expression colonies were inoculated into 7 mL (LB, 100 μg / mL ampicillin) starter cultures, which were vigorously shaken overnight at 37° C. The next day, the starter cultures were inoculated into 750 mL of 2×YBT broth (100 μg / mL ampicillin) and grown to an optical density of 0.8 at 37° C., after which gene expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside, followed by overnight incubation at 17° C. Cells were harvested (6000g, 15 min), resuspended in 100 mL lysis buffer (20 mM HEPES, pH 7.5 RT, 0.5 mM TCEP, 500 mM NaCl, 1 mM PMSF), and then lysed by high-pressure homogenizer. The lysate fraction was clarified by centrifugation at 30000g for 30 min and loaded onto a 5 mL Bio-Scale™ Mini Profinity™ IMAC cartridge (Bio-Rad) pre-equilibrated in binding buffer (20 mM HEPES, pH 7.5 RT, 500 mM NaCl). Bound proteins were eluted by wash buffer (20 mM HEPES, pH 7.5, 500 mM NaCl, 250 mM imidazole). Maltose-binding proteins were cleaved from the eluted proteins by TEV proteinase overnight and captured by a second MBP affinity step. The resulting protein was loaded onto a heparin column and eluted with a gradient of 300-1250 mM NaCl. The fraction of the eluate containing Cas9 was purified by a Bio-Scale™ Mini Bio-Gel® P-6 desalting cartridge pre-equilibrated in storage buffer (20 mM HEPES, pH 7.5, 5% glycerol, 150 mM NaCl, 1 mM TCEP) and the concentration was measured by a NanoDrop 8000 spectrophotometer (Thermo Scientific) (Figure 10). The protein was purified at a constant temperature of 4°C, flash frozen in liquid nitrogen, and stored at -20°C.

[0157] Synthesis of oligonucleotides and sgRNA Oligonucleotide synthesis and purification. All phosphoramidites and DNA synthesis reagents were obtained from Glen Research. The sequences used in this study are listed in Table 2. DNA synthesis was carried out on a MerMade12 oligonucleotide synthesizer (MM12, BioAutomation Inc., Texas, USA) or an ABI394 synthesizer on a 10 μmol scale on controlled pore glass (CPG) beads. All oligonucleotides were purified using 30% NH 4 The product was deprotected from the CPG beads overnight at room temperature using OH. Ammonia was then removed under a stream of nitrogen using an Organomation® Multivap® Nitrogen Evaporator. The remaining solution was filtered through a 0.2 μM filter to remove the CPG beads. A fraction of the filtrate was purified by reversed-phase high-performance liquid chromatography (RP-HPLC, Varian ProStar 210, Agilent Technologies Inc., Palo Alto, CA, USA) to isolate the product. An Agilent Dynamax Microsorb C4 column and a gradient of 0-75% B over 45 min (A=triethylammonium acetate buffer, B=acetonitrile) were used. The collected fractions were lyophilized and redissolved in 20% acetic acid for 2 h, and the cleaved dimethoxytrityl groups were removed by ethyl acetate extraction. Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS; RapiFlex, Bruker) was used to confirm the mass of the oligonucleotides using 2',6'-dihydroxyacetophenone and diammonium hydrogen citrate as matrices. DNA concentrations were determined by measuring the solution absorbance at λ = 260 nm using UV-vis spectroscopy (Cary 5000 UV-vis spectrophotometer, Varian) using the extinction coefficients of the oligonucleotides obtained from the IDT Oligo Analyzer Tool.

[0158] Design and synthesis of sgRNA. Synthetic dsDNA templates of sgRNA with a consensus 5' T7 promoter binding site followed by 20 bp of sgRNA target sequence were in vitro transcribed using MEGAscript™ T7 Transcription Kit (ThermoFisher). Transcription was performed in a buffer containing 20 mM Tris-HCl (pH 8.0), 30 mM MgCl2, 10 mM DTT, 5 mM each NTP, 100 μg / mL T7 polymerase, RNase Inhibitor (Promega), and 100 ng of DNA template. Reactions were incubated at 37°C for approximately 18 hours. In vitro transcribed RNA was precipitated with ethanol, redissolved in water, and sgRNA concentration was finally quantified by Nano Drop8000 spectrophotometer (Thermo Scientific), flash frozen in liquid nitrogen, and stored at -20°C. The sequences are listed in Table 2. DNase I-sgRNA, GRIN2B-sgRNA, Grin2b-sgRNA, and EGFP-sgRNA were used to generate sgRNAs for genome editing or gene silencing at DNase I, GRIN2B, Grin2b, and EGFP sites.

[0159] [Table 4]

[0160] Synthesis and characterization of Cas9 ProSNAs Reaction with Alexa Fluor647 (AF647). Cas9 protein was first modified with amino-active Alexa Fluor™ 647 NHS ester (Thermo Fisher Scientific) (Figure 11). Alexa Fluor™ 647 NHS ester was dissolved in DMSO and analyzed by UV-visible absorbance spectroscopy (ε647 = 270,000 M -1 cm -1) to give a 10 mM stock solution. A 5 equivalent excess of AF-647 was added to the solution of Cas9 protein and the reaction was shaken at 900 rpm overnight. Excess Alexa Fluor647 was monitored at 650 nm and removed by size exclusion chromatography on a Bio-Rad FPLC. The number of Alexa Fluor647 modifications per protein was determined using a Cary-500 UV-vis spectrophotometer and their respective extinction coefficients (εCas9=204,470 M at 280 nm). -1 cm -1 , 324,610M at 260nm -1 cm -1 , εAF-647=270,000 at 650 nm) (Figure 12).

[0161] Surface-accessible lysines and NHS-PEG 4 -Azide reaction. Surface amines were converted to azides at the opposite end by reacting them with a tetraethylene glycol linker containing an N-hydroxysuccinimide (NHS) ester and an azide moiety (NHS-PEG 4 -N 3 , Thermo Scientific) (Figure 13). A solution of Cas9-AF647 was added with 600 equivalents of NHS-PEG 4 -Azide crosslinker was added. The reaction was shaken (900 rpm) overnight at 4°C. After 2 hours, unconjugated azide linkers were removed by size exclusion chromatography on a Bio-Rad FPLC. The number of azide linker modifications was determined by MALDI-MS using sinapinic acid (Thermo Fisher Scientific) as a matrix on a Bruker AutoFlex-III. Each linker conjugation results in a mass increase of 275 m / z (Figure 14).

[0162] DNA conjugation. DNA conjugation was performed immediately after azide-functionalized Cas9 purification. A 300-fold excess of DBCO-terminated DNA was reacted with Cas9-azide by click reaction. The reaction solution was incubated at 4° C. for 72 h with shaking at 900 rpm. After 3 days, unreacted DNA strands were removed by size exclusion chromatography on a Bio-Rad FPLC 650. The number of DNAs per protein was determined by UV-visible absorbance spectroscopy based on the absorbance of the conjugated AlexaFluor dye (FIG. 15). The AF-647 fluorophore was used to calculate the protein concentration because the absorbance overlaps at 260 nm. After subtracting the protein absorbance, the DNA concentration was determined using an online IDT oligo analyzer (ε260=276,000 M -1 cm -1 ) was determined based on the extinction coefficient calculated using (Figure 16).

[0163] Biostability analysis of Cas9 SNA. To demonstrate whether surface conjugation of DNA can protect the protein from proteinase degradation, both native Cas9 protein and Cas9 ProSNA were incubated with trypsin (proteinase) and SDS-PAGE gel was performed. The reaction solution was incubated in NEB buffer 2 at 37° C. for 1 hour. It was observed that the native protein band incubated with trypsin was significantly reduced after 10 minutes. However, no degradation of Cas9 ProSNA was observed, suggesting that the DNA shell could protect the protein from substantial degradation by trypsin (FIG. 17).

[0164] In vitro investigation of Cas9 ProSNAs Cell viability. Cell viability was determined with a standard Cell Counting Kit-8 (CCK-8) assay. The CCK-8 reagent contains WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt), which can freely enter live cells. Upon entering cells, WST-8 (a weakly fluorescent compound) is reduced to an orange formazan dye by cellular dehydrogenases. Specifically, cells were cultured in DMEM medium with 10% FBS overnight in 96-well cell culture plates (1 × 10 per well). 4 ). The cell culture medium was then replaced with 200 μL of medium containing different concentrations of Cas9 ProSNA, followed by further incubation for 24 h. Afterwards, the cells were washed with 1×PBS and replaced with 10% CCK-8 in PBS. The cells were further incubated for 30 min. Finally, the absorbance of CCK at 450 nm was measured by a BioTek Synergy H4 Hybrid Plate Reader. Experiments were performed in triplicate. Cell viability was also assessed by calcein-AM / PI staining. Briefly, cells were plated in 24-well plates (5×10 per well) and incubated for 24 h. 4 ) and cultured overnight, followed by incubation in medium containing Cas9 ProSNA for 24 hours. The cells were then treated with 500 μL of PBS containing 2 μg / mL calcein-AM and 3 μg / mL PI together. Live or dead cells were observed by a Biotek Synergy H4 Hybrid Plate Reader at excitation of 488 nm for calcein-AM and 535 nm for PI (FIG. 18).

[0165] Cellular uptake in HaCat cells by flow cytometry. HaCaT cells were seeded in 48-well plates (60,000 per well) and cultured overnight in DMEM with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The culture medium was then replaced with OPTI-MEM containing Cas9 ProSNA or Cas9 AF647 to obtain a final concentration of 20 nM for different time intervals (0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h). At the end of each treatment, cells were washed with 1×PBS, 300 uL (trypsinized) (Gibco), 300 uL 1×PBS, centrifuged at 300G for 5 min, and then resuspended in 1 mL PBS. 1 μL of live / dead dye was added to the cell suspension. After 30 min, cells were collected by centrifugation and fixed in 4% paraformaldehyde (Thermo Fisher Scientific). Flow cytometry was then performed using a Becton Dickinson LSR II to measure fluorescence (excitation 640 nm, emission 655-685 nm) of 10,000 single cell events per sample. Raw FCS files were gated based on forward and side scatter intensity and analyzed in FlowJo (Figure 19).

[0166] Intracellular confocal microscopy. Intracellular delivery of Cas9 ProSNAs was assessed by confocal laser scanning microscopy (Zeiss LSM 810 microscope). To investigate endosomal escape of Cas9 ProSNAs or Cas9 AF647, HaCat cells (1 × 10 per well) were cultured at 100°C for 1 h. 4) were seeded on borosilicate 8-chamber cover glass slides (Nalge Nunc International). After 8 hours, cells were incubated with lysosome dye (CellLight™ Lysosomes-GFP, BacMam 2.0) at 37°C, incubated with 500 μL OptiMEM containing Cas9 ProSNA or Cas9 AF647 (20 nM) for different time intervals, followed by washing with PBS and staining with nuclear dye (Hoechst, 1 μg / mL) for 10 minutes at room temperature, followed by fixing the cells with 4% PFA for 10 minutes. Live cells were then imaged by fluorescence microscopy at 405 nm for Hoechst, 488 nm for Lysosomes-GFP, and 561 nm for AF647-labeled Cas9 ProSNA or Cas9 AF647, respectively. Nuclear transport efficiency was determined by confocal microscopy as the percentage of nuclei overlapped by AF647, analyzing approximately 100 cells for each sample (n=3) (FIG. 20).

[0167] Surveyor Assay (Figure 21). HaCat, hBMSC, RAW264.7, and A549 / EGFP cells were plated in 48-well plates (5x10 per well). 4 Cells) and cultured overnight at 37°C. Assembled Cas9 ProSNA (50 nM, human DNase I high activity site:

[0168] [Table 5]

[0169] Human GRIN2B site:

[0170] [Table 6]

[0171] Homologous mouse Grin2b site:

[0172] [Table 7]

[0173] and EGFP site:

[0174] [Table 8]

[0175] After incubation in OPTIMEM for 4 h with the target gene (underline indicates genome editing target), cells were replaced with fresh medium and cultured for another 3 days. Cells were then harvested for genomic DNA extraction using Quick Extraction Solution (Epicentre) followed by amplification by PCR. For the idle assay, 250 ng of DNA extract was denatured in combination with 2 μL of NEBuffer2 (NEB) in a total volume of 19 μL, then reannealed at 95 °C for 5 min, thermal cycling from 95 to 85 °C at 2 °C / s and 85 to 20 °C at 0.2 °C / s. The reannealed DNA was incubated with 1 μL of T7 endonuclease I (10 U / μL, NEB) for 15 min at 37 °C. 10 μL of 50% glycerol was added to the T7 endonuclease reaction and 12 μL was analyzed on a PAGE gel (Bio-Rad) electrophoresed at 200 V for 30 min. The Cas9-induced cut and uncut bands were used to calculate genome editing efficiency using ImageJ.

[0176] Lipofectamine CRISPRMAX Cas9 transfection. Lipofectamine CRISPRMAX transfection reagent was used to transfect cells with Cas9-sgRNA complexes according to the provided transfection protocol. Briefly, 1 μL of Cas9 Plus reagent was added to 25 μL of Opti-MEM medium containing Cas9 protein (500 nM) and sgRNA (1 μM), followed by incubation at room temperature for 5 min (tube 1). Furthermore, 1.5 μL of lipofectamine CRISPRMAX reagent was added to 25 μL of Opti-MEM medium, followed by incubation at room temperature for another 5 min (tube 2). Then, the Cas9-sgRNA Plus mixture from tube 1 was mixed with the lipofectamine CRISPRMAX solution from tube 2, followed by incubation at room temperature for 10 min. Then, 50 μL of the prepared Cas9-sgRNA transfection complexes were dropped into each well (48-well plate, 5 × 10 per well). 4 ).

[0177] In vitro gene silencing. HEK293T cells that constantly express EGFP (HEK293T / EGFP) were used to evaluate the gene silencing effect of Cas9 ProSNA. HEK293T / EGFP cells were cultured in 48-well plates (5 × 10 per well). 4 , 0.5 mL) and cultured overnight at 37°C. The medium is then changed to 2% FBS in OPTI-MUM for 5 hours. After 6 hours of incubation with Cas9 ProSNA in OPTI-MUM to target the coding region of EGFP for 24 hours, the cells were replaced with fresh medium and cultured for 3 days. The cells were then digested with trypsin-EDTA solution and resuspended in 0.3 mL PBS for flow cytometry (BD FACSCANTO II, EGFP channel). All data were analyzed using FCS Express flow cytometry data analysis (Figure 22).

[0178] This example demonstrated that Cas9 ProSNA enhanced cellular internalization by up to approximately 45-fold. 10 The use of the sequences allowed for the facile generation of genome editing tools with excellent biocompatibility, proteinase biostability, and excellent genome editing indel efficiency of approximately 45.4%. These observations lead to the design of nucleic acid-functionalized (bio)macromolecular cargoes that can be universally used so that cellular functions can be specifically and transiently manipulated.

[0179] Example 4 This example describes additional experiments using CRISPR / Cas9 ProSNA.

[0180] Functionalization of Alexa Fluor™ 647. Cas9 protein was modified with amino-active Alexa Fluor™ 647 NHS ester (AF647, Thermo Fisher Scientific). AF647 was dissolved in DMSO and analyzed by UV-visible spectrophotometry (ε 647 A stock solution of 10 mM was obtained, as determined by ε = 270,000 M-1 cm-1). Five excess equivalents of AF647 were added to the solution of Cas9 protein and the reaction was shaken overnight at 900 rpm. Excess AF647 was removed by size exclusion chromatography on a Bio-Rad FPLC. Spectra of AF647 modified proteins were collected on a Cary-500 UV-Visible spectrophotometer (Molecular Devices Inc, USA) and the number of modifications was calculated using the respective extinction coefficients (Cas9: ε at 280 nm, ... 280 =204,470M -1 cm -1 and ε at 260 nm 260 =324,610M -1 cm -1 , AF647: ε at 650 nm 650 =270,000). (Figure 23)

[0181] UV-visible spectrum of AF647 fluorophore modified Cas9. Spectra were obtained at ambient temperature on a Cary5000 spectrophotometer. Protein and AF647 concentrations were calculated from absorbance at 280 nm and 650 nm, respectively. The AF647 fluorophore was used to calculate protein concentration after DNA conjugation and to track cellular uptake in both flow cytometry and confocal imaging experiments. (Figure 23)

[0182] Surface-accessible lysines and NHS-PEG 4 -Azide reaction. Surface lysines were converted to azides at the opposite end by reacting them with a tetraethylene glycol linker containing an N-hydroxysuccinimide (NHS) ester and an azide moiety (NHS-PEG 4 -N 3 (Thermo Scientific). A solution of Cas9-AF647 was added with 600 equivalents of NHS-PEG 4 -Azide linker was added. After 2 hours, unconjugated linkers were removed by size exclusion chromatography on a Bio-Rad FPLC. The number of azide modifications was determined by MALDI-MS using sinapinic acid (Thermo Fisher Scientific) as a matrix. Each linker conjugation resulted in a mass increase of 275 m / z. (Figure 23)

[0183] DNA conjugation. The DNA conjugation reaction was performed immediately after purification of azide-modified Cas9. A 300-fold excess of DBCO-terminated DNA was reacted with Cas9-AF647-azide by click reaction. The reaction solution was incubated at 4 °C for 3 days. After 3 days, unreacted DNA strands were removed by size-exclusion chromatography (ENrich™ SEC650 column, Bio-Rad Inc., USA) on a Bio-Rad FPLC. The number of DNA modifications per protein was determined by UV-visible spectrophotometry. The AF647 fluorophore was used to calculate the concentration of protein after DNA conjugation, since both absorbances overlap at 260 nm. After subtracting the protein absorbance, the concentration of DNA was determined using an Integrated DNA Technologies oligo analyzer (ε 260 =276,000M -1 cm -1 ) was determined based on the absorption coefficient obtained from the

[0184] Determination of the number of DNA strands on Cas9 ProSNA by UV-Visible spectrophotometry. Spectra were collected on a Cary5000 spectrophotometer. Protein and DNA concentrations were calculated from the absorbance at 650 nm and 260 nm, respectively. (Figure 23)

[0185] Circular dichroism spectroscopy. Circular dichroism (CD) was used to characterize the intact Cas9 protein structure after DNA functionalization. All samples were buffer exchanged with PBS and CD spectra were collected at room temperature on a Jasco J-1700 spectrophotometer. Cas9 and DNA samples were prepared at concentrations of 500 nM and 7.13 μM, respectively. The theoretical spectrum of Cas9 ProSNA was calculated by summing the spectra of Cas9-AF647 and free DNA. The collected spectrum of Cas9 ProSNA was consistent with the calculated spectrum. (Figure 24)

[0186] Biostability analysis of Cas9 ProSNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to investigate whether surface conjugation of DNA could protect Cas9 protein from trypsin degradation. Both native Cas9 protein and Cas9 ProSNA were incubated with trypsin at 37° C., and 30 μL of protein was loaded for analysis over different time points. It was observed that the band corresponding to Cas9 protein was significantly reduced after incubation with trypsin for as long as 10 minutes. However, Cas9 ProSNA showed little degradation over the time course of this study due to DNA conjugation. (FIG. 25)

[0187] Cell viability. Cell viability was determined with a standard Cell Counting Kit-8 (CCK-8) assay. The CCK-8 reagent contained 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8), which can freely enter live cells. Upon entering the cells, WST-8 was reduced by cellular dehydrogenases to an orange formazan dye (absorbance at 460 nm). Specifically, cells were plated in DMEM medium containing 10% FBS overnight in 96-well cell culture plates (10 per well). 4 ). The cell culture medium was then replaced with fresh medium containing different concentrations of Cas9 ProSNA and incubated for another 24 h. The cells were then washed with PBS and replaced with 10% CCK-8. The cells were incubated for another 30 min and the absorbance values ​​at 460 nm were measured by a BioTek Synergy H4 Hybrid Plate Reader. All experiments were performed in independent triplicates. Cell viability was also assessed by live / dead staining. Briefly, cells were plated in 24-well plates (5 × 10 per well) and incubated for another 24 h. 4) and cultured overnight, followed by incubation in cell culture medium containing Cas9 ProSNA for 24 hours. The cells were then treated with calcein acetoxymethyl (2 μg / mL) and propidium iodide (3 μg / mL) together. Live or dead cells were observed by a Biotek Synergy H4 Hybrid Plate Reader (BioRad, USA) at excitation of 488 nm for calcein-AM and 535 nm for propidium iodide (Figure 26).

[0188] Cellular uptake in HaCat cells by flow cytometry. HaCaT cells were seeded in 48-well plates (60,000 per well) and cultured in DMEM with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. The cell culture medium was then replaced with Opti-MEM containing Cas9 ProSNA or Cas9-AF647 to obtain a final concentration of 20 nM for different time intervals (0.5 h, 1 h, 2 h, 4 h, 6 h, and 8 h). At the end of each treatment, cells were washed with PBS (trypsinized) (Gibco), centrifuged at 800 × g for 5 min, and fixed with fixation buffer (BioLegend). Flow cytometry was then performed using a Becton Dickinson LSR II to measure the fluorescence (excitation 640 nm, emission 655-685 nm) of at least 10,000 single-cell events per sample. All experiments were performed in triplicate. (Fig.27)

[0189] Intracellular delivery analysis by confocal microscopy. The intracellular delivery of Cas9 ProSNA was evaluated by confocal laser scanning microscopy (Zeiss LSM 810, Germany). To investigate the endosomal escape of Cas9 ProSNA or Cas9 AF647, HaCat cells (10 per well) were cultured in 100% PBS. 4) were seeded on borosilicate 8-chamber cover glass slides (Nalge Nunc International). After 8 hours, cells were incubated with endosomal stain (CellLight™ Late Endosomes-GFP, BacMam 2.0) containing Cas9 ProSNA or Cas9 AF647 (20 nM) for different time intervals. Excess protein was washed with PBS and nuclei were stained with Hoechst (1 μg / mL) for 1 min before fixing cells with 4% paraformaldehyde (Thermo Fisher Scientific) for 15 min. Cells were then imaged simultaneously by confocal fluorescence microscopy at 405 nm for Hoechst, 488 nm for Lysosomes-GFP, and 561 nm for Cas9 ProSNA or Cas9 AF647. Nuclear transport efficiency was determined from confocal microscopy images counting nuclei overlapping with AF647, and approximately 100 cells were analyzed for each sample. (Figure 29)

[0190] Surveyor assay to detect genome editing indel efficiency. HaCat, hBMSC, RAW264.7, and HEK293T / EGFP cells were cultured in 48-well plates (5 × 10 per well). 4Cells) and cultured overnight. After 4 h of transfection with assembled Cas9 ProSNA-sgRNA complex (50 nM) in Opti-MEM, cells were replaced with fresh medium and cultured for another 3 days. Next, genomic DNA was extracted from cells using a genomic DNA extraction kit (Quick Extraction Solution, Epicentre) according to the manufacturer's protocol. Briefly, cells were resuspended in QuickExtract solution and incubated at 65 °C for 15 min and 98 °C for 6 min. 5 μL of the extracted solution was then amplified by PCR reaction with target region primers. For the idle formation assay, an aliquot of 5 μL of PCR product was mixed and denatured with T7 endonuclease I (T7EI) buffer in a total volume of 19 μL, and then reannealed by thermal cycling to allow heteroduplex formation (95 °C for 10 min, 95 to 85 °C with a gradient of -2 °C / s, 85 to 20 °C with a gradient of -0.2 °C / s). The reannealed products were incubated with 1 μL of T7EI (10 U / μL, NEB) for 15 min and analyzed on a 4-15% polyacrylamide gel (BioRad). Cas9-induced cleaved and uncleaved bands were visualized with a Gel Doc gel imaging system (BioRad) and quantified using ImageJ densitometry analysis. Genome editing efficiency scheme was determined as shown in Figure 21. The results are shown in Figure 30.

[0191] Lipofectamine CRISPRMAX Cas9-sgRNA complex transfection. Lipofectamine™ CRISPRMAX™ transfection reagent was used to transfect Cas9-sgRNA complex into RAW 264.7 cells according to the manufacturer's transfection protocol. Briefly, Cas9 Plus reagent was added to the medium containing Cas9 protein and sgRNA, followed by incubation at room temperature for 5 minutes to form Cas9-sgRNA complex. Then, lipofectamine CRISPRMAX reagent was mixed with Opti-MEM medium and incubated for another 5 minutes. Then, Cas9-sgRNA mixture was mixed with lipofectamine CRISPRMAX solution, followed by incubation for 10 minutes. Then, 50 μL of prepared Cas9-sgRNA transfection complex (50 nM final concentration) was added and mixed into the cell medium for 4 hours. After treatment of Cas9-sgRNA complex, cells were cultured in the corresponding medium for 3 days. The cells were then harvested for subsequent Surveyor assays, and the results are shown in Figure 31.

[0192] In vitro gene silencing. The gene silencing effect of Cas9 ProSNA was evaluated using HEK293T cells containing the EGFP gene (HEK293T / EGFP). HEK293T / EGFP cells were cultured in 48-well plates (5 × 10 per well). 4 ) and cultured overnight. After 4 hours of incubation with Cas9 ProSNA (50 nM) targeting the coding region of EGFP in Opti-MEM, the cells were replaced with fresh medium and cultured for another 3 days. The cells were then digested with trypsin-EDTA solution and resuspended in live and dead cell suspension. After 30 minutes, the cells were washed with PBS and fixed for flow cytometry (Becton Dickinson LSR II, EGFP channel). All experiments were performed in independent triplicates. The results are shown in Figure 32.

Claims

1. Protein-core globular nucleic acid (ProSNA), (a) a protein core comprising a gene editing protein; (b) a shell of oligonucleotides bound to said protein core.

2. The ProSNA of claim 1 , wherein each oligonucleotide within the shell of oligonucleotides is covalently linked to the protein core.

3. A ProSNA described in claim 1 or 2, comprising a DNA or RNA gene editing factor substrate.

4. The ProSNA of claim 3, wherein the DNA or RNA gene editing factor substrate is a guide RNA comprising a pre-CRISPR RNA (crRNA) and a transactivating transactivating crRNA (tracrRNA).

5. The ProSNA according to any one of claims 1 to 4, wherein each oligonucleotide within the shell of oligonucleotides is linked to the protein core via a linker.

6. The ProSNA of claim 5, wherein the linker is a cleavable linker, a non-cleavable linker, a traceless linker, or a combination thereof.

7. The ProSNA of claim 5 or 6, wherein the linker is a carbamate alkylene dithiolate linker.

8. At least one oligonucleotide within the shell of oligonucleotides is a protein-core-NH-C(O)-O-C ２－５ Alkylene -S-S-C ２－７ Alkylene-oligonucleotide, or protein-core-NH-C(O)-O-CH ２ -Ar-S-S-C ２－７ The ProSNA of claim 7, comprising an alkylene-oligonucleotide, wherein Ar comprises a meta or para substituted phenyl.

9. At least one oligonucleotide within the shell of oligonucleotides is a protein-core-NH-C(O)-O-C(ZA)(ZB)C １－４ Alkylene-C(XA)(XB)-S-S-C(YA)(YB)C １－６ 8. The ProSNA of claim 7, comprising an alkylene-oligonucleotide, wherein ZA, ZB, XA, XB, YA, and YB are each independently H, Me, Et, or iPr.

10. At least one oligonucleotide within the shell of oligonucleotides is protein-core-NH-C(O)-O-C(XA)(XB)-Ar-S-S-C(YA)(YB)C ２－６ 8. The ProSNA of claim 7, comprising an alkylene-oligonucleotide, wherein XA, XB, YA, and YB are each independently H, Me, Et, or iPr.

11. The ProSNA of claim 5 , wherein the linker is an amido alkylene dithiolate linker.

12. At least one oligonucleotide within the shell of oligonucleotides is a protein-core-NH-C(O)-C ２－５ Alkylene -S-S-C ２－７ The ProSNA of claim 11, comprising an alkylene-oligonucleotide.

13. At least one oligonucleotide within the shell of oligonucleotides is a protein-core-NH-C(O)-C １－４ Alkylene-C(XA)(XB)-S-S-C(YA)(YB)C １－６ 12. The ProSNA of claim 11, comprising an alkylene-oligonucleotide, wherein XA, XB, YA, and YB are each independently H, Me, Et, or iPr.

14. The ProSNA of claim 5 , wherein the linker is an amido alkylene thioether linker.

15. At least one oligonucleotide within the shell of oligonucleotides is a protein-core-NH-C(O)-C ２－４ Alkylene-N-succinimidyl-S-C ２－６ The ProSNA of claim 14, comprising an alkylene-oligonucleotide.

16. Spherical nucleic acid (SNA), (a) a nanoparticle core; (b) a shell of oligonucleotides attached to the outer surface of the nanoparticle core; and (c) a gene editing protein.

17. 17. The SNA of claim 16, wherein the nanoparticle core is a liposome core or a lipid nanoparticle core.

18. 18. The SNA of claim 17, wherein the lipid nanoparticle core comprises an ionizable lipid, a phospholipid, a sterol, and a lipid-polyethylene glycol (lipid-PEG) conjugate.

19. 19. The method of claim 18, wherein each oligonucleotide within the shell of oligonucleotides is covalently attached to the exterior of the lipid nanoparticle core via the lipid-PEG conjugate.

20. The SNA according to any one of claims 17 to 19, wherein the gene editing protein is encapsulated in the lipid nanoparticle core.

21. The ProSNA according to any one of claims 15 to 18, wherein the ProSNA according to any one of claims 1 to 13 is encapsulated in the lipid nanoparticle core.

22. The SNA according to any one of claims 17 to 20, wherein a ribonucleoprotein (RNP) complex is encapsulated in the lipid nanoparticle core, the RNP comprising the gene editing protein, clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) and transactivating crRNA (tracrRNA).

23. The SNA of claim 17, wherein the liposome core comprises multiple lipid groups.

24. The SNA of claim 17 or 23, wherein the gene editing protein is encapsulated in the liposome core.

25. The ProSNA according to claim 24, wherein the ProSNA according to any one of claims 1 to 13 is encapsulated in a liposomal nanoparticle core.

26. The SNA of claim 17 or 23, wherein a ribonucleoprotein (RNP) complex is encapsulated in the lipid nanoparticle core, the RNP comprising the gene editing protein, a CRISPR RNA (crRNA), and a transactivating crRNA (tracrRNA).

27. 27. A SNA according to any one of claims 23 to 26, wherein said lipid groups comprise lipids selected from the group consisting of the phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine families of lipids.

28. The lipid may be 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG), 1,2-dioleo ...

28. The SNA of claim 27, which is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE).

29. A SNA according to any one of claims 16 to 28, wherein at least one oligonucleotide within the shell of said oligonucleotides is attached to the exterior of said liposome or said lipid nanoparticle core via a lipid anchor group.

30. 30. The SNA of claim 29, wherein said lipid anchor group is attached to the 5'-terminus or the 3'-terminus of said at least one oligonucleotide.

31. 31. The SNA of claim 29 or 30, wherein the lipid anchor group is tocopherol or cholesterol.

32. The ProSNA according to any one of claims 1 to 15 or the SNA according to any one of claims 16 to 31, wherein the gene editing protein is a CRISPR associated protein (Cas).

33. The ProSNA or SNA according to claim 32, wherein the Cas is Cas9, Cas12, Cas13, or a combination thereof.

34. ProSNA or SNA according to any one of claims 1 to 33, wherein at least one oligonucleotide within the shell of said oligonucleotides is modified with dibenzocyclooctyl (DBCO) at its 5'-terminus and / or 3'-terminus.

35. The ProSNA or SNA according to any one of claims 1 to 34, wherein the shell of oligonucleotides comprises single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, or a combination thereof.

36. ProSNA or SNA according to any one of claims 1 to 35, wherein at least one oligonucleotide within the shell of oligonucleotides is a modified oligonucleotide.

37. The ProSNA or SNA according to any one of claims 1 to 36, wherein the shell of oligonucleotides comprises from about 2 to about 100 oligonucleotides.

38. 38. The ProSNA or SNA of claim 37, wherein the shell of oligonucleotides comprises from about 10 to about 80 oligonucleotides.

39. 38. The ProSNA or SNA of claim 37, wherein the shell of oligonucleotides comprises from about 5 to about 50 oligonucleotides.

40. 38. The ProSNA or SNA of claim 37, wherein the shell of oligonucleotides comprises from about 5 to about 20 oligonucleotides.

41. 38. The ProSNA or SNA of claim 37, wherein the shell of oligonucleotides comprises about 14 oligonucleotides.

42. 38. The ProSNA or SNA of claim 37, wherein the shell of oligonucleotides comprises about 15 oligonucleotides.

43. The ProSNA or SNA according to any one of claims 1 to 42, wherein each oligonucleotide within the shell of oligonucleotides is from about 5 to about 100 nucleotides in length.

44. The ProSNA or SNA of claim 43, wherein each oligonucleotide within the shell of oligonucleotides is from about 10 to about 50 nucleotides in length.

45. One or more of the oligonucleotides within the shell of the oligonucleotides is (GGX) ｎ 45. ProSNA or SNA according to any one of claims 1 to 44, comprising a nucleotide sequence, in which n is between 2 and 20 and X is a nucleobase (A, C, T, G or U).

46. (GGX) ｎ 46. ​​The ProSNA or SNA of claim 45, wherein the nucleotide sequence is at the 5' end of said one or more oligonucleotides.

47. (GGX) ｎ 46. ​​The ProSNA or SNA of claim 45, wherein the nucleotide sequence is at the 3' end of said one or more oligonucleotides.

48. (GGX) ｎ The nucleotide sequence is (GGT) ｎ The ProSNA or SNA according to any one of claims 45 to 47, which is a nucleotide sequence.

49. The ProSNA or SNA according to any one of claims 1 to 48, wherein the diameter of the ProSNA or SNA is from about 1 nanometer (nm) to about 500 nm.

50. 50. The SNA of any one of claims 16 to 49, wherein the diameter of said SNA is less than or equal to about 50 nanometers.

51. A ProSNA or SNA according to any one of claims 1 to 49 or a SNA according to claim 50, wherein at least one oligonucleotide within the shell of said oligonucleotides is a targeting oligonucleotide.

52. A ProSNA or SNA according to any one of claims 1 to 49 or a SNA according to claim 50, wherein the shell of said oligonucleotide comprises an inhibitory oligonucleotide, an immunostimulatory oligonucleotide, a gene editor substrate DNA or RNA, or a combination thereof.

53. 53. The ProSNA or SNA of claim 52, wherein the inhibitory oligonucleotide is an antisense oligonucleotide, a small interfering RNA (siRNA), an aptamer, a short hairpin RNA (shRNA), a DNAzyme, or an aptazyme.

54. 53. The ProSNA or SNA of claim 52, wherein the immunostimulatory oligonucleotide is a CpG motif-containing oligonucleotide, a double-stranded DNA oligonucleotide, or a single-stranded RNA oligonucleotide.

55. 53. The ProSNA or SNA of claim 52, wherein each of said immunostimulatory oligonucleotides is a toll-like receptor (TLR) agonist.

56. 56. The ProSNA or SNA according to claim 55, wherein the TLR is selected from the group consisting of toll-like receptor 1 (TLR1), toll-like receptor 2 (TLR2), toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), toll-like receptor 5 (TLR5), toll-like receptor 6 (TLR6), toll-like receptor 7 (TLR7), toll-like receptor 8 (TLR8), toll-like receptor 9 (TLR9), toll-like receptor 10 (TLR10), toll-like receptor 11 (TLR11), toll-like receptor 12 (TLR12), and toll-like receptor 13 (TLR13).

57. 13. A composition comprising a plurality of protein-core globular nucleic acids (ProSNAs) according to any one of claims 1-15, 32-49 or 51-56.

58. 58. The composition of claim 57, further comprising a guide RNA.

59. 59. The composition of claim 57 or 58, wherein at least two of the ProSNAs comprise different protein cores.

60. A composition comprising a plurality of spherical nucleic acids (SNA) according to any one of claims 16 to 56.

61. 60. The composition of claim 59, wherein at least two of the SNAs comprise different nanoparticle cores.

62. 57. The ProSNA of any one of claims 1 to 15, 32 to 49, or 51 to 56 for delivering a gene editing protein to a cell.

63. The ProSNA described in claim 62, wherein contact of the cell with the ProSNA results in an insertion / deletion (indel) frequency of approximately 30% to 45%.

64. 60. A composition according to any one of claims 57 to 59 for delivering a gene editing protein to a cell.

65. A SNA according to any one of claims 16 to 56 for delivering a gene editing protein to a cell.

66. 62. A composition according to claim 60 or 61 for delivering a gene editing protein to a cell.

67. (i) for treating, ameliorating, and / or preventing a disorder in a subject; (ii) a ProSNA according to any one of claims 1 to 15, 32 to 49 or 51 to 56, (iii) a SNA according to any one of claims 16 to 56, A composition according to any one of claims 57 to 61, or (iv) a combination thereof.

68. 68. The ProSNA, SNA, composition or combination of claim 67, wherein the disorder is cancer, an infectious disease, an autoimmune disease, a neurodegenerative disease, a genetic disease, a cardiovascular disease, or a combination thereof.

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