WO2024196422A1

WO2024196422A1 - Dna nanodevices for specific and efficient delivery of functional payloads to the cytoplasm and methods of their use

Info

Publication number: WO2024196422A1
Application number: PCT/US2023/069910
Authority: WO
Inventors: Hao Yan; Lu Yu; Shuoxing JIANG; Yang Xu; AL-AMIN, Md; Nicholas STEPHANOPOULOS
Original assignee: Arizona State University ASU; Arizona State University Downtown Phoenix campus
Current assignee: Arizona State University ASU; Arizona State University Downtown Phoenix campus
Priority date: 2023-03-21
Filing date: 2023-07-10
Publication date: 2024-09-26
Anticipated expiration: 2025-09-21

Abstract

A novel nanostructured nucleic acid platform that uniquely integrates the advantages of a disulfide moiety for the enhanced cytosolic uptake of a DNA or RNA nanostructure ("DNA origami") which can further comprise a therapeutic agent and a targeting moiety is described herein. The targeting moiety can be an affibody, in particular, a Her2 affibody. The disulfide moiety can be formed from a sulfide-modified oligonucleotide designed to target enhanced cystolic uptake.

Description

DNA NANODEVICES FOR SPECIFIC AND EFFICIENT DELIVERY OF FUNCTIONAL PAYLOADS TO THE CYTOPLASM AND METHODS OF THEIR USE

Related Applications

[001 ] This disclosure claims priority to U.S. Provisional Application No. 63/453,702, filed March 21 , 2023, the contents of which are herein incorporated by reference in their entirety.

SEQUENCE STATEMENT

[002] The instant application contains a Sequence Listing, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said file is named G81 18-03903 SEQ ID LISTING. xml and is 276 kb in size.

Field

[003] The present invention relates to compositions comprising a nanostructured nucleic acid platform engineered for rapid cytosolic uptake. The invention also includes methods of creating a nanostructured nucleic acid platform, compositions comprising said a nanostructured nucleic acid platform, and methods of their use in treating diseases (e.g., cancer).

Background

[004] The development of rapid and efficient cytosolic drug delivery vehicles with target specificity and biosafety holds great potential for a variety of diseases. Various promising drug modalities, including therapeutic oligonucleotides, mRNAs, proteins, plasmids, and antitumor drugs require cytosolic uptake. However, their therapeutic potential can not be fully achieved owing to their poor cell membrane permeability, endo/lysosomal trapping, enzymatic degradation, rapid liver clearance, and off-target effect induced safety concern. Carriers such as virus-like particles, organic nanoparticles (e.g., liposome, synthetic polymer and micelle), and inorganic nanoparticles (e.g., mesoporous silica nanoparticles (MSNs) and gold nanoparticles) have been used to enhance cytosolic uptake but with some off-target cytotoxicity.

[005] Human epidermal growth factor receptor 2 (HER2) is a transmembrane protein which exhibits a plurality of cysteine residues external to the cell membrane and is involved in normal cell growth. Overexpression of HER2 is found in a variety of cancers including breast cancers, gastric/gastroesophageal cancers, and other cancers (ovary, endometrium, bladder, lung, colon, and head and neck cancers).

Summary

[006] This disclosure provides for a nanostructured nucleic acid platform and compositions comprising a disulfide moiety for the enhanced cytosolic uptake for the administration of a therapeutic agent for the treatment of a disease. In some aspects, the disease is selected from Alzheimer’s disease, a Lysosomal Storage disease, or cancer. In some aspects, the disease is cancer (e.g., a solid tumor cancer). In some aspects, nanostructured nucleic acid platform comprises nanostructured DNA.

[007] The platforms and methods if this disclosure overcome typical problems observed with cytosolic uptake carriers such as cytoxicity, which has been a consistent concern for some popular nanocarriers, such as lipid nanoparticles and polymers. Another concern for some popular carriers is that when they are predominantly delivered to cells by the endocytosis pathway, they induce subsequent endo and/or lysosomal trapping resulting in further degradation due to their highly enzymatic and acid environment.

[008] In some aspects, this disclosure addresses these challenges through the development of a novel cytosolic-targeted nanostructured nucleic acid platform that uniquely integrates the advantages of cell-surface targeting moieties (e.g, Her2 moieties) and disulfide moieties to enhance the cytosolic uptake to cell-surface target overexpressed cells (e.g., Her2 overexpressed cells).

[009] In some aspects, this disclosure provides for a cytosolic uptake composition comprising: a. a nanostructured nucleic acid complex; b. an cytosolic uptake moiety; c. a targeting moiety; and d. a therapeutic agent.

[010] In some aspects, the nanostructure nucleic acid complex can be a DNA nanostructure or an RNA nanostructure. In some aspects, the nanostructure nucleic acid complex is a DNA nanostructure. In some aspects, the DNA nanostructure can be selected from a double-stranded DNA sequence, a DNA origami, a DNA nanosheet, a DNA triangle, or a DNA tube. [011 ] The cytosolic uptake moiety can be an oligonucleotide comprising a disulfide moiety. The oligonucleotide comprising a disulfide moiety can further comprise a nucleotide sequence which is partially complementary to a nucleotide sequence of the nanostructured nucleic acid complex.

[012] The targeting moiety can be an aptamer, an antibody, an affibody, an scFv, a lectin, a peptide, a molecule comprising an electrophile, a molecule comprising a nucleophile, or a combination thereof. In some aspects, the affibody can be a HER2 targeting affibody. In some aspects, the affibody can be linked to the nanostructured nucleic acid complex, or an oligonucleotide have a complementary nucleotide sequence which is partially or wholly complementary thereto, by a covalent linkage through a crosslinker moiety.

[013] In some aspects, the therapeutic agent can be an anti-MCI-1 shRNA, an anti-bcl-xl shRNA, or a chemotherapeutic drug, including doxyrubicin. The anti-MCI-1 shRNA or the anti-bcl-xl shRNA can comprise a nucleotide sequence which is partially or completely complementary to a section of the nanostructured nucleic acid complex.

[014] In some aspects, the composition can further comprise an imaging agent. The imaging agent can comprise FAM, including an oligonucleotide comprising FAM.

[015] In some aspects, this disclosure provides for a method of treating cancer in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of the cytosolic uptake composition of this disclosure to said subject. In some aspects, the cancer can be HER2-positive breast cancer. In some aspects, the cancer can be gastric/gastroesophageal cancer.

[016] In some aspects, the nanostructure nucleic acid complex can be a DNA nanostructure or an RNA nanostructure.

[017] In some aspects, the disulfide moiety can be on the 5’ terminus, the 3’ terminus, within the nucleotide sequence, or combinations thereof, within the oligonucleotide. In some aspects, the oligonucleotide comprising a disulfide moiety further can comprise a nucleotide sequence which is partially complementary to a nucleotide sequence of the nanostructured nucleic acid complex.

[018] In some aspects, the targeting moiety can be selected from an aptamer, an antibody, an affibody, an scFv, a lectin, a peptide, a molecule comprising an electrophile, a molecule comprising a nucleophile, or a combination thereof. In some aspects, the targeting moiety can be an aptamer. In some aspects, the targeting moiety can be an affibody, in particular a HER2 targeting affibody. The affibody can be linked to the nanostructured nucleic acid complex by a covalent linkage through a crosslinker (e.g., SMCC). In some aspects, the affibody can be linked to the nanostructured nucleic acid complex by a covalent linkage of the affibody to an oligonucleotide, and wherein said oligonucleotide is partially or completely complementary to a portion of the nanostructured nucleic acid complex.

[019] In some aspects, the therapeutic agent can be selected from siRNA, shRNA, mRNA, a small molecule (e.g., anticancer drug), a protein (e.g., antibody), or combinations thereof. In some aspects, the therapeutic agent can be anti-MCI-1 shRNA or anti-bcl-xl shRNA. In some aspects, the anti-MCI-1 shRNA or anti-bcl-xl shRNA can comprise a nucleotide sequence which is partially or completely complementary to ta section of the nanostructured nucleic acid complex. In some aspects, the therapeutic agent can further comprise an imaging agent, in particular FAM. In some aspects, the imaging agent can be an oligonucleotide comprising FAM.

[020] In some aspects, this disclosure provides for a method of treating a disease in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of the cytosolic uptake composition of this disclosure to said subject. The diseases can be Alzheimer’s disease, a Lysosomal Storage Disease, or cancer. In some aspects, the disease is a solid tumor cancer.

Brief Description of Drawings

[021] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office.

[022] FIG. 1A depicts the design and characterization of cyto-direct DNA nanodevice. Illustration of the construction of the disulfide and HER2 affibody modified cyto-direct DNA nanodevice;

[023] FIG. 1 B is a schematic illustration of the target proximity-indued accelerated thiol-mediated rapid cytosolic uptake process;

[024] FIG. 1 C shows representative negative stain TEM images of DON, DS- DON, and DSHAF-DON. Scale bar, 100nm;

[025] FIG. 2A shows a comparison of target specificity of DS-DON, HFA-DON and DSHAF-DON in HER2 positive and negative breast cancer cells. HER2 overexpressed cell targeting ability of HER2 modified DON affected by the number of modified HER2 affibody. Scale bar, 25pm;

[026] FIG. 2B is a schematic illustration of the interaction between HER2 protein expressed on the SK-BR-3 cell membrane and HER2 affibody modified on DON.; [027] FIG. 2C are representative confocal microscopy images of SK-BR-3 (HER2 positive) and MCF-7 (HER2 negative) cells incubated with FAM labeled DS-DON, HFA- DON and DSHAF-DON. Scale bar, 25pm;

[028] FIG. 3A demonstrates the cellular uptake of a representative DNA nanocarrier with different modifications. Representative confocal microscopy images of HER2 affibody modified DNA nanocarrier with disulfide (top) and without disulfide (bottom) modification at different time points (5min, 0.5h, 3h, and 7h, scale bar 25pm);

[029] FIG. 3B is the flow cytometry analysis of the internalization of DNA nanocarrier with different modifications. FAM labeled with or without disulfide modified hybridized strand (top). Alexa Fluor® 647 labeled staple strands of DNA origami (bottom). Error bars represents mean ± s.d. 0.1234 (ns), 0.0332 (*P), 0.0021 (**P), 0.0002 (***P), <0.0001 (****p) from three independent experiments;

[030] FIG. 3C depicts a proposed four stage internalization process of DSHAF- DON;

[031 ] FIG. 4 shows the cell distribution of a representative DNA nanocarrier with different modifications. Confocal microscopy image of SK-BR-3 cells treated with DSHFA-DON, HAF-DON, and DS-DON for 5h. The nucleus was stained with Hoechst 33342 and the late endosome and lysosome were stained with LysoTracker. Scale bar, 25 pm. Enlarged images are amplified images of the white boxed regions in the merged channels. Scale bar, 5pm. DSHFA-DONs diffused in the cytoplasm of cells;

[032] FIG. 5 shows inhibitory assays for cellular uptake of cyto-direct DNA nanodevice. SKBR3 cells with pre-treatment of different inhibitors, including sodium iodoacetate, nystatin (NYS), chloropromazine (CPZ), and methyl-[3-cyclodextrin. Scale bars: 25pm;

[033] FIG. 6A shows a tumor penetration study of DSHAF-DON and HAF-DON. a. 3D projection images of SK-BR-3 spheroids incubated with DSHAF-DON and HAF- DON for 4h, 8h and 12h, respectively. Scale bar, 200pm;

[034] FIG. 6B shows radial distribution plots of fluorescent molecule labeled DSHAF-DON and HAF-DON in 3D SK-BR-3 tumor spheroid;

[035] FIG. 7A shows the delivery of MCI-1 shRNA and anticancer doxorubicin to SK-BR-3 cells. A schematic illustration of using cytosolic uptake composition to deliver MCI-1 shRNA by DNA-RNA hybridization and deliver doxorubicin by double strand DNA intercalation;

[036] FIG. 7B shows the gene knockdown effect of cytosolic uptake composition delivered MCI-1 shRNA determined by western blot; [037] FIG. 7C shows the comparison of SK-BR-3 cell apoptosis between doxorubicin and cyto-direct DNA nfanodevice delivered doxorubicin by FITC-Annexin V and PI co-staining. Scale bar, 25pm. White arrows in the upper merged channel represent viable cells. Enlarged images are amplified images of the white boxed regions in the merged channels. Scale bar, 10pm;

[038] FIG. 8 depicts the cadnano design of the cyto-direct DNA nanodevice. Lighter strands are the 6SS-DNA capture strands, and the darker strands are the shRNA capture strands, with the medium shaded strand the scaffold nucleotide sequence;

[039] FIG. 9A shows innate curvature in DNA nanosheets through rational addition of different degrees of left-shift at crossover sites in structures with no overhangs, 104 overhangs, and 189 overhangs. The free-energy profile of DNA nanostructure designs is shown as a function of the distance between the two long edges (end-to-end distance) with no overhangs;

[040] FIG. 9B shows the free-energy profile of structures with 104 overhangs;

[041 ] FIG. 9C shows the free-energy profiles of structures with 189 overhangs;

[042] FIG. 9D shows the all-atom electrostatic surface mapping of mean structure from oxDNA2 simulation;

[043] FIG. 10 shows scheme 1 : the synthesis route for the disulfide phosphoramidite unit;

[044] FIG. 1 1 shows the ¹H NMR spectra of compound 1 ;

[045] FIG. 12 shows the ¹H NMR spectra of compound 2;

[046] FIG. 13 shows the ¹H (top) and ³¹ P (bottom) NMR spectra of compound 3;

[047] FIG. 14 shows the ¹H (top) and ³¹ P (bottom) NMR spectra of compound 4;

[048] FIG. 15 shows the MALDI-TOF-MS for the OSS and 6SS disulfide modified FAM labeled DNA oligonucleotide. The observed masses correspond well with the expected mass;

[049] FIG. 16 shows the SDS-PAGE characterization of HER2 affibody-DNA conjugate. Lane 1 , marker; lane 2, Amine-DNA; lane 3, HER2 affibody; lane 4, HER2 affibody-DNA conjugate before purification; lane 5, HER2 affibody-DNA conjugate after purification;

[050] FIG. 17 shows the gel image of 1 % agarose gel electrophoresis of purified DONs and DON loaded with different functional modules;

[051 ] FIG. 18A shows the characterization and yield analysis for DON. Schematic and % conformational state of DON analyzed through nsTEM images;

[052] FIG. 18B shows representative nsTEM images (scale bar 200 nm); [053] FIG. 18C shows representative nsTEM images (scale bar 200 nm);

[054] FIG. 18D shows representative nsTEM images (scale bar 200 nm);

[055] FIG. 19A shows Characterization and yield analysis for DSHAF-DON. Schematic and % state of DSHAF-DON analyzed through nsTEM images;

[056] FIG. 19B are representative nsTEM images (scale bar 200 nm);

[057] FIG. 19C are representative nsTEM images (scale bar 200 nm);

[058] FIG. 19D are representative nsTEM images (scale bar 200 nm);

[059] FIG. 20A shows the characterization and yield analysis for shRNA-DSHAF-

DON. Schematic and % state of shRNA loaded cytosolic uptake composition analyzed through nsTEM images;

[060] FIG. 20B are representative nsTEM images (scale bar 200 nm);

[061 ] FIG. 20C are representative nsTEM images (scale bar 200 nm);

[062] FIG. 20D are representative nsTEM images (scale bar 200 nm);

[063] FIG. 21 A is the AFM characterization of disulfide modified DON before

Tween 80 treatment;

[064] FIG. 21 B is the AFM characterization of disulfide modified DON after Tween 80 treatment;

[065] FIG. 22A shows the loading capacity characterization of a DNA nanostrucuture (e.g., DNA origami). The relationship between measured relative fluorescence intensity and number of input DNA on each origami is indicated. Square block marked line represents the expected DNA density calculated by extending the fluorescence intensity at the density of 26 and 51 of OSS DNA since the density of DNA is accurate in low density. Circle marked line represents the density of DNA without disulfide-modification on each origami. Triangle marked line represents the density of 6- repeat disulfide conjugated ASD density on each origami;

[066] FIG. 22B shows the results of a nuclease resistance study of 1046-disu If ide modified DNA nanostructure (e.g., DNA origami) in HeLa complete medium (with 10% FBS) at 37°C with varied incubation times;

[067] FIG. 23 shows CCK-8 assay analysis of the cytotoxicity of disulfide modification on DON. Disulfide modified DON showed no cytotoxicity, while lipofectamine showed increased cytotoxicity as the dose increased;

[068] FIG. 24 shows the flow cytometry analysis of HER2 expression in SK-BR- 3 and MCF-7 cells;

[069] FIG. 25 shows the data for a time course study of the cellular uptake of DSHAF-DON and HAF-DON for 1 h, 5h, 9h and 12h. Bright shades represents OSS or 6SS modified DON (FAM). Relatively darker shades represents nucleus of SK-BR-3 cells (Hoechst 33342). Images are merged channels;

[070] FIG. 26A shows the cellular uptake difference influenced by the location of disulfide modification. Schematic illustration of the distribution of disulfide (even, central and peripheral);

[071 ] FIG. 26B shows a flow cytometry analysis of the cellular uptake of DON with different disulfide distribution;

[072] FIG. 27 shows the cellular uptake and intracellular distribution of disulfide modified DNA nanostructure (e.g., DNA origami) in HeLa cells. The left panel is the schematic drawing of DNA origami with or without disulfide modification. The right panel is the confocal images of Hela cells that incubated with DNA nanostructure in 2h. Bright shade represents DNA origami; Moderate shade represents endosome or lysosome; Darker shade represents nucleus. Scale bar equals 25pm;

[073] FIG. 28A shows gel images of shRNA gene knockdown effect on HeLa cell lines. Significant gene knockdown is observed for the MCI1 -34 shRNA while almost no knockdown is obserbed for control 0ss-ASD_FAM;

[074] FIG. 28B shows gel images of shRNA gene knockdown effect on and SKBR3 cell lines. Significant gene knockdown is observed for the MCI1 -34 shRNA while almost no knockdown is obserbed for control 0ss-ASD_FAM;

[075] FIG. 29 is the contiguous M13mp18 scaffold nucleotide sequence (5’->3’);

[076] FIG. 30A-I lists the nucleotide sequences for the staple strands for disulfide-DNA capture loading (5’->3’);

[077] FIG. 31 A-G lists the nucleotide sequences for the staple strands for MCI-1 shRNA captures (5’ to 3’);

[078] FIG. 32A lists the nucleotide sequences for the open blocker staple strands (5’ to 3’);

[079] FIG. 32B lists the nucleotide sequences for the edge staple strands (5’ to 3’);

[080] FIG. 33 lists the nucleotide sequences for the staple strands for HER2 affibody-DNA captures (5’ to 3’);

[081 ] FIG. 34 are the nucleotide sequences for the Disulfide modified DNA sequence, HER2 affibody protein sequence, ssDNA sequence for HER2 affibody conjugation, MCI-1 shRNA nucleotide sequence, representative bcl-xl shRNA sequences, and the plasmid sequence for generating the Her2 affibody protein. Detailed Description

[082] Certain Definitions

[083] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[084] Units, prefixes, and symbols are denoted in their System International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[085] The singular forms “a”, “an”, and “the” may refer to plural articles unless specifically stated otherwise.

[086] As used herein, the term “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, comprising monomers (nucleotides) containing a sugar, phosphate and a base that is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues.

[087] As used herein, the terms “nucleotide sequence” and “nucleic acid sequence” refer to a sequence of bases (purines and/or pyrimidines) in a polymer of DNA or RNA, which can be single-stranded or double-stranded. In some embodiments, the nucleotide sequence comprises synthetic, non-natural or altered nucleotide bases, and/or backbone modifications (e.g., a modified oligomer, which can include or exclude a morpholino oligomer, phosphorodiamate morpholino oligomer or vivo-mopholino). The terms “oligo”, “oligonucleotide” and “oligomer” may be used interchangeably and refer to such sequences of purines and/or pyrimidines. The terms “modified oligos”, “modified oligonucleotides” or “modified oligomers” may be similarly used interchangeably, and refer to such sequences that contain synthetic, non-natural or altered bases and/or backbone modifications (e.g., chemical modifications to the internucleotide phosphate linkages and/or to the backbone sugar).

[088] Modified nucleotides can include or exclude alkylated purines; alkylated pyrimidines; acylated purines; and acylated pyrimidines. These classes of pyrimidines and purines can include or exclude pseudoisocytosine; N4, N4-ethanocytosine; 8- hydroxy-N6-methyladenine; 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil; 5- fluorouracil; 5-bromouracil; 5-carboxymethylaminomethyl-2-thiouracil; 5- carboxymethylaminomethyl uracil; dihydrouracil; inosine; N6-isopentyl-adenine; 1 - methyladenine; 1 -methylpseudouracil; 1 -methylguanine; 2,2-dimethylguanine; 2- methyladenine; 2-methylguanine; 3-methylcytosine; 5-methylcytosine; N6- methyladenine; 7-methylguanine; 5-methylaminomethyl uracil; 5-methoxy amino methyl- 2-thiouracil; beta-D-mannosylqueosine; 5-methoxycarbonylmethyluracil; 5- methoxyuracil; 2-methylthio-N6-isopentenyladenine; uracil-5-oxyacetic acid methyl ester; psueouracil; 2-thiocytosine; 5-methyl-2 thiouracil, 2-thiouracil; 4-thiouracil; 5- methyluracil; N-uracil-5-oxyacetic acid methylester; uracil 5-oxyacetic acid; queosine; 2- thiocytosine; 5-propyluracil; 5-propylcytosine; 5-ethyluracil; 5-ethylcytosine; 5-butyluracil; 5-pentyluracil; 5-pentylcytosine; and 2, 6, -diaminopurine; methylpsuedouracil; 1 - methylguanine; 1 -methylcytosine. Backbone modifications can include or exclude chemical modifications to the phosphate linkage. The chemical modifications to the phosphate linkage can include or excludee.g. phosphorodiamidate, phosphorothioate (PS), N3’phosphoramidate (NP), boranophosphate, 2’,5’phosphodiester, amide-linked, phosphonoacetate (PACE), morpholino, peptide nucleic acid (PNA), inverted linkages (5’-5’ and 3’-3’ linkages)) and sugar modifications (e.g., 2’-O-Me, UNA, LNA).

[089] The oligonucleotides described herein may be synthesized using solid or solution phase synthesis methods. In some embodiments, the oligonucleotides are synthesized using solid-phase phosphoramidite chemistry (U.S. Patent No. 6,773,885) with automated synthesizers. Chemical synthesis of nucleic acids allows for the production of various forms of the nucleic acids with modified linkages, chimeric compositions, and nonstandard bases or modifying groups attached in chosen places through the nucleic acid’s entire length. In some embodiments, the oligonucleotides described herein may be synthesized using enzymatic methods which can include adding single-bases via an enzyme.

[090] By “portion” or “fragment,” as it relates to a nucleic acid molecule, sequence or segment of the invention, when it is linked to other sequences for expression, is meant a sequence having at least 80 nucleotides, at least 150 nucleotides, or at least 400 nucleotides. If not employed for expressing, a “portion” or “fragment” means at least 9, at least 12, at least 15, or at least 20, consecutive nucleotides, e.g., probes and primers (oligonucleotides), corresponding to the nucleotide sequence of the nucleic acid molecules of the invention.

[091 ] The term “complementary” as used herein refers to the broad concept of complementary base pairing between two nucleic acids aligned in an antisense position in relation to each other. When a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other, then the nucleic acids are considered to be complementary to each other at this position. Two nucleic acids are substantially complementary to each other when at least about 50%, at least about 60%, or at least about 80% of corresponding positions in each of the molecules are occupied by nucleotides which normally base pair with each other (e.g., A:T (A:U for RNA) and G:C nucleotide pairs).

[092] As used herein, the term “DNA nanostructure” refers to a nanoscale structure made of DNA, wherein the DNA acts both as a structural and function element. DNA nanostructures can also serve as a scaffold for the formation of other structures. DNA nanostructures may be prepared by methods using one or more nucleic acid oligonucleotides. For example, in certain embodiments, the DNA nanostructure is a DNA rectangle nanostructure, self-assembled from single-stranded DNA molecules using “staple strands.” In some embodiments, the DNA nanostructure has the shape of a flat sheet (e.g., rectangle), tube, triangle, or gridiron form. In some embodiments, the DNA nanostructure is a DNA origami. In some embodiments, the DNA nanostructure is a double-stranded DNA sequence.

[093] The length of the single stranded DNA scaffold strand is variable and depends on, for example, the type of nanostructure. In certain embodiments, the DNA scaffold strand is comprised of multiple oligonucleotide strands. In certain embodiments, the DNA scaffold strand is comprised of a single oligonucleotide strand. In certain embodiments, the DNA scaffold strand is about 1000 nucleotides in length to about 10000 nucleotides in length. In some embodiments, thbe DNA scaffold strand is M13 phage DNA. [094] In some embodiments, the staple strand nucleic acids can be synthesized de novo using any of a number of oligonucleotide synthesis methods procedures. For example, the cyanoethyl phosphoramidite method (Beaucage, S. L., and Caruthers, M. H., Tet. Let. 22:1859, 1981 ); nucleoside H-phosphonate method (Garegg et al., Tet. Let. 27:4051 -4054, 1986; Froehler et al., Nucl. Acid. Res. 14:5399-5407, 1986; Garegg et al., Tet. Let. 27:4055-4058, 1986, Gaffney et al., Tet. Let. 29:2619-2622, 1988). These chemistries can be performed by a variety of commercial oligonucleotide synthesizers, including the use of an in vitro transcription method. Alternatively, the staple strand oligonucleotide sequences can be obtained from commercial vendors (e.g., IDT Technologies, Coralville, IA).

[095] In some embodiments, the DNA nanostructure has increased nuclease resistance (e.g., as compared to a control, such as an unfolded ssDNA molecule comprising the same nucleic acid sequence as the DNA nanostructure). In certain embodiments, nuclease resistance of the DNA nanostructure is 10%, 20%, 30%, 40%, 50% , 60%, 70%, 80%, 90%, 95% or more than a control.

[096] In some embodiments, the DNA nanostructure is assembled using a single stranded DNA molecule as an initial scaffold. In certain embodiments, the DNA nanostructure comprises both single stranded and double stranded regions.

[097] In certain embodiments, the present invention provides a DNA nanostructure comprising: a single stranded DNA scaffold strand of about 5000 to 10,000 bases in length; a plurality of staple strands of DNA of about 32 bases in length, wherein each staple strand has a unique nucleotide sequence and is hybridized to a specific position on the DNA scaffold strand, wherein the plurality of staple strands hybridized to the DNA scaffold form a rectangular sheet having a top surface and a bottom surface, and having four corners; one or more fastener strands of DNA, wherein the one or more fastener strands of DNA is capable of configuring the rectangular sheet into a tubeshaped structure; and one or more DNA capture strands, wherein each capture strand is operably linked to a therapeutic agent.

[098] In certain embodiments, the tube-shaped DNA nanostructure has a diameter of about about 19 nm. In some embodiments, the DNA nanostructures can be comprised of DNA, RNA, or both.

[099] In certain embodiments, the DNA nanostructure further comprises DNA targeting strands, wherein each targeting strand is operably linked to a targeting moiety. The targeting moiety can be selected from an antibody, nanobody, scFv, affibody, aptamer, or lectin. In some embodiments, the targeting moiety is an affibody. In some embodiments, the targeting moiety is an anti-HER2 affibody.

[0100] In certain embodiments, the DNA nanostructure further comprises DNA imaging strands, wherein each imaging strand is operably linked to an imaging agent. In certain embodiments, the imaging agent is fluorescent dye.

[0101 ] As used herein, the term “operably linked” means that the operably linked nucleic acid sequences maintain their ability to partially or completely hybridize to their complementary sequence while conjugated at either the 3’, 5’, or internally within the sequence with a modified linker base. The modified linker base is covalently conjugated via a linker to the linked moiety. The linked moiety can be an imaging agent, a targeting moiety, a therapeutic agent, or a staple strand.

[0102] As used herein, the term “staple strands” refers to short single-stranded oligonucleotides of about 20 to about 40 nucleotides in length, such as 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length, wherein one end of the staple strand hybridizes with a region of the scaffold strand, and the second end of the staple strand hybridizes with another region of the scaffold strand, thereby “stapling” the two regions of the scaffold strand. In certain embodiments, the dimension of the rectangular sheet is about 90 nmxabout 60 nmx2 nm.

[0103] As used herein, the term “polynucleotide” refers to a nucleic acid molecule containing multiple nucleotides and generally refers both to “oligonucleotides” (a polynucleotide molecule of 18-25 nucleotides in length) and polynucleotides of 26 or more nucleotides. In some embodiments, oligonucleotides comprise a length of 18-25 nucleotides (e. g., 18-mers, 19-mers, 20-mers, 21 -mers, 22-mers, 23-mers, 24-mers, or 25-mers), or medium-length polynucleotides having a length of 26 or more nucleotides (e. g„ polynucleotides of 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, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 1 10, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, or about 300 nucleotides), or long polynucleotides having a length greater than about 300 nucleotides (e. g., polynucleotides of between about 300 to about 400 nucleotides, between about 400 to about 500 nucleotides, between about 500 to about 600 nucleotides, between about 600 to about 700 nucleotides, between about 700 to about 800 nucleotides, between about 800 to about 900 nucleotides, between about 900 to about 1000 nucleotides, between about 300 to about 500 nucleotides, between about 300 to about 600 nucleotides, between about 300 to about 700 nucleotides, between about 300 to about 800 nucleotides, between about 300 to about 900 nucleotides, or about 1000 nucleotides in length, or even greater than about 1000 nucleotides in length. Where a polynucleotide is double-stranded, its length can be similarly described in terms of base pairs.

[0104] As used herein, the term “affibody” refers to an engineered small protein which can be used as a ligand towards a target protein. In some embodiments, affibodies comprise a three-helix bundle based on the scaffold of one of the IgG-binding domains of staphylococcal aureus Protein A. The affibody scaffold has excellent features as an affinity ligand to a target protein. Affibodies can comprise 58 amino acids, 13 of which are randomized to generate libraries with a large number of ligand variants. Thus, affibody libraries can be generated which comprise a multitude of protein ligands with an identical backbone and variable surface-binding properties. A single affibody amino acid sequence can be selected from said library using conventional protein production and isolation methods. Affibodies mimic monoclonal antibodies in their specific targeting, but differ from monoclocal antibodies by their small size. Affibodies can have a molecular weight as low as 6kDa, compared to the molecular weight of antibodies, which is around 150kDa. Affibodies can quickly accululate at HER2-expressing tumor cells and are also rapidly cleared in the blood because of their small size (De A, Kuppusamy G, Karri VVSR. Affibody molecules for molecular imaging and targeted drug delivery in the management of breast cancer. Int J Biol Macromol. 2018;107(Pt A): 906-919; Gebauer M, Skerra A. Engineering of binding functions into proteins. Curr Opin Biotechnol. 2019;60:230-41 ).

[0105] As used herein, the term “therapeutically effective amount,” in reference to treating a disease state/condition, refers to an amount of a therapeutic agent that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease state/condition when administered as a single dose or in multiple doses. Such effect need not be absolute to be beneficial.

[0106] The terms “treat’ and “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or decrease an undesired physiological change or disorder. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “T reatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0107] The terms “inhibiting” or “reducing” or any variation of these terms includes any measurable decrease or complete inhibition to achieve a desired result. The terms “promote” or “increase” or any variation of these terms includes any measurable increase or production of a protein or molecule to achieve a desired result.

[0108] The term “preventing” or any variation of this term means to slow, stop, or reverse progression toward a result. The prevention may be any slowing of the progression toward the result.

[0109] As used herein, the term “therapeutic agent” includes an agent that directly in or indirectly causes the prevention or decrease of an udesired physiological change or disorder. Therapeutic agents can include or exclude: shRNA, siRNA, mRNA, a protein, a small molecule (e.g., chemotherapeutic drug), a plasmid, microRNA, and combinations thereof. In some embodiments, the therapeutic agent is shRNA. In some embodiments, the shRNA is MCI-1 shRNA. In some embodiments, the MCI-1 shRNA has the following nucleotide sequence:

GUGCUACUCCAGUUCGGUUUGGCAUAUCUAAUAAUUUUCUCUUAUUAGAUAUG CCAAACCUUUUCUUGAAGGUGGCAUCAGGAAUGUUUUCUUCAUUCCUGAUGCC ACCUUC (SEQ ID NO: 236). In some embodiments, the short hairpin RNA (shRNA) can also function as short interfering RNA (siRNA) under appropriate conditions. In some embodiments, when the therapeutic agent is a nucleic acid sequence, part or all of the nucleic acid sequence can be complementary to part of the DNA nanostructure such that the therapeutic agent is “loaded” onto the DNA nanostructure by hybridization.

[0110] As used herein, the term “cancer” refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. “Cancer” as used herein refers to primary, metastatic and recurrent cancers. In some embodiments, the cancer is selected from: Adenoid Cystic Carcinoma, Adrenal Gland Cancer, Amyloidosis, Anal Cancer, Ataxia- Telangiectasia, Atypical Mole Syndrome, Basal Cell Carcinoma, Bile Duct Cancer, Birt Hogg Dube Syndrome, Bladder Cancer, Bone Cancer, Brain Tumor, Breast Cancer, Carcinoid Tumor, Cervical Cancer, Colorectal Cancer, Ductal Carcinoma, Endometrial Cancer, Esophageal Cancer, Gastric Cancer, Gastrontestinal Stromal Tumor - GIST, HER2-Positive Breast Cancer, Islet Cell Tumor Juvenile Polyposis Syndrome, Kidney Cancer, Laryngeal Cancer, Leukemia - Acute Lymphoblastic Leukemia, Leukemia - Acute Lymphocytic (ALL), Leukemia - Acute Myeloid AML, Leukemia - Adult, Leukemia - Childhood, Leukemia - Chronic Lymphocytic - CLL, Leukemia - Chronic Myeloid - CML, , Liver Cancer, Lobular Carcinoma, Lung Cancer, Lung Cancer - Small Cell, Lymphoma - Hodgkin's, Lymphoma - Non-Hodgkin's, Malignant Glioma, Melanoma, Meningioma, Multiple Myeloma, Myelodysplastic Syndrome (MDS), Nasopharyngeal Cancer, Neuroendocrine Tumor, Oral Cancer, Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Pancreatic Neuroendocrine Tumors, Parathyroid Cancer, Penile Cancer, Peritoneal Cancer, Peutz-Jeghers Syndrome, Pituitary Gland Tumor, Polycythemia Vera, Prostate Cancer, Renal Cell Carcinoma, Retinoblastoma, Salivary Gland Cancer, Sarcoma, Sarcoma - Kaposi, Skin Cancer, Small Intestine Cancer, Stomach Cancer, Testicular Cancer, Thymoma, Thyroid Cancer, Uterine (Endometrial) Cancer, Vaginal Cancer, and Wilms' Tumor. Is some embodiments, the cancer is HER2- overexpressing cancer. In some embodiments, the cancer is HER2-Positive Breast cancer.

[011 1 ] As used herein, the term “chemotherapeutic drug” can include or exclude any of the following: Adriamycin (Doxorubicin), Afinitor (Everolimus), Alecensa (Alectinib), Alimta (PEMETREXED), Aliqopa (Copanlisib), Alkeran Injection (Melphalan), Alunbrig (Brigatinib), Aredia (Pamidronate), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arzerra (Ofatumumab), Avastin (Bevacizumab), Bavencio (Avelumab), Beleodaq (Belinostat), Besponsa (Inotuzumab Ozogamicin), Bexxar (Tositumomab), BiCNU (Carmustine), Blenoxane (Bleomycin), Bosulif (Bosutinib), Braftovi (Encorafenib), Busulfex (Busulfan), Cabometyx (Cabozantinib), Calquence (Acalabrutinib), Camptosar (Irinotecan), Caprelsa (Vandetanib), Casodex (Bicalutamide), CeeNU (Lomustine), CeeNU Dose Pack (Lomustine), Cerubidine (Daunorubicin), cisplatin, Cinqair (Reslizumab), Clolar (Clofarabine), Cometriq (Cabozantinib), Copiktra (Duvelisib), Cosmegen (Dactinomycin), Cotellic (Cobimetinib), CytosarU (Cytarabine), Cytoxan (Cytoxan), Cyclophosphamide, Dacogen (Decitabine), DaunoXome (Daunorubicin Lipid Complex), Daurismo (Glasdegib), Decadron (Dexamethasone), DepoCyt (Cytarabine Lipid Complex), Dexamethasone Intensol (Dexamethasone), Dexpak Taperpak (Dexamethasone), Docefrez (Docetaxel), Doxil (Doxorubicin Lipid Complex), DTIC (Decarbazine), Eligard (Leuprolide), Ellence (Ellence (epirubicin)), Eloxatin (Eloxatin (oxaliplatin)), Elspar (Asparaginase), Emcyt (Estramustine), Emend (Fosaprepitant), Empliciti (Elotzumab), Erbitux (Cetuximab), Erivedge (Vismodegib), Erleada (Apalutamide), Erwinaze (Asparaginase Erwinia chrysanthemi), Ethyol (Amifostine), Etopophos (Etoposide), Eulexin (Flutamide), Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), Femara (Letrozole), Firmagon (Degarelix), FloPred (Prednisolone), Fludara (Fludarabine), Folex (Methotrexate), Folotyn (Pralatrexate), FUDR (FUDR (floxuridine)), Gemzar (Gemcitabine), Gilotrif (Afatinib), Gleevec (Imatinib Mesylate), Halaven (Eribulin), Herceptin (Trastuzumab), Hexalen (Altretamine), Hycamtin (Topotecan), Hycamtin (Topotecan), Hydrea (Hydroxyurea), Ibrance (Palbociclib), Iclusig (Ponatinib), Idamycin PFS (Idarubicin), Idhifa (Enasidenib), Ifex (Ifosfamide), Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imlygic (Talimogene Laherparepvec), Inlyta (Axitinib), Intron A alfab (Interferon alfa-2a), Iressa (Gefitinib), Istodax (Romidepsin), Ixempra (Ixabepilone), Jakafi (Ruxolitinib), Jevtana (Cabazitaxel), Kadcyla (Ado-trastuzumab Emtansine), Keytruda (Pembrolizumab), Kisqali (Ribociclib), Kyprolis (Carfilzomib), Lanvima (Lenvatinib), Leukeran (Chlorambucil), Leukine (Sargramostim), Leustatin (Cladribine), Lorbrena (Lorlatinib), Lupron (Leuprolide), Lynparza (Olaparib), Lysodren (Mitotane), Matulane (Procarbazine), Megace (Megestrol), Mekinist (Trametinib), Mektovi (Binimetinib), Mesnex (Mesna), Mustargen (Mechlorethamine), Mutamycin (Mitomycin), Myleran (Busulfan), Mylotarg (Gemtuzumab Ozogamicin), Navelbine (Vinorelbine), Nerlynx (Neratinib), Neulasta (filgrastim), Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (Sorafenib), Nilandron (Nilandron (nilutamide)), Ninlaro (Ixazomib), Nipent (Pentostatin), Nolvadex (Tamoxifen), Odomzo (Sonidegib), Oncaspar (Pegaspargase), Oncovin (Vincristine), Opdivo (Nivolumab), Panretin (Alitretinoin), Paraplatin (Carboplatin), Perjeta (Pertuzumab), Platinol (Cisplatin), PlatinolAQ (Cisplatin), Pomalyst (Pomalidomide), Portrazza (Necitumumab), Proleukin (Aldesleukin), Purinethol (Mercaptopurine), Reclast (Zoledronic acid), Revlimid (Lenalidomide), Rituxan (Rituximab), RoferonA alfaa (Interferon alfa-2a), Rubex (Doxorubicin), Rubraca (Rucaparib), Rydapt (Midostaurin), Sandostatin (Octreotide), Soltamox (Tamoxifen), Sprycel (Dasatinib), Stivarga (Regorafenib), Sutent (Sunitinib), Sylvant (Siltuximab), Synribo (Omacetaxine), Tabloid (Thioguanine), Taflinar (Dabrafenib), Tagrisso (Osimertinib), Talzenna (Talazoparib), Tarceva (Erlotinib), Targretin Capsules (Bexarotene), Tasigna (Decarbazine), Taxol (Paclitaxel), Taxotere (Docetaxel), Tecentriq (Atezolizumab), Temodar (Temozolomide), Tepadina (Thiotepa), Thioplex (Thiotepa), Tibsovo (Ivosidenib), Toposar (Etoposide), Torisel (Temsirolimus), Treanda (Bendamustine hydrochloride), Trelstar (Triptorelin), Tykerb (lapatinib), Unituxin (Dinutuximab), Valstar (Valrubicin), Varubi (Rolapitant), Vectibix (Panitumumab), Velban (Vinblastine), Velcade (Bortezomib), Venclexta (Venetoclax), Vepesid (Etoposide), Vepesid (Etoposide Injection), Verzenio (Abemaciclib), Vesanoid (Tretinoin), Vidaza (Azacitidine), Vincasar PFS (Vincristine), Vincrex (Vincristine), Vistogard (Uridine Triacetate), Vitrakvil (Larotrectinib), Vizimpro (Dacomitinib), Votrient (Pazopanib), Vumon (Teniposide), Wellcovorin IV (Leucovorin), Xalkori (Crizotinib), Xeloda (Capecitabine), Xospata (Gilteritinib), Xtandi (Enzalutamide), Yervoy (Ipilimumab), Yescarta (Axicabtagene), Yondelis (Trabectedin), Zaltrap (Ziv-aflibercept), Zanosar (Streptozocin), Zejula (Niraparib), Zelboraf (Vemurafenib), Zevalin (Ibritumomab Tiuxetan), Zoladex (Goserelin), Zolinza (Vorinostat), Zometa (Zoledronic acid) Zortress (Everolimus), ,Zydelig (Idelalisib), Zykadia (Ceritinib), and Zytiga (Abiraterone).

[0112] In certain embodiments, the methods of this disclosure can treat cancer in a relevant model. Mouse models for Her2-expressing cancers to which the methods of this disclosure are expected to demonstrate the treatment of cancer are described in Herreros- Villanueva et al., Mouse models of pancreatic cancer World J Gastroenterol. (2012) Mar 28; 18(12): 1286-1294. doi: 10.3748/wjg.v18.i12.1286, PubMed ID: 22493542).

[0113] As used herein, the term “subject” refers to mammals. In some embodiments, the mammal includes humans, higher non-human primates, domestic, cows, mice, rabbits, rats, other rodents, horses, pigs, sheep, dogs and cats. In one embodiment, the subject is a human.

[0114] DHA-DOG design and characterization

[0115] Rationally designed DNA nanostructures enable the programmable engineering to assemble these multiple entities and combinations thereof. Described herein are DNA nanostructure compositions comprising disulfide and HER2 affibody modified DNA nanostructure (DHA-DOG) which can efficiently induce fast cytosolic uptake of delivered therapeutic agents to targeted cancer cells. The disulfide units can induce fast cytosolic uptake by interaction with cellular membrane-linked cysteine moieties. The HER2 affibody serves both as a targeting domain and as a regulator to bring disulfide units on the DNA nanostructure to the cell surface thiol groups in close proximity which can vastly increase the reaction rate of disulfide exchange and hence subsequent cytosolic uptake.

[0116] In some embodiments, this disclosure provides for a cytosolic uptake composition which comprises a DHA-DOG and a pharmaceutically acceptable carrier.

[0117] In some embodiments, the nanostructured nucleic acid platforms of this disclosure comprise a targeting moiety. The targeting moiety can be a HER2 affibody. In some embodiments, the amino acid sequence of the HER2 affibody is: VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQAPK CGGSMSENLYFQHHHHHHHH (SEQ ID NO: 234).

[0118] With the HER2 affibody ability to target and bind overexpressed HER2 on cancer cells affords the delivery of the of the nanostructured nucleic acid platform to the cancer cell. The nucleic acid platforms comprising a HER2 affibody can exhibit diffused distribution over the cells instead of forming large localized punctate bright spots which has always been a problem for conventional drug delivery vehicles. In some embodiments, the nucleic acid platforms of this disclosure comprose DHA-DOG. DHA- DOG has high cytosolic uptake in HER2 overexpressed cancer cells. Therefore, the DHA-DOG is an appropriate platform for the cytosolic delivery of a large variety of therapeutic agents for treating versatile diseases with overexpressed HER2 on the cell surface.

[0119] There are three major components in the DHA-DOG design (FIG. 1 A). First, to deliver cargo solely to the cytosol of targeted cancer cells in a highly controlled way, self-assembled DNA nanostructure (which can include or exclude DNA origami) is chosen to be the platform due to its biocompatibility, programmability, highly controllable size, shape and surface modification. Second, disulfide units are modified on DNA nanostructure to mediate rapid cytosolic uptake. It has been reported that disulfide modified oligonucleotides can be efficiently internalized in the cytoplasm within 10 mins through disulfide exchange reactions with the thiol groups on the cell membrane. Third, HER2 affibody serves both as targeting module and as regulator to bring surface thiol group and disulfide units on DNA nanostructure into proximity which will dramatically increase the disulfide exchange rate and mediate fast cytosolic uptake to the targeted cancer cell. The DHA-DOG therefore has the ability to deliver cargo (e.g., a therapeutic agent) directly to the cytoplasm of the targeted cells efficiently. Following the process (FIG. 1 B): 1 , DHA-DOG target to the targeted cancer cell membrane by HER2 affibody, and then bring disulfide units on DNA origami and cell surface thiol group into proximity; 2, disulfide units on DNA nanostructure easily react with surface thiol groups by dynamic covalent disulfide exchange; 3, followed by direct translocation through micellar pores; and 4, internal release by disulfide exchange with glutathione (GSH) in the cytoplasm.

[0120] Methods of Treatment Using cytosolic uptake composition

[0121 ] Many diseases originate from changes in biological processes at the nanoscale level, including gene mutation, protein misfolding, and virus or bacterial infections. These molecules are often nanometers in size and might be protected by barriers in nanometer size. The aim of nanomedicine is to diagnose and treat diseases at the molecular level by utilizing the unique characteristics and physical properties of nanomaterials. While there has been great progress in the development of nanocarriers for drug delivery, the safe and rapid delivery of drugs to targeted cells in the cytosol remains a challenge. Two critical problems faced by most current strategies are the lack of target specificity and compromised therapeutic effect due to the late endosomal/lysosomal entrapment. To overcome these challenges, we developed a rapid cytosolic drug delivery system to target cancer cells and deep tissue. The cytosolic uptake compositions described herein are based on a DNA nanostructure template and modified with two important modules: a cytosolic uptake module (disulfide units) and a targeting and regulating module (HER2 affibody). This system is widely applicable to other targeted cell types by changing the targeting module. In contrast to conventional intracellular delivery strategies that mostly rely on endocytosis-dependent pathways, the synergistic effect of the cytosolic uptake compositions of this disclosure enables direct translocation of the DNA nanocarrier to the cytoplasm of targeted cells and tissues.

[0122] The synergistic effect of disulfide and HER2 affibody modification on the DNA nanostructure template results in rapid cytosolic uptake and excellent tumor penetration ability for targeted drug delivery, leading to high levels of therapeutic efficacy. The combination of the unique properties of disulfide units and HER2 affibody onto the biocompatible and programmable DNA nanostructure template enables cytosolic delivery to targeted cells. The disulfide modified DNA nanostructure displayed a tubular shape due to the hydrophobic effect of the disulfide protection groups. HER2 affibody modification endowed the DNA nanocarrier with target specificity to HER2 overexpressed breast cancer cells, leading to the adjacence of disulfide units and cell surface thiol groups, followed by an accelerated disulfide exchange reaction. DNA nanostructure modified with both disulfide units and HER2 affibody demonstrated significantly higher cellular uptake efficiency than with single module modification or without modification. Distinct stages of internalization process were observed from a time-course study, although the precise entry process is still under investigation, cytosolic uptake compositions of this disclosure exhibited a diffused distribution inside the cytoplasm, which solves the challenging problem of using DNA nanostructure as a drug delivery platform. Access to deep tissue has always been challenging, as it requires a balance combination between transcytosis and cytosolic release. The excellent tumor penetration capacity and the mechanism study for the cytosolic uptake compositions of this disclosure further supports the thiol-mediated direct translocation. [0123] The cytosolic uptake compositions can be used as a drug delivery system that can deliver therapeutic agents rapidly and directly to the cytoplasm of targeted cells and deep tissue to treat a disease. The delivery of payloads to the cytoplasm enables treatment for a a variety of diseases associated with cytosolic defects (Bareford LM, Swaan PW. Endocytic mechanisms for targeted drug delivery. Adv Drug Deliv Rev. 2007 Aug 10;59(8):748-58. doi: 10.1016/j.addr.2007.06.008. Epub 2007 Jun 28. PMID: 17659804; PMCID: PMC2000329). Appropriate selection of the targeting moiety (e.g., cell-penetrating peptide, disulfide moiety, or receptor mimic) therefore allows for controlled delivery to the lysosomes or endosomes to alleviate conditions associated with these individual organelles to treat a disease. In some embodiments, the cytosolic uptake compositions of this disclosure can be used to treat a disease selected from Alzheimer’s Disease, a Lysosomal Storage disease, or cancer. In some embodiments, the cancer type can be those as described herein. In some embodiments, the cancer is a solid tumor cancer. The Lysosomal Storage disease can be selected from: GM1 gangliosidosis, Wolman disease, Niemann-Pick A, Niemann-Pick B, , Niemann-Pick C1 and C2 (perinatal), Alfa 1 antitrypsin deficiency Tyrosinemia, Idiopathic neonatal cholestasis, Congenital infections, Niemann-Pick A, Gaucher disease, Niemann-Pick C1 and C2 (Childhood), GM 2 gangliosidosis, Other LSDs, Organic acidemias, Maple syrup urine disease, Mitochondrial disorders, Wilson Disease, ADHD, Dystonias, Niemann- Pick C1 and C2 (Adult), Progressive supranuclear palsy, Frontotemporal dementia, Tay Sachs Disease, Sandhoff disease, Cerebral Neuronal Lipofuscinosis(CLN), Leigh syndrome, Niemann-Pick, CLN(Juvenile and Adult types), Leigh syndrome, Gaucher Disease, Niemann-Pick diseases, Pompe disease, Hurler disease(MPS I), Fabry Disease, Schindler disease, Gaucher disease, Fucosidosis, Erythromelalgia, Metachromatic leukodystrophy(MLD), ASA pseudodeficiency, Multiple sulfatase deficiency(MSD), Chronic inflammatory demyelinating polyneuropathy(CIDP), Guillain Barre syndrome(GBS), Lysosomal Acid Lipase Deficiency (LALD), Wolman disease, Niemann-Pick diseases, Galactosemia, Fructosemia, Aminoacid metabolism disorder, Chamarin Dorfman syndrome, Krabbe Disease, Adrenal leukodystrophy(ALD), MLD, Canavan's leukodystrophy, Alexander disease, CLN 2 ( Late infantile Neuronal Ceroid Lipofuscinosis 2)/ [tripeptyl peptidase 1 (TPP1 ) D eficiency], Doose syndrome, Dravet syndrome, Lennox-Gestault syndrome, Lafora disease, , Unverricht-Lundborg disease, Chanelopathies( SCN1A or SCN2A) , CLN 5,6,7,8,10 diseases, Sialidosis, Galactosialidosis, Pompe disease( Glycogen Storage Disease II /GSD 2), Werdnig Hofmann syndrome (Spinal muscular atrophy I), Danon disease, Endomyocardial fibroelastosis, Other GSDs, Fascioscapulohumeral, Becker, and Duchenne muscular dystrophies, McArdle(GSD V), Hers(GSD VI) diseases, Cystinosis, Conditions causing Fanconi syndrome, Lowe syndrome, Wilson disease, Tyrosinemia type 1 , Galactosemia, GSDs, LSDs, Diabetes Insipidus, MPS IV A (Morquio A), MPS IVb, GM 1 gangliosidosis, Other MPS, Mucolipidosis, Legg Calve-Perthes disease, Spondyloepiphyseal dysplasia, MPS VI (Maroteaux-Lamy Syndrome), MPS I. II, IVa , and VII, Multiple sulfatase deficiency, Mucolipidosis, Sialidosis, MPS VII(Sly Syndrome), Mucolipidosis, MPS I (Hurler (severe), Scheie (mild), Hurler-Scheie (intermediate), MPS II and other MPS, Multiple sulfatase deficiency, l-cell disease, MPS II ( Hunter disease- Neuronopathic and Non-neuronopathic), MPS I, Mucolipidosis, Mannosidosis, or Multiple sulfatase deficiency.

[0124] Therapeutic agents

[0125] In some embodiments, the therapeutic agent can be a moiety which treats a selected disease or condition. In some embodiments, the therapeutic agent can be a chemotherapeutic drug, anticancer oligonucleotide, or anticancer nucleic acid which targets a cancer. In some embodiments, the chemotherapeutic drug can be the chemotherapeutic agents described herein.

[0126] Targeting moiety

[0127] In some embodiments, the targeting moiety can be a moiety which increases the rate at which the cytosolic uptake composition is in contact or relative proximity to a targeted cell. In some embodiments, the targeting moiety is also a regulatory molecule for the targeted cell. The targeting moiety can comprise a Her2- directed agent which targets cells overexpressing Her2 relative to housekeeping cells. In some embodiments, the targeting moiety is selected from an aptamer, affibody, antibody, scFv, or lectin which targets a part or all of Her2.

[0128] Cytosolic uptake moiety

[0129] In some embodiments, the cytosolic uptake moiety is a molecule or portion thereof which reacts with a cell-suface thiol and enables cell-surface exchange of a DNA nanostructure linked to said molecule. In some embodiments, the cytosolic uptake moiety comprises an oligonucleotide comprising one or a plurality of disulfide units. In some embodiments, the disulfide units are components of a modified oligonucleotide base. In some embodiments, there are one or a plurality of cytosolic uptake moieties in the DNA nanostructure complex.

[0130] Administration [0131 ] As described herein, methods of the invention comprise administering a DNA nanostructure composition described herein, and optionally, a therapeutic agent to a subject. Such compositions may be formulated as a pharmaceutical composition and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, intraperitoneal or topical or subcutaneous routes.

[0132] The DNA nanostructure composition may be administered intravenously or intraperitoneally by infusion or injection. Solutions of the DNA nanostructure composition or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0133] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0134] Sterile injectable solutions are prepared by incorporating the DNA nanostructure compositions of this disclosure in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0135] Useful dosages of the DNA nanostructure compositions of this disclosure can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.

[0136] The amount of the DNA nanostructure, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition cellbeing treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

[0137] The DNA nanostructure composition may be conveniently formulated in unit dosage form. In one embodiment, the invention provides a composition comprising a compound formulated in such a unit dosage form. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations. In some emodiments, the amount of the DNA nanostructure in the unit dosage form can be between 0.5 micrograms to 1000 milligrams of the DNA nanostructure.

[0138] Certain Methods

[0139] Certain embodiments of the invention also provide a method of treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a cytosolic uptake composition as described herein.

[0140] In certain embodiments, a method of the invention further comprises administering at least one therapeutic agent to the subject.

[0141 ] The at least one therapeutic agent may be administered in combination with the cytosolic uptake composition. As used herein, the phrase “in combination” refers to the simultaneous or sequential administration of the cytosolic uptake composition and the at least one therapeutic agent. For simultaneous administration, the cytosolic uptake composition and the at least one therapeutic agent may be present in a single composition or may be separate (e.g., may be administered by the same or different routes).

[0142] Certain embodiments of the invention provide a cytosolic uptake composition as described herein for use in medical therapy. [0143] Certain embodiments of the invention provide the use of a cytosolic uptake composition as described herein for the manufacture of a medicament for inducing an immune response in a subject (e.g., a mammal, such as a human).

[0144] Certain embodiments of the invention provide the use of a cytosolic uptake composition as described herein for the manufacture of a medicament for inducing an immune response in a subject (e.g., a mammal, such as a human), in combination with at least one therapeutic agent.

[0145] Certain embodiments of the invention provide a cytosolic uptake composition as described herein for inducing an immune response.

[0146] Certain embodiments of the invention provide a cytosolic uptake composition as described herein for inducing an immune response, in combination with at least one therapeutic agent.

[0147] Certain embodiments of the invention provide the use of a cytosolic uptake composition as described herein for the manufacture of a medicament for treating a disease or disorder in a subject.

[0148] Certain embodiments of the invention provide the use of cytosolic uptake composition as described herein for the manufacture of a medicament for treating a disease or disorder in a subject, in combination with at least one therapeutic agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent as described herein.

[0149] Certain embodiments of the invention provide a cytosolic uptake composition as described herein for the prophylactic or therapeutic treatment a disease or disorder.

[0150] Certain embodiments of the invention provide a cytosolic uptake composition as described herein for the prophylactic or therapeutic treatment of a disease or disorder, in combination with at least one therapeutic agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent as described herein.

[0151] In certain embodiments, the disease or disorder is a condition that requires a boost of the host immunity. In certain embodiments, the disease or disorder is a hyperproliferative disorder, such as cancer. In certain embodiments, the disease or disorder is an infectious disease.

[0152] In certain embodiments, the cancer is carcinoma, lymphoma, blastoma, sarcoma, or leukemia. In certain embodiments, the cancer is a solid tumor cancer.

[0153] In certain embodiments, the cancer is squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, renal cell carcinoma, gastrointestinal cancer, gastric cancer, esophageal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (e.g., endocrine resistant breast cancer), colon cancer, rectal cancer, lung cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, melanoma, leukemia, or head and neck cancer. In certain embodiments, the cancer is breast cancer.

[0154] Pharmaceutical Compositions

[0155] Pharmaceutical compositions of this disclosure may comprise a DNA nanostructure complex, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers including neutral buffered saline, phosphate buffered saline and the like; carbohydrates including glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids including glycine; antioxidants; chelating agents including EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of this disclosure are in one aspect formulated for intravenous administration.

[0156] Pharmaceutical compositions of this disclosure may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration is determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

[0157] Suitable pharmaceutically acceptable excipients can include or exclude phosphate buffered saline (e.g. 0.01 M phosphate, 0.138 M NaCI, 0.0027 M KCI, pH 7.4), an aqueous solution containing a mineral acid salt including a hydrochloride, a hydrobromide, a phosphate, or a sulfate, saline. Ringer's solution, a solution of glycol or ethanol, and a salt of an organic acid including an acetate, a propionate, a malonate or a benzoate. In some embodiments, an adjuvant including a wetting agent or an emulsifier, and a pH buffering agent can also be used. In some embodiments, the pharmaceutically acceptable excipients described in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991 ) is appropriately used. The composition of this disclosure is formulated into a known form suitable for parenteral administration, for example, injection or infusion. In some embodiments, the composition of this disclosure may comprise formulation additives including a suspending agent, a preservative, a stabilizer and/or a dispersant, and a preservation agent for extending a validity term during storage.

[0158] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0159] Pharmaceutical compositions for use in accordance with the present invention may be formulated using one or more physiologically acceptable carriers or excipients. Any suitable concentration of the cytosolic uptake compositions may be used, and any active pharmaceutical ingredient will be administered in an amount effective to achieve its intended purpose.

[0160] A variety of suspending fluids or carriers may be employed to suspend the cytosolic uptake compositions. Such fluids include without limitation: sterile water, saline, buffer, or complex fluids derived from growth medium or other biological fluids. Preservatives, stabilizers and antibiotics may be employed in the cytosolic uptake compositions.

[0161] Methods of making a pharmaceutical composition include admixing at least one active compound or agent, as defined above, together with one or more other pharmaceutically acceptable ingredients, such as carriers, diluents, excipients, and the like. When formulated as discrete units, such as tablets or capsule or suspension, each unit contains a predetermined amount of the active compound or agent.

[0162] Suitable formulations will depend on the method of administration. The pharmaceutical composition is preferably administered by intradermal administration, but other routes of administration include for example oral, buccal, rectal, parenteral, intramuscular, subcutaneous, intraperitoneal, transdermal, intrathecal, nasal, intracheal. The polyvalent vaccine can also be administered to the lymph nodes such as axillary, inguinal or cervial lymph nodes. The active agent may be systemic after administration or may be localized by the use of regional administration, intramural administration, or use of an implant that acts to retain the active dose at the site of implantation.

[0163] Pharmaceutical compositions described herein may be administered directly, they may also be formulated to include at least one pharmaceutically- acceptable, nontoxic carriers of diluents, adjuvants, or non-toxic, nontherapeutic, fillers, buffers, preservatives, lubricants, solubilizers, surfactants, wetting agents, masking agents, and coloring agents. Also, as described herein, such formulation may also include other active agents, for example, other therapeutic or prophylactic agents, nonimmunogenic stabilizers, excipients and the like. The compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents and detergents.

[0164] Useful dosages of cytosolic uptake compositions can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949, herein incorporated by reference.

[0165] The amount of the cytosolic uptake composition, or an active salt thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

[0166] The cytosolic uptake compositions described herein may be conveniently formulated in unit dosage form. In one embodiment, the invention provides a composition comprising a cytosolic uptake compositions formulated in such a unit dosage form. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.

EXAMPLES

[0167] Methods and Materials

[0168] Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references including: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Current Protocols in Molecular Biology (Ausebel et al., Wiley-lnterscience, 1988. New York), and PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.).

[0169] Reagents

[0170] Scaffold M13mp18 plasmid was purchased from Bayou Biolabs (USA). Staple strands and DNA oligonucleotides were purchased from Integrated DNA Technologies (USA). DNA sequence details are listed in Supplementary Table 1. Lysotracker and Hoechst 33342 were purchased from Thermo Fisher Scientific Inc (USA). Anti-MCI-1 monoclonal antibody (D35A5) was purchased from Cell Signaling Technology Inc (USA). Secondary antibody (Goat anti-Rabbit IgG antibody/HRP, AP187P and Goat anti-mouse IgG antibody/HRP, AP181 P) were obtained from MilliporeSigma (USA).

[0171 ] oxDNA simulation of DNA origami with different designed curvatures

[0172] A coarse-grained model was used to optimize the system for encapsulation of cargos in representative DNA nanostrucutres (DNA origami, “DON”). Fifteen DONs were simulated and analyzed using oxDNA2 molecular dynamics software GPU implementations. In order to cancel the electrostatic repulsion of DNA overhangs that force the DNA sheet to curve towards exposing cargos, we introduced different curvatures by left-shift crossovers of DNA origami (5, 10, 13, 15, and 18 bases). Unbiased simulations were run with no overhangs, 104 overhangs, and 189 overhangs. When running the simulations, a time step of 0.003 internal oxDNA units was used, translating to 9.09 fs. The simulations were run with a salt concentration of 1 M and a temperature of 293.15 K. An Anderson-like thermostat was applied to the simulations, ensuring the simulation was run at a constant temperature. A diffusion coefficient of 2.5 simulation units was used, which effectively enables the nanotube structures to sample timescales greater than that implied by the number of simulation steps. OxView was used to add overhang extensions to the nanostructure designs, oxDNA analysis tools preformed the mean and centroid structure analysis, and tacoxDNA converted the mean oxDNA files into PDB format for ChimeraX electrostatic surface rendering. To quantitatively analyze the curvature of the structures, umbrella sampling simulations were performed.

[0173] Agarose gel electrophoresis.

[0174] Different DONs were loaded on 1 % agarose gel for native gel electrophoresis in 1 xTAE-Mg²⁺ at 100 V for 2.5 h. The gel was stained with ethidium bromide and visualized by Gel DocTM XR+ imaging system (Bio-Rad, USA).

[0175] Negative-stain TEM and image processing

[0176] 5pl of above purified sample (2 nM) was adsorbed on a commercially supplied formvar stabilized carbon type-B, 400mesh copper grids (Ted Pella, part number 01814-F) that was glow discharged for 1 min at 15mA using a Pelco easiGlow glow-discharge system (Ted Pella, Redding, CA, USA) and stained using 5pl of a freshly prepared 2% aqueous uranyl formate solution containing 25 mM sodium hydroxide (NaOH). Samples were incubated for 60 to 90 Sec depending on sample conditions. Excess liquid was wicked away with Whatmann filter paper 1 and grids left to dry for 30- 60 min prior to imaging. Images were acquired on a Talos microscope (thermos fisher LMT) operated at 120 kV accelerating voltage using a charge-coupled device (CCD) camera at 73000x magnification. Particles were manually picked and class averaged using Relion3.0 software without CTF correction.

[0177] Serum stability test

[0178] DNA nanostructure (e.g., DNA origami) loading disulfide modified DNA was incubated with McCoy’s 5A medium supplemented with 10% FBS and 1 % penicillinstreptomycin at 37°C for 0, 10min, 30min, 1 h, 2h, 4h, 9h, 12h, 24h and 52h, respectively. The samples were loaded into 1 % agarose gel and stained with EB buffer in 1xTBE with 12.5mM Mg²⁺, running at 100V for 2.5h. Gel was observed under UV irradiation and analyzed by Imaged.

[0179] Cell culture

[0180] All cell lines were purchased from American Type Culture Collection (ATCC). HeLa cells were cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS) (ATCC, USA) and 1 % penicillinstreptomycin solution (Gibco, USA). SK-BR-3 cells were cultured in McCoy’s 5A medium supplemented with 10% FBS and 1 % penicillin-streptomycin solution. MCF-7 cells were cultured with Dulbecco’s Eagle’s medium supplemented with 10% FBS and 1 % penicillinstreptomycin solution. All the cells were cultured in a humidified incubator of 5% CO2 at 37°C. All cell experiments were based on live cells.

[0181 ] Cellular CLSM imaging

[0182] 2.0x10^A5 cells/ml of SK-BR-3 cells were seeded in p-Slide 18 wells (ibidi, USA) for 48 h. When the confluency was reaching 80-90%, different groups of DONs (3nM) were added and incubated at 37°C for different time points. Nuclear staining was performed by incubating cells with Hoechst 33342 (Invitrogen, USA) at 37°C for 25min. Late endosome and lysosome of cells were incubated with 100nM LysoTrackerTM Red DND-99 (Invitrogen, USA) at 37°C for 1 h. Cells were imaged on ZEISS LSM 880 with Airyscan. The images were analyzed by Imaged software.

[0183] Flow cytometry analysis

[0184] 2.4x10^A5 cells/ml of SK-BR-3 cells were seeded in 24-well plates for 48 h. When the confluency was reaching 80-90%, different groups of DONs (3nM) were added and incubated at 37°C for 5 h. Cells were then washed twice with PBS and collected for flow cytometry (ThermoFisher Attune NxT - Cell Analyzer, USA) analysis. Data was quantified by Flow Jo software.

[0185] Tumor spheroid formation and imaging

[0186] 1 ,200 SK-BR-3 cells were seeded per well in a 96 well round bottom ultralow attachment plate (Corning) for 5 d to form tumor spheroids. Tumoroids were incubated with FAM labeled 10nM disulfide and HER2 affibody modified DONs and FAM labeled 10nM HER2 affibody modified DONs for different time points (4 h, 8 h and 12 h, respectively). Following incubation, tumoroids were directly imaged on ZEISS LSM 880 with Airyscan and analyzed by Imaged.

[0187] Pathway inhibition experiment

[0188] 2.0x10^A5 cells/ml of SK-BR-3 cells were seeded in p-Slide 18 wells (ibidi, USA) for 48 h after passage. When the confluency was reaching 80-90%, the cells was preincubated with 1.2mM sodium iodoacetate for masking cell surface thiol at 37°C for 30 min , 10pM chlorpromazine for inhibiting clathrin-mediated endocytosis , and 25 pg/ml Nystain for inihibiting Caveolin-mediated endocytosis, at 37°C for 1 h, respectively. Afterwards, the cells were washed with PBS twice and incubated with disulfide and HER2 affibody modified DNA origami for 5h. Cells were then stained by Hoechst 33342 at 37°C for 25min and imaged on ZEISS LSM 880 with Airyscan.

[0189] Western blot assay

[0190] 2.4x10^A5 cells/ml of SK-BR-3 cells were seeded in 24-well plates for 48 h and treated with different groups of DONs for 24 h. Then, cells were lysed with cell lysis buffer (EMD Miliipore, USA). The protein concentration was measured by BCA protein assay kit (ThermoFisher Scientific, USA). 25pg protein extracts were loaded onto 10% SDS-PAGE gel and ran at 70V for 1 h and 200V for 25 min. Proteins were then transferred on nitrocellulose membrane (Bio-Rad), running at 15V for 20 min. Nonspecific binding sites were blocked by EveryBlot Blocking Buffer (Bio-Rad) for 10 min. The primary antibody for MCI-1 was incubated overnight at 4°C. After washing with TBST for 3 times, the membrane was incubated with goat anti-Rabbit IgG antibody/HRP for 1 h and imaged on Amersham ImageQuantTM 800 system (Cytiva, USA) and analyzed by Imaged.

[0191 ] Cytotoxicity assay

[0192] Hela cells were cultured in 96-well plate, and treated with DNA origami (1 nM, 3nM and 6nM) with or without disulfide modification, as well as lipofectamine for 48h. After incubation, cells were incubated with EMEM medium supplemented with 10% FBS and 10% Cell Counting Kit-8 reagent (Dojindo, Japan) for 4h. The absorption at 450nm was measured for the analysis.

[0193] Statistical analysis

[0194] The significance of data differences between groups was analyzed by two- way ANOVA, performed by GraphPad Prism 9.0 (San Diego, CA). Data values with 0.1234 (ns), 0.0332 (*P), 0.0021 (**P), 0.0002, (***P), <0.0001 (****P).

[0195] Data statistics analysis

[0196] In vitro experiments will be performed at least in triplicates, and the replicate number will be increased according to the expected level of effect or sample variation. Prism 8.0 software will be used for statistical analyses. For cross-sectional analyses of two groups, T-tests will be performed, and when greater than two groups, ANOVA with Turkey’s test will be used for multiple comparisons between all experimental groups. For interpretation of survival data, Kaplan-Meier survival analysis will be used for all survival studies, and the groups will be compared using the long-rank test.

[0197] Sample size justification for animal studies

[0198] The primary outcomes for the mouse studies are animal overall survival and biodistribution. This can be performed using 6 mice per group to assess the outcome of the overall animal survival and biodistribution, including four doses and three-time points. The DNA or RNA nanostructure compositions will be assembled with near-IR fluorescent dyes and imaged in vivo using LI-COR imaging of near-infrared fluorescence intensity. The primary analysis will compare biodistribution between the DNA nanostructure. This can be performed using 12 mice per group to assess the overall animal survival, including 8 groups and three repeats. The primary analysis will compare mouse weight, animal overall survival, and tissue IHC of organs between the control and DNA nanostructure treatment group using a linear mixed-effects model, with an interaction term to determine whether the anticancer effect differs from the effect of either treatment alone. To justify the sample size, consider the difference between the treated and control groups at the end of the experiment. A sample size of 12 mice per group gives a power of 89%. This sample size justification assumes a single comparison at the end of the study. Survival will be analyzed using Kaplan-Meier curves with a Cox proportional hazards model to assess treatment effects (and their interaction). To investigate potential sex-based differences, the activity of the treatments using data disaggregated by sex will be compared.

[0199] Example 1. Preparation of a Representative DHA-DOG [0200] To prepare the DHA-DOG (FIG. 2A), a linear tertbutyl disulfide with 6 repeats was introduced to the 5’ end of single strand DNA, HER2 affibody was conjugated to single strand DNA through a cross-linker sulfo-SMCC, and DNA origami with 24-helices was synthesized. One representative HER2 affibody is commercially available (Abeam PN ab31889), but other HER2 affibodies can be used. The extended complementary strands on the DNA origami were then able to hybridize with disulfide modified DNA strands and HER2 affibody modified DNA strands, allowing them to be anchored onto the surface of DNA nanostructured complex. MCL-1 belongs to the anti- apoptotic protein of Bcl-2 family whose knockdown will induce cell apoptosis. Therefore, MCL-1 shRNA is a representative therapeutic moiety to target cancer cells and introduce tumor death. MCL-1 shRNAs were also hybridized onto the DHA-DOG by anchor strand hybridization, where the MCL-1 -shRNA comprises a single stranded RNA portion which is complementary to a selected region of the DNA nanostructured complex. Each component can be loaded onto DNA nanostructured complex as characterized by agarose gel electrophoresis (FIG. 2A). Interestingly, the morphology of disulfide modified DNA nanostructured complex is tubular under transmission electron microscopy (TEM) and atomic force microscopy (AFM) (Fl. 2B, FIG. 2C). The hydrophobicity introduced by tertbutyl protection group of the disulfide units therefore is causative of the tubular morphology retention. The hydrophobicity can also protect the therapeutic agents inside of the tubular structure to some extent, and can further improve the cellular uptake by increasing the interaction between cell membrane and DHA-DOG. Collectively, these results demonstrated the successful self-assembly of a DHA-DOG, and its loading potential with a cargo moieties (e.g., therapeutic agent, targeting moiety, and disulfide moiety).

[0201 ] HER2 affibody carrier synthesis

[0202] The HER2 affibody-DNA was synthesized by attaching amine-DNA to HER2 affibody molecules expressed from E. coli cells through cross-linker sulfo-SMCC (sulfosuccinimidYL-4-[N-maleimidomethyl]cyclohexane-1 -carboxylate). HER2 affibody- DNA was purified by FPLC and characterized by SDS-PAGE (FIG. 16).

[0203] The HER2 affibody was recombinantly expressed in E. Coli from the following plasmid nucleotide sequence:

CCATGGGTTGACAACAAATTCAACAAAGAAATGCGTAACGCGTACTGGGAAATCG CGCTGCTGCCGAACCTGAACAACCAGCAGAAACGTGCGTTCATCCGTTCTCTGTA CGACGACCCGTCCCAGTCTGCGAACCTGCTGGCGGAAGCGAAAAAACTGAACGA CGCGCAGGCGCCGAAATGCGGTGGTTCTATGTCTGAAAACCTGTACTTCCAGCAC CACCACCATCACCACCACCACTAACTCGAG (SEQ ID NO: 237).

[0204] The created HER2 affibody had the following amino acid sequence: VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQAPK CGGSMSENLYFQHHHHHHHH (SEQ ID NO:234).

[0205] In particular, HER2 affibody production was performed using the following method:

[0206] Preparation of DNA-HER2 affibody conjugate

[0207] The plasmid of HER2 affibody

(VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPS- QSANLLAEAKKLNDAQAPKCGGSMSENLYFQHHHHHHHH (SEQ ID NO:234)) was purchased from Bio Basic company (Canada). The HER2 affibody was expressed in BL21 competent E. coli cells and purified by Ni-NTA column and characterized by SDS- PAGE.

[0208] A amine-modified DNA strand (/5AmMC6/TCAGCATTCTAATAGCAGCT) (SEQ ID NO:235) was treated with 40-fold excess of 200mM sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1 -carboxylate), followed by adding 1 M NaHCO3 to adjust pH to around 8.0 and the mixture was gently shaken for overnight at room temperature. The DNA-sulfo-SMCC conjugate was purified by ethanol precipitation to remove the excess sulfo-SMCC. Before conjugation, HER2 affibody was treated with 8-fold excess of 100mM TCEP-HCI (Tris(2-carboxyethyl)phosphine hydrochloride) for 2h at room temperature with gentle shaking, followed by washing with 1 xPBS, and then using NAP-10 column to remove excess TCEP-HCI. A 4-fold excess of DNA-sulfo-SMCC conjugate was mixed with TCEP-HCI treated HER2 affibody for overnight at room temperature with gentle mixing. To purify HER2-DNA conjugate, the sample was first run through an anion exchange column (Mono QTM 4.6/100PE, Cytiva, USA) by fast protein liquid chromatography (FPLC) to remove excess HER2 affibody and then run through Ni-NTA column to remove excess DNA. Purified HER2-DNA conjugate was dialyzed with 1 xPBS overnight at 4°C and characterized by SDS-PAGE (FIG. 16)

[0209] 6SS-DNA and HER2 affibody-DNA were loaded onto a DNA nanosheet through DNA-DNA hybridization with extended overhangs on DNA nanosheet. After assembly, cytosolic uptake composition was characterized by native agarose gel electrophoresis. Discrete bands with expected mobility shift were observed for nanostructures without disulfide modification, suggesting the formation of designed nanostructures. However, 6SS-DNA loaded DON moved faster than 0SS-DNA loaded DON and has a dimer band, indicating a change in morphology (FIG. 17). This was further verified by transmission electron microscopy (TEM). Most DONs showed rectangular morphology, while approximately 15% displayed tubular structure. Interestingly, all DON exhibited tubular shape after disulfide modification, with around 30% dimer, which was attributed to the hydrophobic effect introduced by the tertbutyl protection groups of disulfide unit (FIG. 1 C, FIGs. 18-21 ). This morphology can protect payloads inside of the tubular structure to some extent, and the hydrophobic effect can also improve cellular uptake by increasing the interaction between cell membrane and the cyto-direct DNA nanodevice. Importantly, the designed curvature helped avoid the formation of aggregates. Tubular disulfide modified DON unfolded in the presence of the surfactant Tween 80 from AFM images, suggesting the tubular conformation was induced by hydrophobic actuation (FIG. 21 A, FIG. 21 B). Overall, these results demonstrated the successful self-assembly of the cyto-direct DNA nanodevice.

[0210] Synthesis of MCI-1 shRNA

[021 1 ] The DNA template of MCI-1 shRNA was purchased from IDT and purified by denaturing PAGE. MCI-1 shRNA

(rGrUrGrCrUrArCrUrCrCrArGrUrUrCrGrGrUrUrUrGrGrCrArUrArUrCrUrArArU- rArArUrUrUrUrCrUrCrUrUrArUrUrArGrArUrArUrGrCrCrArArArCrCrUrUrUrUrCrUrllrGrA rArGrGrUrGrGrCrArUrCrArGrGrArArUrGrUrUrUrUrCrUrUrCrArUrUrCrCrUrGrArUrGrCr CrArCrCrUrUrC) (SEQ ID NO:236) was transcribed by transcription kit (5x ribomax buffer, NTPs mix, SUPERase IN, PPase, DNA template of MCI-1 , T7 pol, T7 promoter and distilled water) and incubated at 30°C overnight. Obtained shRNA was purified by denaturing PAGE.

[0212] Self-assembly of DNA origami and hybridization of different functional elements

[0213] DNA origami were prepared by mixing scaffold DNA (p7249) with 5-fold staple strands in the buffer containing 40mM of Tris base, 20mM acetic acid, 12.5mM magnesium acetate, at pH 8.3. The mixture was then annealed with thermocycler (Life Technologies, USA), annealing from 95°C to 25°C in 10.5h. Excessive staples were removed by using 100 KDa Amicon centrifugal filters (EMD Miliipore, USA). 1.3-fold HER2 affibody DNA strands were loaded onto purified DNA origami by incubating the mixture at 35°C for 2h, followed by adding disulfide modified DNA strands under the same hybridization condition.

[0214] To investigate whether 0SS-DNA or 6SS-DON can be quantitively loaded onto DON, fluorescence spectroscopy was performed to measure the fluorescence intensity of DNA origami. DNA origami was synthesized with different numbers of capture staple strands (0, 26, 51 , 101 and 189) and then loaded with the corresponding amount of FAM-labeled OSS-DNA or 6SS-DNA. Each DNA origami was labeled with 4 Alexa Fluor 647 fluorophores to normalize the fluorescence result. Therefore, by comparing the relative fluorescence intensity of samples and expected results, the amount of DNA loaded on DNA origami could be determined. The excitation wavelength of FAM was 490nm and the excitation wavelength of Alexa Fluor 647 was 650nm. As shown in FIG. 22A, DNA origami with or without disulfide modification had a similar fluorescence intensity compared with the expected result under different amounts of DNA which indicated the amounts of OSS-DNA or 6SS-DNA loaded on DNA origami can be quantitatively controlled. The stability of disulfide modified DON was also investigated by incubating with cell complete medium (with 10% FBS), and it can remain intact for at least 24h (FIG. 22B). The cytotoxicity of disulfide modified DON was assessed by using cell counting kit-8 (CCK-8) assay (FIG. 23). No cytotoxicity to Hela cells was observed, demonstrating the disulfide modified DON’S biocompatibility. In contrast, commercial lipofectamine exhibited obvious cytotoxicity to cells (FIG. 23).

[0215] Plasmid transformation.

[0216] The plasmid of HER2 affibody was purchased from Bio Basic company. 50ng of HER2 affibody plasmid DNA was added to the BL21 competent E. coli cells and placed on ice for 30 minutes. Cells were heat shock at 42 °C for 10 seconds and then placed on ice for 5 minutes. 950 l of SOC was added to the mixture and then shaken vigorously at 37 °C for 60 minutes. Cells were then centrifuged at 1000 rpm and discarded the supernatant. Add 200ul SOC broth. Different dilutions of cells were spread onto the quartered selection plate (Kana+) and incubated overnight at 37 °C.

[0217] Express protein.

[0218] One colony was picked and inoculated in 10ml LB (with Kana) at 37 °C overnight. 10ml from overnight cells were added to 1 L LB broth. When the ODeoo reached 0.6, IPTG was added and continued for another 4-6 h incubation. Cells were harvested by centrifugation (4700 rpm for 15 minutes). Supernatant was discarded.

[0219] Protein isolation and purification.

[0220] Cell pellet was resuspended with lysis buffer (25mM Tris-HCI, 300mM NaCI, pH 8) and sonicated for 5 min and then spined down with JA-10 rotor at 4700 rpm for 30min. Supernatant was collected and filtered with a 0.45 m syringe filter. Protein was purified by Ni-NTA column and characterized by SDS-PAGE. [0221 ] The MCI-1 shRNA was prepared by the following method: The DNA template of MCI-1 shRNA was purchased from IDT and purified by denaturing PAGE. MCI-1 shRNA was transcribed by transcription kit (5x ribomax buffer, NTPs mix, SUPERase IN, PPase, DNA template of MCI-1 , T7 pol, T7 promoter and distilled water) and incubated at 30 °C overnight. Obtained shRNA was purified by denaturing PAGE.

[0222] Example 2. Demonstration of the Selectivity of DHA-DOG to HER2 overexpressed SKBR3 breast cancer cells

[0223] To investigate the selectivity of DHA-DOG, both HER2 overexpressed breast cancer cell (SKBR3) and HER2 negative breast cancer cell (MCF-7) were studied. Fluorescein (FAM) was labeled on DNA origami to track the location of DHA-DOG. The expression of HER2 protein in SK-BR-3 cells was found to be significantly higher than in MCF-7 cells (~ 20.4-fold), as examined by flow cytometry (FIG. 24). The amount of HER2 affibody (0, 1 , 3 and 5) on DON loaded with 0SS-DNA (OSS-DON) and DON loaded with 6SS-DNA (6SS-DON) was qualitatively tested by confocal laser scanning microscopy (CLSM). Three HER2 affibody on each DNA origami were found to be strong enough for targeting HER2 overexpressed cell (FIG. 2A). The targeting ability of cytosolic uptake composition (DSHAF-DON), HER2 affibody modified DNA origami (HAF-DON) and disulfide modified DNA origami (DS-DON) were compared in SK-BR-3 and MCF-7 cells by CLSM (FIG. 2B, FIG. 2C). Fluorescein (FAM) was labeled on DON to track the location of DON. Confocal imaging showed that both cyto-direct DON and HAF-DON can bind to the surface of SK-BR-3 cells, but they barely bound to MCF-7 cells, indicating the selectivity of HER2 affibody modified DON for HER2 overexpressed cells. However, without HER2 affibody modification, only negligible green fluorescent was observed, further confirming the target specificity was induced by HER2 affibody. This unprecedented advantage over other traditional delivery systems made cytosolic uptake composition much safer and more efficient for cancer treatment. In summary, these experimental results demonstrate that a representative DHA-DOG nanocarrier of this disclosure has selectivity for breast cancer cells over normal tissue.

[0224] Example 3. Demonstration of DHA-DOG uptake by SKBR3 cells

[0225] The cellular uptake DHA-DOG was demonstrated qualitatively and quantitatively by confocal laser scanning microscopy (CLSM) and flow cytometry, respectively. A time-course study (0.08h, 0.5h, 1 h, 3h, 5h, 7h, 9h and 12h) was conducted to examine the cellular uptake overtime by CLSM (FIG. 3a=A, FIG. 25). Considering that HAF-DON also bound to SK-BR-3 cells quickly (FIG. 2C), HAF-DON was used as a control to focus on the study of disulfide modification impact and eliminate the interference by HER2 affibody on the cellular uptake, cytosolic uptake composition (DSHAF-DON) began binding to SKBR3 cells in 5 minutes, and upon prolonging time to 30 min, cytosolic uptake composition accumulated more around the cell membrane and initiated the translocation process. At about 3 to 7 hours, most of the cytosolic uptake composition was released from the internal cell membrane, as observed by the decreased fluorescence intensity on the cell membrane and their distribution throughout the cytosol, which continued even after 12 hours (FIG. 25). Most of the cytosolic uptake composition was predominately distributed evenly inside of the cytoplasm while having a few punctate bright spots, which is a distinctive feature of late endo/lysosomal sequestration. In contrast, HAF-DONS were hardly up-taken inside the SK-BR-3 cells but remained on the cell membrane, further confirmed the significant influence of disulfide modification in the cellular uptake. Consistent results were also observed in flow cytometry experiment. The cellular uptake of DON, DS-DON, DSHAF-DON and cytosolic uptake composition was compared, and FAM was labeled onto the loaded 6SS or OSS modified captured strands (FIG. 3B). A 5.3-fold increase in the uptake in SK-BR-3 cells for cytosolic uptake composition was observed, compared to HAF-DON, an 1 1.3-fold increase in cytosolic uptake composition than DS-DON, and about 55-fold increase in cytosolic uptake composition than DON. These results indicated that both HER2 affibody and disulfide modification were critical to the cellular uptake. The combination of HER2 affibody and disulfide modification of DOG displayed the strongest fluorescent compared to other groups, indicating their synergistic effect. To rule out the possibility that only disulfide modified strand got internalized but not DNA origami. Four staple strands of DNA origami with another fluorophore of Alexa Fluor 647 were labelled (FIG. 3B). In agreement with FAM labeled DNA origami, a similar trend was observed in Alexa Fluor 647 labeled DNA origami, cytosolic uptake composition also showed significantly higher cellular uptake efficiency. In addition, to further illustrate the influence of disulfide distribution on DON since the hydrophobic effect of disulfide modification can lead to the morphology of DON, three different distributions of disulfide modification were designed (even distribution, central distribution, and peripheral distribution) on DON and their cellular uptake difference was measured by flow cytometry (FIG. 26A, FIG. 26B). Central and even distribution showed similar cellular uptake efficiency, while peripheral showed decreased cellular uptake efficacy. The cytosolic uptake composition was designed based on the central distribution of disulfide units. In summary, these results suggested that disulfide and HER2 affibody had a synergistic effect, and the enhanced cellular uptake was attributed to the target-induced accelerated disulfide exchange reaction. Based on CLSM observation, there are four sequential stages during the internalization of cytosolic uptake composition into SK-BR-3 cells, as illustrated in schematic model (FIG. 3C). Stage I: binding with the surface membrane by targeting the HER2 protein; Stage II: proximity-induced efficient disulfide exchange reaction; Stage III: direct translocation of DiHA-DOG to the cytoplasm; and Stage IV: GSH-assisted nanocarrier release from cell membrane.

[0226] Example 4. Demonstration of Subcellular distribution of DHA-DOG

[0227] The cellular distribution of cyto-direct DNA nanodevice, HAF-DON and DS- DON was analyzed by CLSM. SK-BR-3 cells were stained with Lysotracker™ to visualize the co-localization phenomenon of DNA origami with late endosomes and lysosomes. As depicted in FIG. 4, the fluorescence (visually observed to be green) was diffusely distributed inside the cells for the cytosolic uptake composition treated cells, with only a few punctate bright spots that were co-localized with observed red color labeled late endosome or lysosomes. This indicates that the cellular uptake of cytosolic uptake composition occurred through an endocytosis-independent pathway. In contrast, without disulfide modification, HA-DOG can hardly be internalized inside the cells but remained on the cell membrane due to the binding with cell surface HER2 protein. Moreover, DON without a targeting module was barely taken up by the cells. Altogether, these findings indicated that cytosolic uptake composition can be efficiently internalized inside the cell through endocytosis-independent pathway. The co-localization of disulfide modified DON with late endosomes and lysosomes in Hela cells was also measured (FIG. 27). Compared with OSS-DON, 6SS-DON exhibited higher cellular uptake and didn’t colocalize with late endosomes or lysosomes, indicating the endocytosis independent internalization. However, most of the fluorescence signal still behaved as punctate spots which implied the sequestrating in vesicles. Altogether, these findings suggested that the DHA-DOG is efficiently internalized inside the cell through an endocytosis-independent pathway.

[0228] Example 5. Analysis of the Internalization Mechanism of DHA-DOG

[0229] To explore the underlying mechanism of the highly efficient cellular uptake of DHA-DOG, the cells were pre-treated with different inhibitors, including cell surface thiol inhibitor (sodium iodoacetate), clathrin-mediated endocytosis inhibitor (chlorpromazine, CPZ), methyl-[3-cyclodextrin (M[3CD, a lipid raft inhibitor), and caveolin- mediated endocytosis inhibitor (nystatin, NYS). DNA nanostructures are predominantly delivered inside cells through clathrin and caveolin-mediated endocytosis pathways. Inhibition studies showed that cytosolic uptake composition exhibited thiol-mediated internalization in SK-BR-3 cells because the treatment of cells with sodium iodoacetate resulted in almost completely reduction in uptake, whereas the blockade of other pathways (i.e., caveolin, clathrin and lipid raft) did not affect cellular uptake obviously (FIG. 5). The effect of sodium iodoacetate is to mask the surface thiol group, which apparently blocks the internalization of DHA-DOG, suggesting that the mechanism of DHA-DOG uptake is thiol-mediated uptake.

[0230] Example 6. Demonstration of T umor penetration of a Representative DHA- DOG in Tumor 3D Multicellular Spheroid

[0231 ] This experiment demonstrates the tumor penetration ability of the cytosolic uptake composition in deep tissue as a drug delivery system. Thiol-mediated uptake is an intrinsic property of cytosolic delivery into deep tissue (Martinent, R. et al. Dithiolane quartets: thiol-mediated uptake enables cytosolic delivery in deep tissue. Chem Sci 12, 13922-13929 (2021 )). Multicellular spheroid has emerged as an attractive model for studying the penetration effect of nanoparticles across deep tissues and has also been applied to study the tumor penetration ability for DNA nanostructures (Wang, Y. et al. DNA Origami Penetration in Cell Spheroid Tissue Models is Enhanced by Wireframe Design. Adv Mater 33, e2008457 (2021 )). The tumor penetration ability of cytosolic uptake composition versus HAF-DON in SK-BR-3 tumor spheroids was compared in vitro. Spheroids of SK-BR-3 cells with an average size of approximately 400pm were prepared. SK-BR-3 spheroids were incubated with 3nM cytosolic uptake composition and HAF-DON for 4h, 8h and 12h, respectively (FIG. 6A, 6B). Their tumor penetration ability was tracked by 3D confocal microscopy. The 3D projection of fluorescence scanning showed a distinct distribution pattern between cytosolic uptake composition and HAF- DON. Specifically, the distribution of FAM signals derived from cytosolic uptake composition was penetrate gradually to the central region of tumor spheroid over time and predominately localized to the central region after 12h incubation, while FAM from HAF-DON confined to the marginal areas and decreased after 12h. These results together showed excellent tumor penetration ability of cyto-direct DNA nanodevice.

[0232] Example 7. Demonstration of a Therapeutic Oligonucleotide and Small Molecule Anticancer Drug Delivery by cytosolic uptake composition

[0233] To assess the intracellular drug delivery efficiency of the cytosolic uptake compositions of this disclosure, two types of anti-cancer drugs (FIG. 7A) were used as representative therapeutic agents to be included with the cytosolic uptake compositions: the therapeutic oligonucleotide MCI-1 shRNA, and a small molecular chemotherapy drug doxorubicin. MCI-1 shRNA tandem was designed to further increase the gene knockdown effect of siRNA and its effect was testified in Hela and SK-BR-3 cells transfected by lipofectamine (FIG. 28A, FIG. 28B). MCI-1 shRNA was delivered by cytosolic uptake composition through DNA-RNA hybridization. The gene knockdown effect of cytosolic uptake composition delivered MCI-1 shRNA in SK-BR-3 cells was evaluated by western blot assay. After 24h incubation with different treatment methods, MCI-1 shRNA delivered by cytosolic uptake compositionshowed the strongest gene knockdown effect than other groups (FIG. 7B). Notably, the extent of protein inhibition effect mediated by cytosolic uptake composition delivered shRNA was even greater than that achieved with commercial lipofectamine, indicating the excellent delivery efficacy of cyto-direct DNA nanodevice. shRNA delivered by DNA origami without any modification or disulfide modified DNA origami showed weak gene knockdown effect. In addition, shRNA delivered by HER2 affibody modified DNA origami showed negligible gene silencing effect, indicating the tight binding of HER2 affibody to the cell membrane HER2 protein which increases the cell surface retention time and lead to the degradation of loaded shRNA. These results demonstrated the efficient delivery of MCI-1 shRNA delivered by cytosolic uptake composition to the target SK-BR-3 cells.

[0234] In addition to demonstrating the successful delivery of shRNA, other small molecular chemotherapy drugs (as representative therapeutic agents) can also be efficiently delivered to targeted cancer cells. Doxorubicin was loaded onto the cytosolic uptake composition according to the previous report (Wang, Z. et al. A Tubular DNA Nanodevice as a siRNA/Chemo-Drug Co-delivery Vehicle for Combined Cancer Therapy. Angew Chem Int Ed Engl 60, 2594-2598 (2021 )). The amount of doxorubicin intercalation in cytosolic uptake composition was determined by measuring the absorption at 487nm. It was calculated that each DNA nanodevice contains around 1850 doxorubicin molecules from absorption analysis. The apoptosis of free doxorubicin to cytosolic uptake composition loaded doxorubicin on SK-BR-3 cell line was compared (FIG. 7C). Propidium iodide (PI) and FITC-Annexin V co-staining assay was used to evaluate the cell conditions (viable, apoptotic, or necrotic). Compared with free doxorubicin treated SK-BR-3 cells, the confocal microscopy images of cyto-direct nanodevice delivered doxorubicin treated SK-BR-3 cells showed very clear halo view of the apoptotic membranes labelled with FITC-Annexin V. Meanwhile, PI also efficiently labeled the nuclei of these cells demonstrating the damage of membrane integrity. However, most of the cells in free doxorubicin treated cells still maintained well-stretched morphology (health condition). Taken together, these results indicate that cytosolic uptake composition is capable of delivering doxorubicin (as a representative small molecule chemotherapy drug) to the target cancer cells efficiently and initiate the apoptosis process of target cancer cells.

[0235] Example 8. Synthesis of DNA-Poly disulfide

[0236] Disulfide unit modification of DNA strands was achieved by phosphoramidite chemistry. Linear tertbutyl disulfide with 6-repeats (6SS) was introduced to the 5’ end of single strand DNA. 6SS-DNA was purified by HPLC and characterized by MALDI-TOF-MS. A representative disulfide moiety of this disclosure was prepared by the following route.

[0237] Synthesis of 3-[Bis(4-methoxyphenyl)phenylmethoxy]-1 -propanol (compound 1 )

[0238] To a cooled (0-5 °C) solution containing 1 ,3-propanediol (19.02 g, 250 mmol, 20.0 eq.) in pyridine (75 mL) was added dropwise a solution containing DMT-CI (4.23 g, 12.5 mmol) in pyridine (30 mL). The mixture was stirred at r.t. under argon for 18 h. The solvent was evaporated under diminished pressure and the residue was partitioned between water (120 mL) and EtOAc (100 mL). The organic phase was washed with water (50 mL), brine (50 mL), dried (MgSO5) and evaporated under diminished pressure. The residue was purified on a silica gel column (6 x 14 cm), eluting with 1 :1 hexane-EtOAc. The product was obtained as yellow syrup: yield 4.40 g (93%). 1 H NMR (500 MHz, CDCI3) 5 7.43 - 7.38 (m, 1 H), 7.34 - 7.16 (m, 4H), 6.86 - 6.79 (m, 2H), 3.77 (s, 5H), 3.26 (t, J = 5.8 Hz, 1 H), 2.23 (s, 1 H), 1 .84 (p, J = 5.8 Hz, 1 H).

[0239] Synthesis of 3-[(2-Methyl-2-propanyl)disulfanyl]-1 -propanol (compound 2)

[0240] To a solution containing 3-mercaptopropanol (1.73 mL, 1.84 g, 20 mmol) and 2-methyl-2-propanethiol (22.4 mL, 18.0 g, 200 mmol) in absolute EtOH (10 mL) was added dropwise a solution containing iodine (5.08 g, 20 mmol) in EtOH (50 mL). The mixture was stirred under argon at r.t. for 18 h (pale yellow solution). The reaction was quenched by dropwise addition of satd. aq. NaHCO3 (100 mL), stirred for 1 h then EtOH was evaporated under diminished pressure. The residue was suspended in EtOAc (150 mL) and washed successively with 10% aq. NaHSO3 (3 x 100 mL) and brine (100 mL). The organic layer was dried (MgSO4) and evaporated under diminished pressure. The residue was purified on a silica gel column (6 x 1 1 cm), eluting with 2:1 hexane-EtOAc (700 mL) and 1 :1 hexane-EtOAc (300 mL). The disulfide was obtained as a pale yellow oil: yield 3.19 g (88%). 1 H NMR (500 MHz, CDCI3) 5 7.02 (t, J = 6.2 Hz, 2H), 6.10 (t, J = 7.1 Hz, 2H), 5.21 (tt, J = 7.2, 6.1 Hz, 2H), 4.62 (s, 10H). [0241 ] Synthesis of 3-(4,4’-Dimethoxytrityloxy)propyl-1 -[3’-[(2’-Methyl-2’- propanyl)disulfanyloxy]-propyl-l’ -(N,N-diisopropyl)]-phosphoramidite (compounds 3 and 4)

[0242] To a cooled (0-5 °C) solution containing N,N-bis-

(diisopropylamino)chlorophosphine (415 mg, 1 .55 mmol, 1.15 eq.) and triethylamine (217 uL, 157 mg, 2.2 mmol, 1.15 eq.) in anhydrous CH2CI2 (12 mL) was added dropwise a solution containing DMT-monoprotected 1 ,3-propanediol (512 mg, 1.35 mmol) in anhydrous CH2CI2 (2 mL). The resulting mixture was stirred at r.t. under argon for 1 h (or until TLC showed the reaction to be complete, hexane-EtOAc-Et3N 70:25:5). 1 H NMR (500 MHz, CDCI3) 5 7.49 - 7.37 (m, 1 H), 7.35 - 7.30 (m, 2H), 7.22 - 7.14 (m, 1 H), 6.84 - 6.77 (m, 2H), 3.78 (s, 4H), 3.66 (dt, J = 7.3, 6.4 Hz, 1 H), 3.46 (dh, J = 10.7, 6.8 Hz, 2H), 3.17 (t, J = 6.6 Hz, 1 H), 1.91 (p, J = 6.5 Hz, 1 H), 1 .10 (dd, J = 17.8, 6.8 Hz, 14H). P NMR (202 MHz, CDCI3) 5 123.24

[0243] ter-Butyldisulfide propanol (244 mg, 1.35 mmol, 1.0 eq.) in CH2CI2 (1 mL) was added to the reaction mixture followed by diisopropylammonium tetrazolide (232 mg, 1 .35 mmol, 1 .0 eq.). The mixture was stirred at r.t. for 30 min., quenched by addition of satd. aq. NaHCO3 (40 mL) and the phases separated. The organic layer was washed with brine (30 mL), water (50 mL), dried (MgSO4) and evaporated under diminished pressure. The residue was purified on a silica gel column (2.5 x 12 cm), eluting with 15:1 hexane-EtOAc containing 2% Et3N. The product was obtained as a colorless syrup: yield 494 mg (53%). 1 H NMR (500 MHz, C6D6) 5 7.76 - 7.59 (m, 2H), 7.53 - 7.44 (m, 4H), 7.20 (td, J = 7.8, 1 .8 Hz, 2H), 7.12 - 7.05 (m, 1 H), 6.85 - 6.71 (m, 4H), 3.74 - 3.52 (m, 2H), 3.33 (s, 9H), 2.87 - 2.62 (m, 2H), 2.05 - 1 .84 (m, 4H), 1 .26 - 1 .09 (m, 20H). P NMR (202 MHz, CDCI3) 5 145.61

[0244] Disulfide modified oligonucleotides synthesis

[0245] Two oligonucleotides were synthesized based on standard solid phase oligonucleotide synthesis on a controlled pore glass (CPG, 1 um). Standard DNA phosphoramidites, solid supports and additional reagents were purchased from Glen Research. The oligonucleotides were synthesized on an Applied Biosystems 3400 automated DNA/RNA synthesizer using a standard 1 .0 pmole phosphoramidite cycle of acid-catalyzed detritylation, activating and coupling, capping, and iodine oxidation. Stepwise coupling efficiencies and overall yields were determined by automated trityl cation conductivity monitoring. All [3-cyanoethyl phosphoramidite monomers were dissolved in anhydrous acetonitrile to a concentration of 0.1 M immediately prior to use. 480 mg of tert-butyldisulfidep phosphonamidite was dissolved in 3.75 ml of anhydrous CH3CN and 4 ml anhydrous dichloromethane to give a 0.09 M solution. Cleavage of the oligonucleotides from the solid support and deprotection was achieved by exposure to concentrated aqueous ammonia solution for 60 min at room temperature followed by heating in a sealed tube for 4 h at 65 °C. The cleavage solutions were diluted with water and removed the ammonia by washing with water using an amicon filter. The oligonucleotides were purified using reverse-phase HPLC and characterized by using MALDI-Mass spectrometry.

[0246] 5’- SSSS SSS CCA GCC TTC CAG CTC CTT -3’ (SEQ ID NO:233)

[0247] 5’- SSS SSS CCA GCC TTC CAG CTC CTT FAM -3 (SEQ ID NO:233)

[0248] Example 9. Demonstration of Therapeutic efficiacy of DNA nanostructured complexs of this disclosure

[0249] DNA nanostructured complexes are administered by tailvane injection as doses described herein to mouse cohorts which are subjected to Her2 expressing breast cell xenografts. Cohorts treated with the DNA nanostructured complexs of this disclosure are shown to exhibit higher overall survival, and/or burden compared to cohorts treated with vehicle alone. Further experiments can be demonstrated with mouse cohorts which are subjected to Her2 expressing gastric/gastroesophageal cancer xenografts, confirming that the DNA nanostructured complexes of this disclosure can be used to treat gastric/gastroesophageal cancer.

[0250] Although the foregoing specification and examples fully disclose and enable the present invention, they are not intended to limit the scope of the invention, which is defined by the claims appended hereto.

[0251 ] While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.

[0252] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0253] Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIMS What is claimed is:

1 . A cytosolic uptake composition comprising: a. a nanostructured nucleic acid complex; b. a cytosolic uptake moiety; c. a targeting moiety; and d. a therapeutic agent.

2 The cytosolic uptake composition of claim 1 , wherein the nanostructure nucleic acid complex is a DNA nanostructure or an RNA nanostructure.

3 The cytosolic uptake composition of claim 2, wherein the DNA nanostructure is selected from a double-stranded DNA sequence, a DNA origami, a DNA nanosheet, a DNA triangle, a DNA gridiron, or a DNA tube.

4 The cytosolic uptake composition of claim 1 , wherein the cytosolic uptake moiety is an oligonucleotide comprising a disulfide moiety.

5 The cytosolic uptake composition of claim 4, wherein the oligonucleotide comprising a disulfide moiety further comprises a nucleotide sequence which is partially complementary to a nucleotide sequence of the nanostructured nucleic acid complex.

6 The cytosolic uptake composition of claim 1 , wherein the targeting moiety is selected from an aptamer, an antibody, an affibody, an scFv, a lectin, a peptide, a molecule comprising an electrophile, a molecule comprising a nucleophile, or a combination thereof.

7 The cytosolic uptake composition of claim 6, wherein the targeting moiety is an aptamer.

8 The cytosolic uptake composition of claim 7, wherein the targeting moiety is an affibody.

9 The cytosolic uptake composition of claim 8, wherein the affibody is a HER2 targeting affibody.

10 The cytosolic uptake composition of claim 8, wherein the affibody is linked to the nanostructured nucleic acid complex by a covalent linkage through a crosslinker moiety.

1 1. The cytosolic uptake composition of claim 8, wherein the affibody is linked to the nanostructured nucleic acid complex by a covalent linkage of the affibody to an oligonucleotide, and wherein said oligonucleotide is partially or completely complementary to a portion of the nanostructured nucleic acid complex.

12. The cytosolic uptake composition of claim 1 , wherein the therapeutic agent is anti-MCI-1 shRNA or anti-bcl-xl shRNA.

13. The cytosolic uptake composition of claim 12, wherein the anti-MCI-1 shRNA or anti-bcl-xl shRNA comprises a nucleotide sequence which is partially or completely complementary to a section of the nanostructured nucleic acid complex.

14. The cytosolic uptake composition of claim 1 , wherein the therapeutic agent is a chemotherapeutic drug.

15. The cytosolic uptake composition of claim 14, wherein the chemotherapeutic drug is doxyrubicin.

16. The cytosolic uptake composition of claim 1 , further comprising an imaging agent.

17. The cytosolic uptake composition of claim 16, wherein the imaging agent comprises FAM.

18. The cytosolic uptake composition of claim 17, wherein the imaging agent is an oligonucleotide comprising FAM.

19. A method of treating cancer in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of the cytosolic uptake composition of claim 1 to said subject.

20. The method of claim 19, wherein the cancer is HER2-positive breast cancer or HER2-positive gastric/gastroesophageal cancer.