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WO2025049691A1 - Molécules de revêtement à base de peptides pour stabiliser des nanostructures d'adn et conférer une activité biologique - Google Patents

Molécules de revêtement à base de peptides pour stabiliser des nanostructures d'adn et conférer une activité biologique Download PDF

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Publication number
WO2025049691A1
WO2025049691A1 PCT/US2024/044349 US2024044349W WO2025049691A1 WO 2025049691 A1 WO2025049691 A1 WO 2025049691A1 US 2024044349 W US2024044349 W US 2024044349W WO 2025049691 A1 WO2025049691 A1 WO 2025049691A1
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Prior art keywords
peptide
dna
composition
peg
endolysosomal
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Nicholas STEPHANOPOULOS
Skyler Jennifer WRAY HENRY
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Arizona State University ASU
Arizona State University Downtown Phoenix campus
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Arizona State University ASU
Arizona State University Downtown Phoenix campus
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • DNs are promising biomedical tools, their use is limited by their stability in physiological media.
  • the self-assembly of DNs often necessitates the close packing of DNA helices, resulting in electrostatic repulsion between their negatively charged phosphate backbones.
  • high concentrations e.g., 5–20 mM
  • divalent cations such as Mg 2+
  • DNs are particularly susceptible to the activity of nucleases and risk degradation in cell media and in vivo.
  • FBS fetal bovine serum
  • a key limitation of this approach is that it is only applicable to in vitro cell culture conditions. More experimental studies must be done regarding the in vivo behavior and integrity of DNs if they are to be used for biomedical purposes. What is needed are peptide-based coating molecules for stabilizing DNA nanostructures and imparting biological activity in vitro and in vivo.
  • a nanoparticle composition comprising a DNA nanostructure (DN) functionalized with an endolysosomal escape peptide coating.
  • the DN comprises a 6-helix bundle (6HB) nanostructure.
  • the 6HB nanostructure comprises six different double-stranded DNA helices, each DNA helix comprising a nucleotide sequence having at least 90–99% identity to any one of SEQ ID NO: 1–6.
  • the 6HB nanostructure comprises six different double-stranded DNA helices, each DNA helix comprising a nucleotide sequence selected from any one of SEQ ID NO: 1–6.
  • the 6HB nanostructure is a rigid and monomeric assembly roughly 7 ⁇ 6 nm 2 in size.
  • the endolysosomal escape peptide coating comprises one or more endolysosomal escape peptides having an amino acid sequence comprising at least 90–95% identity to any one of SEQ ID NO: 7–23.
  • the endolysosomal escape peptide coating comprises one or more endolysosomal escape peptides comprising an amino acid sequence of any one of SEQ ID NO: 7–23.
  • the endolysosomal escape peptide comprises a lysine10 (K10) peptide (SEQ ID NO: 7).
  • the endolysosomal escape peptide comprises an aurein 1.2 peptide (SEQ ID NO: 16).
  • the endolysosomal escape peptide coating binds the DN through electrostatic interactions at a nitrogen/phosphate ratio of about 0.8 to about 1.5. In another aspect, the nitrogen/phosphate ratio is about 1. In another aspect, the composition is stable in intracellular lysosomal compartments for up to 24 hr of incubation. In another aspect, the composition further comprises a therapeutic agent. Another embodiment described herein is a method for improving cellular uptake of a DNA nanostructure (DN) through enhanced endolysosomal escape, the method comprising delivering to a cell a nanoparticle composition comprising a DN functionalized with an endolysosomal escape peptide coating.
  • DN DNA nanostructure
  • Another embodiment described herein is a method for enhancing the delivery of a therapeutic agent to a subject, the method comprising administering to the subject a nanoparticle composition comprising a DNA nanostructure (DN) functionalized with an endolysosomal escape peptide and the therapeutic agent.
  • Another embodiment described herein is the use of a nanoparticle composition comprising a DNA nanostructure (DN) functionalized with an endolysosomal escape peptide to enhance the delivery of a therapeutic agent to a subject.
  • DESCRIPTION OF THE DRAWINGS 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 upon request and payment of the necessary fee. FIG.
  • FIG. 1A–E show electrostatic coating strategies for in vivo DNA nanostructure stabilization.
  • FIG. 1A shows a schematic of PEGylated oligolysine coating and subsequent crosslinking by glutaraldehyde.
  • FIG. 1B shows a lock copolymer protection strategy.
  • DNA origami is degraded in the absence of polymer but protected from nucleases after polymer complexation. Decomplexation is made possible via competition with dextran sulfate.
  • FIG.1C shows a schematic view of the two types of peptoid surface coatings of octahedral DNA origami (brush-type and block-type) and their chemical structures.
  • FIG.1D shows lipid micellization of an octahedral DN with lipid-conjugated DNA handles in a solution of surfactant and lipids (liposomes, DOPC, and PEG-PE).
  • FIG. 1E shows a schematic of BSA conjugated to a synthetic binding domain (G2) and its dendritic chemical structure.
  • FIG. 2 shows a schematic depicting the general steps of Fmoc solid-phase peptide synthesis (SPPS). Deprotection, activation, and coupling occur in an iterative fashion until the full-length peptide is synthesized, at which point the peptide is cleaved from the resin.
  • FIG.3A–D show schematics of common bio-orthogonal conjugation techniques.
  • FIG.3A shows a copper-catalyzed strain-promoted azide/alkyne cycloaddition (CuAAC).
  • FIG.3B shows a strain-promoted azide/alkyne cycloaddition (SPAAC).
  • FIG.3C shows a N-hydroxysuccinimide (NHS) ester/amine reaction.
  • FIG.3D shows a maleimide/thiol “click” reaction.
  • FIG.4A–C show the design and coating of a DNA 6-helix bundle (6HB).
  • FIG.4A shows a strand diagram showing the six oligonucleotides that comprise the six-helix bundle (6HB) DN.
  • FIG. 4B shows schematics of two peptides used for coating DNs: a decalysine (“K10”) peptide and K10 flanked by two copies of the aurein 1.2 endolysosomal escape (“aurein” or “EE”) peptide.
  • FIG.4C shows schematics of the DNs coated with either the K10 peptide (top) or an 80:20 mixture of EE/K10 (bottom).
  • FIG.5 shows a schematic of the intracellular fate of coated 6-helix bundles (6HB) coated with K10-EE peptides and the cellular internalization pathways taken under differing cell culture conditions.
  • FIG.6A–D show a general scheme for the coating, design, and synthesis of K 10 -PEG 1K - EE.
  • FIG.6A shows a representative example of an origami structure with an mRNA scaffold and DNA staples.
  • FIG.6B shows two proposed synthetic routes to the production of the K 10 -PEG 1K - EE bioconjugate.
  • FIG.6C–D show chemical structures and graphical illustrations of K 10 -(PEG 1K - EE) 2 (FIG.6C) and K 10 -EE (FIG.6D).
  • FIG.7A–G show a summary of bioconjugation reactions.
  • FIG.7A shows a diagram of a SPAAC reaction scheme to produce K 10 -PEG 1K -Mal diblock polymer.
  • FIG.7B shows a schematic depicting two attempted methods of K10-DBCO production: (1) NHS ester-Lys bioconjugation and (2) SPPS attachment of HO 2 C-DBCO.
  • FIG.7C shows a representative MALDI-TOF MS of the reaction mixture from FIG.7A. Peak regions corresponding to unreacted, degraded K 10 (1679 Da) and K 10 -DBCO ( ⁇ 1950 Da) are boxed. Region corresponding to unreacted polydisperse PEG monomer is boxed ( ⁇ 1050 Da). Region in which product peaks are missing ( ⁇ 3000 Da) is boxed.
  • FIG.7D shows a diagram of a CuAAC reaction scheme to produce K 10 -PEG 1K -maleimide diblock polymer.
  • FIG.7E shows a representative MALDI-TOF MS of the reaction mixture from FIG.7D. Regions corresponding to unreacted polydisperse PEG monomer are boxed ( ⁇ 1000 and 2000 Da). Region in which product peaks are missing is boxed ( ⁇ 2650 Da).
  • FIG.7F shows a diagram of a thiol-maleimide click reaction scheme to produce N 3 -PEG 1K -EE.
  • FIG. 7G shows a representative MALDI-TOF MS of the reaction mixture from FIG.7F. Region corresponding to unreacted polydisperse PEG monomer is boxed ( ⁇ 1000 and 2000 Da). Peak corresponding to unreacted EE-Cys peptide is boxed ( ⁇ 1843 Da).
  • FIG. 8A–C show SPPS production of K 10 -PEG NK -EE.
  • FIG. 8A shows the chemical structure of K 10 -PEG NK -EE synthesized by SPPS.
  • FIG. 8B shows an illustration of the Fmoc- based SPPS route utilized to produce K 10 -PEG 1K -EE.
  • FIG. 8C shows a mass spectrum of successfully synthesized, pure K 10 -PEG 1K -EE and K 10 -PEG 2K -EE.
  • FIG. 8A–C show SPPS production of K 10 -PEG NK -EE.
  • FIG. 8A shows the chemical structure of K 10 -PEG NK -EE synthesized by SPPS.
  • FIG. 8B shows an illustration of the Fmoc- based SPPS route utilized to produce K 10 -PEG 1K -EE.
  • FIG. 8C shows a mass spectrum of successfully synthesized, pure K 10 -PEG 1K -EE and K 10 -
  • FIG. 9A–E show DNA barrel origami coating and stability.
  • FIG. 9A shows agarose gel electrophoresis (AGE) depicting fully annealed DNA barrel origami before and after purification.
  • FIG. 9B shows a TEM image of an annealed origami structure. Inset shows the design and dimensions of the barrel structure.
  • FIG.9C shows AGE depicting a barrel coated with pure K 10 - PEG 1K -EE at various nitrogen:phosphate (N:P) ratios.
  • FIG. 9D shows AGE of coated barrel origami after 0.5, 1, and 2 h of incubation with DNaseI.
  • FIG.9E shows AGE of a comparison with K 10 -EE and K 10 -PEG 5K after 1 h incubation with DNaseI.
  • FIG.10A–C show mRNA-decorated DNA origami for GFP expression.
  • FIG.10A shows a coated DNA origami-enabled mRNA delivery vehicle diagram.
  • FIG. 10B shows a square block DNA origami (SQB) design GFP-expressing mRNA strand attachment scheme.
  • FIG.10C shows transfection of RAW 264.7 and THP-1 cells by unstructured GFP mRNA linked to DNA-origami leader domain versus lipofectamine control. Bar graphs show percentage of cells exhibiting GFP fluorescence after transfection with SQB-mRNA coated with K 10 -EE for endosomal escape.
  • FIG. 11A–G show square block origami (SQB) featuring oligo-EE “patches” and K 10 - PEG 1K -EE coating for mRNA Expression.
  • FIG. 11A shows a conjugation reaction scheme for DNA-DBCO and DNA-peptide.
  • FIG. 11B–E show mass spectra of PLC-purified DNA-EE conjugate variation (N)EE-5′DNA (FIG.11B); (C)EE-5′DNA (FIG.11C); (N)EE-3′DNA (FIG.11D), and (C)EE-3′DNA (FIG. 11E).
  • FIG. 11F shows GFP expression of mRNA-SQB coated using various strategies and FIG.11G shows corresponding confocal microscope images.
  • FIG.12A–E show characterization of K 10 -(EK) 5 .
  • FIG.12A shows a diagram of K 10 -(EK) 5 , synthesized by SPPS.
  • FIG.12B shows the chemical structure of K 10 -(EK) 5 .
  • FIG.12C shows a mass spectrum depicting the correct mass (2699 Da) for K 10 -(EK) 5 .
  • FIG.12E shows an agarose electrophoresis image of K 10 -(EK) 5 -coated DNA barrel origami after 1 h and 20 h under different physiological buffer conditions and incubation at 37 °C.
  • FIG. 13A–D show antibody-oligonucleotide conjugation.
  • FIG. 13A shows the reaction scheme and conditions for antibody-DNA conjugation. Not drawn to scale.
  • FIG.13B shows SDS- PAGE images depicting stain-free protein.
  • FIG. 13C shows TAMRA fluorescence of antibody- DNA conjugates and precursors.
  • FIG.13D shows TEM micrographs of antibody-decorated DNA barrel structures. Insets show a zoomed in view of single barrel-Ab structures. White arrows indicate antibody position.
  • FIG.14A–D show square block origami featuring varying ratios of K10, K10-EE, and K10- PEG1K-EE coatings for mRNA Expression.
  • FIG. 14A shows the protocol for coating a single DNA origami sample with multiple molecules.
  • FIG.14B shows GFP expression of mRNA-SQB coated with various ratios of K10-EE to K10-PEG5K.
  • FIG.14C shows GFP expression of mRNA- SQB coated with various ratios of K10-EE, K10-PEG5K-EE, and K10-PEG5K.
  • FIG.14D shows corresponding confocal microscope images. DETAILED DESCRIPTION Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control.
  • amino acid amino acid
  • ribonucleic acid deoxyribonucleic acid
  • polynucleotide vector
  • polypeptide protein
  • Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.
  • Nucleic acids may be single stranded or double stranded or may contain portions of both double stranded and single stranded sequence.
  • the nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid thereof, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine.
  • Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.
  • variants can include, but are not limited to, those that include conservative amino acid (AA) substitutions, SNP variants, degenerate variants, and biologically active portions of a gene or nucleotide.
  • a “degenerate variant” as used herein refers to a variant that has a mutated nucleotide sequence, but still encodes the same polypeptide due to the redundancy of the genetic code.
  • nucleotide or peptide described herein may be modified, for example, to facilitate or improve identification, activity, expression, isolation, storage and/or administration, so long as such modifications do not reduce the function of the nucleotide or peptide to an unacceptable level.
  • substantially identical of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 25% sequence identity compared to a reference sequence as determined using programs known in the art (e.g., Basic Local Alignment Search Tool (BLAST)). In preferred embodiments, percent identity can be any integer from 25% to 100%.
  • BLAST Basic Local Alignment Search Tool
  • More preferred embodiments include polynucleotide sequences that have at least about: 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity compared to a reference sequence. These values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.
  • polynucleotides of the present disclosure encoding a protein or polypeptide of the present disclosure include nucleic acid sequences that have substantial identity to the nucleic acid sequences that encode the proteins or polypeptides of the present disclosure.
  • substantially identical of amino acid sequences (and of peptides or polypeptides having these amino acid sequences) means that an amino acid sequence comprises a sequence that has at least 25% sequence identity compared to a reference sequence as determined using programs known in the art (e.g., BLAST). In preferred embodiments, percent identity can be any integer from 25% to 100%.
  • More preferred embodiments include amino acid or polypeptide sequences that have at least about: 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity compared to a reference sequence.
  • Polypeptides that are “substantially identical” share amino acid sequences except that residue positions which are not identical may differ by one or more conservative amino acid changes. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Exemplary conservative amino acid substitution groups include valine-leucine-isoleucine, phenylalanine-tyrosine, lysine- arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. Accordingly, peptides, polypeptides, or proteins, encoded by the polynucleotides of the present disclosure, include amino acid sequences that have substantial identity to the amino acid sequences of the reference polypeptide sequences.
  • the terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.”
  • the present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
  • the term “a,” “an,” “the” and similar terms used in the context of the disclosure are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.
  • “a,” “an,” or “the” means “one or more” unless otherwise specified.
  • the term “or” can be conjunctive or disjunctive. As used herein, the term “and/or” refers to both the conjunctive and disjunctive. As used herein, the term “substantially” means to a great or significant extent, but not completely. As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system.
  • the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ⁇ 10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol “ ⁇ ” means “about” or “approximately.” All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1–2.0 includes 0.1, 0.2, 0.3, 0.4 .
  • the terms “active ingredient” or “active pharmaceutical ingredient” refer to a pharmaceutical agent, active ingredient, compound, or substance, compositions, or mixtures thereof, that provide a pharmacological, often beneficial, effect.
  • the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells.
  • the term “dose” denotes any form of an active ingredient formulation or composition, including cells, that contains an amount sufficient to initiate or produce a therapeutic effect with at least one or more administrations. “Formulation” and “composition” are used interchangeably herein.
  • the term “prophylaxis” refers to preventing or reducing the progression of a disorder, either to a statistically significant degree or to a degree detectable by a person of ordinary skill in the art.
  • the terms “effective amount” or “therapeutically effective amount,” refers to a substantially non-toxic, but sufficient amount of an action, agent, composition, or cell(s) being administered to a subject that will prevent, treat, or ameliorate to some extent one or more of the symptoms of the disease or condition being experienced or that the subject is susceptible to contracting. The result can be the reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
  • An effective amount may be based on factors individual to each subject, including, but not limited to, the subject’s age, size, type or extent of disease, stage of the disease, route of administration, the type or extent of supplemental therapy used, ongoing disease process, and type of treatment desired.
  • the term “subject” refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), non- human primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a primate. In one embodiment, the subject is a human. As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments.
  • the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.
  • “treatment” or “treating” refers to prophylaxis of, preventing, suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of biological process including a disorder or disease, or completely eliminating a disease.
  • a treatment may be either performed in an acute or chronic way.
  • the term “treatment” also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease.
  • “Repressing” or “ameliorating” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject after clinical appearance of such disease, disorder, or its symptoms.
  • “Prophylaxis of” or “preventing” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject prior to onset of the disease, disorder, or the symptoms thereof.
  • “Suppressing” a disease or disorder involves administering a cell, composition, or compound described herein to a subject after induction of the disease or disorder thereof but before its clinical appearance or symptoms thereof have manifest.
  • administering refers to providing, contacting, and/or delivery of an action, agent, composition, or cell(s) by any appropriate route to achieve a desired effect.
  • the term “administering” may also refer to the placement of a compound or a composition as disclosed herein into a subject by a method or route that results in at least partial localization of the compound or composition at a desired site in the subject.
  • Administration may include, but is not limited to, oral, sublingual, parenteral (e.g., intravenous, intracardiac, infusion (e.g., cardiac catheter infusion), subcutaneous, intracutaneous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional or intracranial injection), enteral, transdermal, topical, buccal, rectal, vaginal, nasal, ophthalmic, via inhalation, and implants.
  • parenteral e.g., intravenous, intracardiac, infusion (e.g., cardiac catheter infusion), subcutaneous, intracutaneous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional or intracranial injection
  • enteral transdermal
  • topical buccal
  • rectal vaginal
  • nasal ophthalmic
  • implants via inhalation, and implants.
  • the compound or composition may be in the form of solutions or suspensions for
  • the compound or composition may be in the form of capsules, gel capsules, tablets, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, or microspheres, nanospheres, lipid vesicles, or polymer vesicles allowing for controlled release.
  • the compound or composition may be in the form of an aerosol, spray, powder, lotion, cream, paste, gel, ointment, oil, suspensions, solutions, or emulsions.
  • DNA tetrahedro Some structures, such as the DNA tetrahedro, have been shown to be stable under cell culture conditions without any stabilizers, making them a favorable and often-implemented design for intracellular studies with DNs. This stability may be due to the lower-density packing of DNA helices, in concert with the relatively short length of the tetrahedral edges, which limits the available space for nuclease binding.
  • the simple geometry and small size of DNA tetrahedrons limit their capacity for multi-functionalization and internal cargo loading, necessitating methods to be developed for structural stabilization of alternative DN architectures.
  • nucleic acid vaccines and gene therapies utilize both physical methods and particulate formulations to overcome the challenges associated with their cellular delivery.
  • Physical methods including direct injection, gas decompression, sonoporation, and electroporation, are nonspecific procedures that generate mostly localized effects.
  • particle-based systems such as viral vectors, virus-like particles, conjugation to metal nanoparticles, micellization in polymers and lipids, and cell penetrating peptide (CPP) modification, offer a more targeted approach.
  • CPP cell penetrating peptide
  • Many methods for DN protection and delivery take inspiration from these established approaches designed for unstructured nucleic acids (i.e., nucleic acids whose structures are not user-defined).
  • Cationic Polymers Therapeutically oriented DN research has developed several approaches to address the challenge of DN stability in denaturing or enzymatic hydrolyzing environments.
  • Cationic block copolymers were synthesized to electrostatically adhere to and encase DNA origami structures. These polymer-coated DNs exhibited very limited cell toxicity after nine hours of incubation with A549 human epithelial cells.
  • luciferase proteins encapsulated by DNs demonstrated limited enzyme activity when coated compared to their bare counterparts due to the restriction of substrate accessibility. The extent of substrate inaccessibility was dependent upon polymer structure, suggesting that these coatings may serve future applications in tuning enzymatic reaction rates in addition to providing physiological stability.
  • the coated structures also showed a 5-fold greater half-life in vivo in murine models compared to the uncoated structures, likely due to their increased resistance to nuclease degradation and denaturation. Even greater stability (>48 h of incubation with DNase I) was achieved in a follow-up investigation in which oligolysine amines were covalently cross-linked with glutaraldehyde after adherence to DNs, which was hypothesized to decrease mobility and/or dissociation of the coating (FIG.1A). It was also shown that this crosslinked coating is internalized into cells more readily than its non-crosslinked counterpart.
  • a comparable cationic PEG- polylysine polymeric coating molecule was shown to have similar protective benefits when incubated with 10% FBS, DNase I, or in low salt concentrations (namely, 20 mM NaCl and an absence of Mg 2+ ).
  • this study illustrated that the coating can be removed from DNs via competitive complexation of polyanionic dextran sulfate (FIG.1B).
  • Molecular dynamics simulations of a non-PEGylated oligolysine decamer (K 10 ) interacting with a DNA origami rectangle showed that the peptide binds nonspecifically and reversibly to the structure, stabilizing it by binding across adjacent helices with its flexible cationic side chains.
  • cryo-EM cryo-electron microscopy
  • Peptoids are peptidomimetic N- substituted glycine oligomers that offer many of the same benefits as peptides, including biocompatibility, low-cost synthesis, and high chemical addressability, but without susceptibility to proteases.
  • a series of PEGylated peptoid molecules were designed and tested for their ability to protect octahedral DNs from biological adversaries.
  • peptoids with two different binding modes were designed: brush-type and block-type. Brush-type peptoids exhibited alternating amine (Nae) and PEG (Nte) side chains and bound to DNs with the full backbone against the DN structure.
  • the block-type peptoids were designed with Nae and Nte clustered into blocks of repeating units that resulted in binding of the Nae end of the chain to the DNs while the Nte end protruded out and away from the structure (FIG.1C). It was determined that brush-type peptoids exhibited the greatest DN protection against physiological conditions, including 1.25 mM MgCl 2 or the presence of DNase I or 10% FBS over 24 hours. Furthermore, peptoid-coated DNs conferred a 26% increase in protection of their encapsulated protein cargo when incubated with trypsin compared to uncoated DN encapsulation, demonstrating that these modifications can serve purposes beyond protection of the scaffold alone.
  • peptoid-coated DNs may serve as a multifunctional platform for biological applications.
  • Lipid and Protein-Based Coatings Although cationic polymers present a promising solution to the issue of DN stability, other methods have shown similar potential.
  • Previous studies, for example, have developed a method of lipid micellization of DNA nanostructures by utilizing lipid-DNA conjugates that anneal to external DNA handles on the structure in a surfactant solution (FIG. 1E).
  • the virus-inspired encapsulation strategy resulted in up to 85% of the encapsulated DNs being protected from DNase I digestion over 24 hours.
  • micellized DNs were decreased by two orders of magnitude compared with controls, as measured by inflammatory cytokine secretion and uptake into splenocytes.
  • lipid micellization was shown to significantly increase the biodistribution and pharmacokinetic half-life of DNs in murine models.
  • this encapsulation strategy was not explicitly tested for efficacy of DN protection in low- salt conditions, their behavior in murine models suggests that the nanostructures were not denatured at physiological salt concentrations.
  • Another concern regarding the use of DNA nanostructures as intracellular delivery vehicles is their transfection efficiency.
  • a protein-based coating was designed to enhance DN transfection and biological stability by chemically modifying bovine serum albumin (BSA) with a cationic dendrimer to enable nonspecific DNA binding (FIG.1D).
  • BSA bovine serum albumin
  • FOG.1D nonspecific DNA binding
  • This coating not only yielded a 2.5-fold enhancement of transfection into human embryonic kidney (HEK293) cells, but it notably reduced the levels of inflammatory cytokine secretion by primary mouse splenocytes.
  • HEK293 human embryonic kidney
  • Another interesting observation is that the BSA-coated structures seemed to exhibit enhanced endosome escape capabilities compared to the uncoated structures, a useful attribute if cytosolic delivery of DN therapeutic cargo is required.
  • Hybrid Biomaterials for Drug Delivery Hybrid biomaterials take inspiration from nature, often composed of synthetic materials coupled to bioactive motifs or composed of different types of biomolecules synthetically linked together.
  • the hybrid design of these materials permits considerable chemical versatility and enhanced biofunction that could not be achieved by one type of material alone.
  • the flexibility of their design allows for tuning physical, mechanical, and biological properties. Due to their versatility and biological compatibility and functionality, hybrid biomaterials are powerful tools for the design of drug delivery platforms. Many variations of these materials have been constructed to facilitate self-assembly, mitigate toxicity and off-target effects, and to enhance water solubility, specificity, cargo protection, and pharmacokinetics of delivery platforms.
  • Peptides are a common and easily synthesized component of hybrid biomaterials.
  • the 20 canonical amino acid monomers that make up these biological polymers offer excellent chemical and structural variety of the material.
  • Peptides also play a large role in many biological processes which are determined by their amino acid sequence and composition. Examples of the widespread biological functions of peptides include cell penetration, innate immune modulation, tissue targeting, antimicrobial, antioxidative, antihypertension, mineral binding, and more. Because of this, they are useful tools for imparting function to many kinds of therapeutics. Additionally, the facile incorporation of noncanonical amino acids confers additional chemical motifs for conjugation with other materials, such as DNA, proteins, or other synthetic biostable polymers, that can help modulate the function, pharmacokinetics, or safety of a therapeutic.
  • peptides are manufactured via solid-phase peptide synthesis (SPPS), as depicted in FIG.2.
  • SPPS solid-phase peptide synthesis
  • the peptide backbone is anchored onto an insoluble polymeric support resin to facilitate the C- to N-terminal stepwise addition of amino acids.
  • the resin enables a physical separation of the product from the reaction mixture without the need for cumbersome purifications after each synthetic step.
  • Synthesis begins with the piperidine deprotection of base-labile 9- fluorenylmethoxycarbonyl (Fmoc) protected rink amide resin, resulting in a reactive amide group for carbodiimide-mediated coupling with the first amino acid in the sequence.
  • Amino acid coupling refers to the formation of a peptide bond between the amine group of the growing peptide chain to the carboxy group of the next amino acid in the sequence.
  • the coupling reagent employed for peptide synthesis in this research N,N′-diisopropylcarbodiimide (DIC), is used in conjunction with the additive ethyl cyanohydroxyiminoacetate (oxyma), which suppresses product racemization and accelerates coupling.
  • Copper-Catalyzed Azide/Alkyne Cycloaddition Copper-catalyzed azide/alkyne cycloaddition (CuAAC), also known as “copper click” chemistry, has the ability to react two molecular building blocks together quickly, efficiently, and selectively under a variety of solvent conditions.
  • CuAAC Copper-catalyzed Azide/Alkyne Cycloaddition
  • CuAAC Copper-catalyzed azide/alkyne cycloaddition
  • CuAAC Copper-catalyzed azide/alkyne cycloaddition
  • the solvent for this reaction can, remarkably, be aqueous or organic, making this a suitable method for both biochemical conjugation and organic synthesis.
  • Cupric salts such as Cu 2 SO 4
  • Cu 2 SO 4 are often used as the source of copper, but must be reduced to form the Cu(I) species; one commonly used reducing agent for this purpose is sodium ascorbate.
  • ligands are often used to accelerate the reaction rate.
  • Water soluble tris- hydroxypropyltriazolylmethylamine (THPTA) in varying ratios is a popular accelerating ligand which protects biomolecules from hydrolysis by Cu(II) byproducts and intercepts oxidants formed by O 2 /Cu/ascorbate that react with amino acid residues such as histidine.
  • THPTA Water soluble tris- hydroxypropyltriazolylmethylamine
  • SPAAC can be a preferential to CuAAC for bioconjugation because it does not involve biologically toxic Cu(I) species and does not require additives to prevent unwanted byproducts.
  • SPAAC is an especially attractive route due to its simplicity, selectivity, and ability to be carried out under the aqueous physiological conditions required by most biomolecules.
  • the triazole-linked product between organic azide and cyclooctyne-functionalized biomolecules is highly stable.
  • Amine-Reactive Crosslinkers NHS Esters N-hydroxysuccinimide (NHS) esters are popular amine-specific functional groups used for bioconjugation reactions with proteins (FIG.3C).
  • CPPs cell penetrating peptides
  • CPP examples include TAT, gp41, SP, penetratin, and Aurein 1.2, to name a few.
  • Aurein 1.2 is a 13-amino acid alpha helical, antimicrobial peptide known to be a potent facilitator of cytosolic delivery by disintegrating the endosomal membrane in a detergent-like manner, termed the “carpet mechanism.”
  • a small six-helix bundle was designed (“6HB-DN,” FIG.4A) and coated with a K 10 peptide that featured the Aurein 1.2 (“EE,” for endosomal escape) sequence on both its N- and C-termini (FIG.4B–C). It was demonstrated that 6HB-DNs coated with K 10 -EE peptides successfully exit endosomal compartments in hepatic cell lines when administered under serum-free conditions.
  • K 10 -PEG-EE an alternative oligolysine-based coating for endosomal escape, termed K 10 -PEG-EE, was designed and synthesized in serum-containing cell media.
  • the modification to previous K 10 -EE designs is the poly(ethylene glycol) (PEG) moiety incorporated between the oligolysine and EE sequences (refer to FIG.6 for an overview of the coating process and proposed molecular design).
  • PEGylation is a widely used strategy in nanoparticle and therapeutic formulations.
  • PEG can be utilized for its ability to increase water solubility, decrease nonspecific protein binding (e.g., protein corona formation), reduce immunogenicity and toxicity, shield cargo from enzymatic degradation, and modify the pharmacodynamics of therapeutics.
  • PEGylated K 10 has demonstrated DN protection from nuclease degradation, a feature not observed when using K 10 alone. It is hypothesized that the smaller, more compact nature of the 6-helix bundle nanostructure is already less prone to enzymatic degradation than more large, open structures, contributing to their apparent stability even without the PEG moiety in the coating. Additionally, the brush-like EE portion of the coating molecule may further decrease nuclease accessibility to the DNA helices.
  • FIG.6A illustrates the workflow for nanostructure coating.
  • the nucleic acid structure is assembled in its native annealing buffer. Then, it is mixed with an oligolysine variant at a defined ratio of positive charges (nitrogen (N) from the amines on lysine residues) and negative charges (phosphates (P) on DNA backbones), deemed the N:P ratio.
  • N nitrogen
  • P phosphates
  • a non-limiting exemplary structure analogous to a PEG-modified version of K 10 -EE would include two copies of each PEG and EE, as depicted in FIG.6C–D. Due to the large size of these molecules, the first attempt at creating this peptide-PEG-peptide polymer includes only one copy of PEG-EE on the C-terminus of the K 10 sequence.
  • a one kilodalton PEG unit was chosen in part due to its more manageable size for synthesis compared to a five kilodalton PEG, but also because there is precedent for a K 10 -PEG 1K sufficiently protecting DNA origami in cellular experiments and even promoting greater internalization than K 10 -PEG 5K .
  • Two general synthetic routes were developed for producing K 10 -PEG 1K -EE. The first method was to generate the entire molecule on-resin using Fmoc-based SPPS, in which Fmoc- PEG 1K -COOH would be used to couple PEG to the growing amino acid chain in the same manner as normal amino acid coupling. Initial attempts to produce the product with this method failed but were later revisited.
  • the second proposed route was to conjugate the individual components together (K10, PEG, and EE) using heterobifunctional linkers for site-specific attachment (FIG. 6B).
  • the synthesis strategies for generating the disclosed K 10 -PEG-EE molecules via the bioconjugation routes are described herein. Table 1. Sequences of the DNA strands that make up the 6-helix bundle DN.
  • the DN comprises a 6-helix bundle (6HB) nanostructure.
  • the 6HB nanostructure comprises six different double-stranded DNA helices, each DNA helix comprising a nucleotide sequence having at least 90–99% identity to any one of SEQ ID NO: 1–6.
  • the 6HB nanostructure comprises six different double-stranded DNA helices, each DNA helix comprising a nucleotide sequence selected from any one of SEQ ID NO: 1–6.
  • the 6HB nanostructure is a rigid and monomeric assembly roughly 7 ⁇ 6 nm 2 in size.
  • the endolysosomal escape peptide coating comprises one or more endolysosomal escape peptides having an amino acid sequence comprising at least 90–95% identity to any one of SEQ ID NO: 7–23.
  • the endolysosomal escape peptide coating comprises one or more endolysosomal escape peptides comprising an amino acid sequence of any one of SEQ ID NO: 7–23.
  • the endolysosomal escape peptide comprises a lysine10 (K10) peptide (SEQ ID NO: 7).
  • the endolysosomal escape peptide comprises an aurein 1.2 peptide (SEQ ID NO: 16).
  • the endolysosomal escape peptide comprises a lysine10 (K10) peptide, one or more polyethyleneglycol (PEG) linkers, and an aurein 1.2 peptide.
  • the endolysosomal escape peptide comprises one or more of K10-PEG1K-EE (SEQ ID NO: 21), K10- PEG2K-EE (SEQ ID NO: 22), or K10-PEG5K-EE (SEQ ID NO: 23).
  • the diameter of the functionalized DN is from about 15 nm to about 28 nm.
  • the endolysosomal escape peptide coating binds the DN through electrostatic interactions at a nitrogen/phosphate ratio of about 0.8 to about 1.5. In another aspect, the nitrogen/phosphate ratio is about 1. In another aspect, the composition is stable in intracellular lysosomal compartments for up to 24 hr of incubation. In another aspect, the composition further comprises a therapeutic agent. Another embodiment described herein is a method for improving cellular uptake of a DNA nanostructure (DN) through enhanced endolysosomal escape, the method comprising delivering to a cell a nanoparticle composition comprising a DN functionalized with an endolysosomal escape peptide coating.
  • DN DNA nanostructure
  • endolysosomal escape efficiency is determined by a protein corona.
  • cellular uptake efficiency of the functionalized DN is linearly dependent on the cell size.
  • the cell is a hepatoblastoma cell or a hepatocellular carcinoma cell.
  • the endolysosomal escape peptide coating facilitates enhanced endolysosomal escape without concomitant disruption of a cell membrane and without cytotoxicity to the cell.
  • the composition further comprises a therapeutic agent and the method is used to deliver the therapeutic agent to a cell.
  • Another embodiment described herein is a nanoparticle composition comprising a DNA nanostructure (DN) functionalized with one or more antibodies.
  • Another embodiment described herein is a method for enhancing the delivery of a therapeutic agent to a subject, the method comprising administering to the subject a nanoparticle composition comprising a DNA nanostructure (DN) functionalized with an endolysosomal escape peptide and the therapeutic agent.
  • a nanoparticle composition comprising a DNA nanostructure (DN) functionalized with an endolysosomal escape peptide to enhance the delivery of a therapeutic agent to a subject.
  • compositions, apparata, assemblies, and methods provided are exemplary and are not intended to limit the scope of any of the disclosed embodiments. All the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations.
  • the scope of the compositions, formulations, methods, apparata, assemblies, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences described herein.
  • the compositions, formulations, apparata, assemblies, or methods described herein may omit any component or step, substitute any component or step disclosed herein, or include any component or step disclosed elsewhere herein.
  • composition of clause 1, wherein the DN comprises a 6-helix bundle (6HB) nanostructure.
  • Clause 3 The composition of clause 1 or 2, wherein the 6HB nanostructure comprises six different double-stranded DNA helices, each DNA helix having a nucleotide sequence having at least 90–99% identity to any one of SEQ ID NO: 1–6.
  • Clause 4. The composition of any one of clauses 1–3, wherein the 6HB nanostructure comprises six different double-stranded DNA helices, each DNA helix having a nucleotide sequence selected from any one of SEQ ID NO: 1–6.
  • Clause 5. The composition of any one of clauses 1–4, wherein the 6HB nanostructure is a rigid and monomeric assembly roughly 7 ⁇ 6 nm 2 in size.
  • Clause 6 The composition of any one of clauses 1–5, wherein the endolysosomal escape peptide coating comprises one or more endolysosomal escape peptides having an amino acid sequence having at least 90–95% identity to any one of SEQ ID NO: 7–23.
  • Clause 7. The composition of any one of clauses 1–6, wherein the endolysosomal escape peptide coating comprises one or more endolysosomal escape peptides having an amino acid sequence of any one of SEQ ID NO: 7–23.
  • Clause 8. The composition of any one of clauses 1–7, wherein the endolysosomal escape peptide comprises a lysine10 (K10) peptide (SEQ ID NO: 7).
  • any one of clauses 1–10, wherein the endolysosomal escape peptide comprises one or more of K10-PEG1K-EE (SEQ ID NO: 21), K10-PEG2K-EE (SEQ ID NO: 22), or K10-PEG5K-EE (SEQ ID NO: 23).
  • Clause 12 The composition of any one of clauses 1–11, wherein the diameter of the functionalized DN is from about 15 nm to about 28 nm.
  • Clause 13 The composition of any one of clauses 1–12, wherein the endolysosomal escape peptide coating binds the DN through electrostatic interactions at a nitrogen/phosphate ratio of about 0.8 to about 1.5.
  • a nanoparticle composition comprising a DNA nanostructure (DN) functionalized with one or more antibodies.
  • DN DNA nanostructure
  • Clause 24 A method for enhancing the delivery of a therapeutic agent to a subject, the method comprising administering to the subject a nanoparticle composition comprising a DNA nanostructure (DN) functionalized with an endolysosomal escape peptide and the therapeutic agent.
  • Clause 25 Use of a nanoparticle composition comprising a DNA nanostructure (DN) functionalized with an endolysosomal escape peptide to enhance the delivery of a therapeutic agent to a subject.
  • CHCA ⁇ -Cyano-4-hydroxycinnamic acid
  • HPA 3-hydroxypicolinic acid
  • DCTB ⁇ (2E)-2-Methyl-3-[4-(2-methyl-2-propanyl)phenyl]-2-propen-1- ylidene ⁇ malononitrile
  • DNs DNA barrel origami structures were annealed and purified either by 100 kDa MWCO filtration or 25–30% ethanol precipitation. Six-helix bundle nanostructures were annealed and used without further purification. DN Coating and Characterization DNs were coated as described previously. All coated DNs run on agarose gels utilized a fluorescein labeled K 10 at 20 mol % of the total K 10 concentration. Fluorescein was employed for fluorescent imaging. Barrel origami structures were electrophoresed in 2% agarose gels at 60 V for 3.5 h at 4 °C. 6-helix bundle nanostructures were electrophoresed in 1.5% agarose gels at 65 V for 60 min.
  • nuclease reaction buffer 100 mM Tris, 25 mM MgCl 2 , 1 mM CaCl 2
  • RPMI-1640 medium Gibco
  • FBS heat-inactivated at 56 ⁇ °C by the vendor
  • Peptide-DNA Conjugation Amine modified oligonucleotides from IDT (Coralville, IA), 1 ⁇ mol, were dissolved in 1 ⁇ PBS buffer (pH 7.5) and reacted with five equivalents of NHS ester-sulfo-DBCO at RT for 4 h. Another five equivalents of NHS-sulfo-DBCO was added and the mixture was incubated at RT overnight.
  • the DBCO-modified DNA was purified using reverse phase HPLC (Agilent 1220), using a Zorbax Eclipse C18 column with 50 mM triethylammonium acetate and methanol buffers.
  • a gradient of 0–70% methanol was applied over 45 min while monitoring the absorbance at both 260 and 309 nm, the peak absorbance wavelengths of the DNA and DBCO, respectively.
  • the peak displaying an absorbance at both wavelengths was collected and exchanged into water using a 3 kDa molecular weight cut off (MWCO) filter.
  • the DBCO-modified DNA was then mixed with the azidolysine-modified peptide in a 1:4 ratio (DNA:peptide) in 1 ⁇ PBS buffer (pH 7.5), and incubated at RT overnight.
  • the DNA-peptide conjugate was purified using the same HPLC method. Conjugates were characterized by MALDI-TOF MS.
  • Antibody-DNA Conjugation Briefly, 1 ⁇ mol of amine-modified oligonucleotide from IDT (Coralville, IA) was reacted with NHS-PEG 4 -N 3 (4-fold excess) in 1 ⁇ PBS (pH 7.5) at RT for 2 h. The product was purified using a 3 kDa MWCO filter. Separately, antibodies (Ab) were reacted at 1 mg/mL in 1 ⁇ PBS (pH 6.5) with NHS-sulfo-DBCO at 50 ⁇ M for 60 min at RT.
  • the product was purified and concentrated using a 30 kDa MWCO filter and the number of DBCO molecules per antibody were estimated with a UV/Vis spectrometer, as described previously. Then, azido-DNA and DBCO-Ab were reacted in 1 ⁇ PBS (pH 7.2) in a 3:1 mol ratio (DNA:Ab) for 10 h at 37 °C. Excess DNA was removed using a 30 kDa MWCO filter and ratio of DNA:Ab in the final product was estimated by UV/Vis spectrometry.
  • Ab-DNA conjugates were visualized by stain-free FastCast PAGE (BioRad) at 150 V for 1 h under either denaturing (1 ⁇ Tris/glycine/SDS, at RT) and nondenaturing PAGE (1 ⁇ TAE with 12.5 mM Mg 2+ , at 4 °C) conditions.
  • TAMRA-labelled DNA-N 3 conjugates were used for Typhoon gel scanner fluorescent identification of Ab-DNA conjugates.
  • Transmission Electron Microscopy Micrographs were taken on a Phillips CM12 transmission electron microscope from the Eyring Materials Center at Arizona State University. Samples were prepared on Formvar/Carbon 400 mesh copper grids from Ted Pella Inc. (Redding, CA) and stained with 2% uranyl acetate.
  • Example 2 Bioconjugation Synthesis The synthesis of K 10 -PEG 1K -EE via bioconjugation of peptide and PEG units proved a difficult task. As depicted in FIG.6B, the multistep process would begin by conjugating either a K 10 -DBCO or a K 10 -propargylalanine to the azido group on heterobifunctional N 3 -PEG 1K - maleimide.
  • this newly formed product would be reacted with the EE peptide (Aurein 1.2 sequence) outfitted with a cysteine on its C-terminus for later conjugation with the maleimide group of K 10 -PEG 1K -maleimide, forming K 10 -PEG 1K -EE.
  • SPAAC was the preferred method to react the PEG and K 10 units due to its presumed simplicity (FIG. 7A).
  • K 10 -DBCO must be reacted with PEG on-resin before cleavage.
  • the EE peptide was not soluble in ethanol, even at lower concentrations of ethanol or peptide.
  • DMF reactions were carried out ⁇ 10mM triethylamine acetate (TEAA) to provide a base for the reaction.
  • TEAA triethylamine acetate
  • the reducing agents used in these reactions can perform side reactions that sabotage the conjugation moieties involved.
  • DTT dithiothreitol
  • TCEP tris(2-carboxyethyl)phosphine
  • Fmoc-PEG 12 -CO 2 H Two successive additions of Fmoc-PEG 12 -CO 2 H would contribute ⁇ 1200 Da of PEG mass to the final product, very near the original target mass of 1000 Da. Furthermore, this PEG unit is of monodisperse length, facilitating product identification with mass spectrometry.
  • the anniversary attempt to synthesize K 10 -PEG 1K -EE using Fmoc-PEG 12 -CO 2 H monomers resulted in a high yield of product, as revealed by mass spectrometry (FIG.8C). Furthermore, it was easily purified using RP-HPLC.
  • K 10 -PEG 2K -EE PEG 2K includes four additions of PEG 12 , or 42 total PEG units, for an ⁇ 2.4 kDa PEG
  • PEG 1K -EE PEG 1K -EE
  • K 10 -PEG 2K -EE was designed to enhance the protection and water solubility of DNs compared to K 10 -PEG 1K -EE, given that longer PEG lengths have been associated with these traits.
  • K 30 -PEG 2K -peptide where the epitope targets either VCAM-1 or fibrin.
  • the success of this two-kilodalton PEG and peptide-based targeting to protect and deliver microRNA inhibitors to target cells supports the notion that K 10 -PEG 2K -EE holds similar potential for success.
  • a benefit of this barrel nanostructure design is the existence of both outer and inner parallel helices with defined positions to enable the complex 3D arrangement of molecules within and on the surface of the barrel. Furthermore, the barrel architecture holds the capacity for multimerization, allowing individual barrels to be coaxially stacked end-to-end in specific orientation with the addition of connector strands.
  • the DNA barrel has dimensions of 30 nm in diameter and 60 nm in height, much larger than the 6-helix bundle (6HB) nanostructure implemented in studies with K 10 -EE. Successful coating strategies using the large DNA barrel vs. the small 6HB will demonstrate the universality of this method.
  • FIG.9 shows the characterization of DNA barrel formation by agarose gel electrophoresis and negative-stain transmission electron microscopy (TEM).
  • FIG. 9C demonstrates K 10 -PEG 1K -EE/Barrel coating to varying degrees.
  • the degree of coating is determined by the ratio of positively charged oligolysine residue amines (N) to negatively charged DNA backbone phosphates (P), also known as the N:P ratio. Therefore, a higher N:P ratio designates a greater degree of coating.
  • N positively charged oligolysine residue amines
  • P DNA backbone phosphates
  • Coated structures exhibit reduced electrophoretic mobility on an agarose gel compared to their bare counterparts due to the negative charge screening by the coating molecule. Additionally, the coating tends to exclude DNA dye molecules from staining the nanostructures, necessitating the addition of 20 mol% fluorescein labelled K 10 , such that UV excitation can allow visualization both the uncoated DNA barrels via SYBR Safe and coated DNA barrels using fluorescence.
  • Example 5 Peptide Conjugates for Cytosolic Delivery of mRNA-Loaded DNA Nanostructures
  • electrostatic adherence i.e., an oligolysine-based approach
  • direction covalent linkage via chemical conjugation of the peptide to an oligonucleotide hybridized to the structure
  • This SQB as a “leader” domain tethered to “follower” domains consisting of unstructured mRNA encoding GFP (FIG. 11A).
  • Poly-dT DNA handles extrude from the surface of the SQB for hybridization of mRNAs via their 3′ poly-A tails (FIG.11B).
  • mRNA-SQB complexes have the ability to transfect cells such as RAW 264.7 macrophages and THP-1 monocytes, with a comparable efficiency to lipofectamine-complexed mRNA (FIG.10C).
  • unstructured mRNA complexed with K 10 -EE also had limited ability to transfect cells, although at a twenty-fold lower efficiency compared to tethering it to a DNA-origami leader domain (data not shown).
  • mRNA-SQB with this “first-generation” endosome escape peptide (K 10 -EE) led to significant aggregation of the particles.
  • the design parameters can be adjusted to minimize unwanted multimerization/aggregation, enabling improved transfection performance.
  • the versatility of the SQB’s multifaceted architecture was utilized when redesigning the mRNA delivery vehicle. On one face of the SQB, mRNA was hybridized to the structure. On another face, EE peptides were covalently linked to the SQB, forming an additional, spatially distinct functional peptide “patch” on the structure.
  • the conjugation strategy (FIG.11A) involved NHS ester attachment of an amine-modified oligonucleotide to NHS-sulfo-DBCO and subsequent SPAAC conjugation of this oligo-DBCO to an azide-modified Aurein 1.2 (synthesized by SPPS).
  • K 10 -PEG 5K K 10 -PEG 5K , a 1:1 ratio of K 10 -EE/K 10 -PEG 5K , and K 10 - PEG 1K -EE.
  • a 3:1 ratio of K 10 -EE/K 10 -PEG 5K was also tested, but in absence of an EE patch on the SQB.
  • K 10 -PEG 5K was used in conjunction with K 10 -EE to mitigate SQB aggregation caused by coating with this peptide.
  • K 10 -PEG 1K -EE exhibits less aggregation than K 10 -EE, obviating the need for supplementary K 10 -PEG 5K in the coating mixture.
  • K 10 -PEG 1K - EE also enhances GFP expression compared to K10-PEG 5K almost 2-fold and demonstrates similar expression levels to lipofectamine alone.
  • K 10 -PEG 2K -EE has yet to be tested, but the longer PEG moiety is likely to provide even more protection and further mitigate aggregation- related problems. It is also hypothesized that a denser coating of the EE, such as with a (PEG 2K - EE)-K 10 -(PEG 2K -EE) that features two copies of EE per molecule, could further enhance GFP expression.
  • SQB-mediated mRNA expression seems more punctate compared to lipofectamine-mediated expression, which appears dispersed throughout the cell.
  • the SQB may be processed through a different cellular internalization pathway compared to lipofectamine, or a distinct mode of cytosolic delivery. Future experiments will incorporate a nuclear stain to better visualize this differential mode of GFP expression. Endolysosomal staining would also shed a better light on the trafficking of these particles into the cytosol from these compartments.
  • the contribution of the EE patch should not be understated. It is not yet known what the cytosolic delivery capacity of the K 10 -PEG 1K -EE coating alone is. Thus far, the EE patch on the SQB improves GFP expression in all samples tested, which can be attributed to either greater cellular uptake or enhanced liberation from endosomal compartments. Thus far, these studies have also garnered insights into the way that differential peptide- DNA conjugation schemes can affect biological function. For example, there may be a preferred EE sequence orientation (e.g., the N-terminus facing outward and thus more accessible for interaction with endolysosomal membranes) for optimal performance.
  • a preferred EE sequence orientation e.g., the N-terminus facing outward and thus more accessible for interaction with endolysosomal membranes
  • PEG is susceptible to oxidation in the presence of oxygen and transition metals, which causes it to lose its antifouling properties. More importantly, there is an increasing amount of research demonstrating the generation of anti-PEG IgM antibodies in patients after their second dose of PEG-containing drugs, inducing quick clearance of these drugs by the immune system and limiting their efficacy. Thus, PEG alternatives have been developed to mitigate these issues.
  • Zwitterionic polymers which exhibit both cationic and anionic groups while maintaining a net neutral charge, have shown much potential for this purpose. Zwitterionic polymers typically demonstrate inherently low immunogenicity in addition to abating issues with nanoparticle aggregation, precipitation, and in vivo clearance.
  • oligolysine-based zwitterion-oligolysine polymer in lieu of a PEG- based oligolysine copolymer include lower immunogenicity and cytotoxicity, as well as facile and cost-efficient synthesis owing to the fact that they can be fabricated via SPPS using canonical amino acid monomers.
  • an oligolysine-based peptide with a zwitterionic region was generated using five repeats of alternating glutamine-lysine residues, termed K 10 -(EK) 5 (FIG. 12A–B).
  • K 10 -(EK) 5 was successfully synthesized on-resin, yielding a molecular weight of 2699 Da (expected 2700 Da, FIG.12C).
  • Example 7 Antibody-Oligonucleotide Conjugates for DN Tissue Targeting
  • This method utilized SPAAC to achieve the desired conjugate (FIG.13A), featuring an azide-modified oligonucleotide and DBCO-modified mAb.
  • Fc constant
  • Fv variable
  • the molar concentrations of DBCO and antibody were determined using their extinction coefficients at A 309 and A 280 , respectively, and the number of DBCO molecules per antibody were calculated by dividing the molar concentration of DBCO by the molar concentration of antibody after MWCO filtration of excess DBCO was performed. An average number of four DBCO groups per antibody were attached when 0.29 mg mAb was reacted with 50 uM NHS-sulfo-DBCO. Three different antibodies were tested with this method: human ⁇ -EGFR, human ⁇ -HER2, and IgG2a isotype control. SDS-PAGE analysis demonstrated an upward shift in mobility for Ab species successfully conjugated to DNA (FIG. 13B).
  • Fluorescent imaging revealed the presence of a TAMRA-labelled DNA band, which aligns with the shifted Ab-DNA band seen in the Stain-Free image, suggesting that the mobility shift is a result of successful DNA conjugation to the antibodies (FIG.13C).
  • the presence of multiple laddered bands, especially for the ⁇ -HER2-DNA and IgG2a isotype control-DNA suggests some antibody species were attached to multiple oligonucleotides, though the major product for each is that of a single conjugation. Reagent stoichiometry in future Ab-DNA reactions should be optimized to minimize this phenomenon.
  • nucleic acid nanostructures can be decorated and modified to suit a myriad of biological purposes.
  • the field of DNA nanotechnology has drawn inspiration from a vast pool of well-established methodologies developed for intracellular nucleic acid delivery. Because these nanostructures have the same chemical composition of unstructured DNA or RNA used for vaccines and gene therapies, they face very similar challenges when administered to cells, including the need for protection, tissue targeting, and intracellular trafficking.
  • the benefit of DNA nanostructures lies in their capability as multifunctional devices along with their addressability at the nanoscale.

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Abstract

L'invention concerne des molécules de revêtement à base de peptides pour stabiliser des nanostructures d'ADN et conférer une activité biologique. Dans un mode de réalisation, un revêtement dense de peptides le long de l'extérieur de la nanostructure d'ADN facilite l'échappement endolysosomal et améliore la distribution cellulaire. L'invention concerne également des compositions de nanoparticules comprenant une nanostructure d'ADN fonctionnalisée avec un revêtement peptidique d'échappement endolysosomal ou un ou plusieurs anticorps.
PCT/US2024/044349 2023-08-31 2024-08-29 Molécules de revêtement à base de peptides pour stabiliser des nanostructures d'adn et conférer une activité biologique Pending WO2025049691A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020034520A1 (en) * 1998-07-29 2002-03-21 Massimo Porro Vaccine for prevention of gram-negative bacterial infections and endotoxin related diseases
US20130330335A1 (en) * 2010-03-23 2013-12-12 Iogenetics, Llc Bioinformatic processes for determination of peptide binding
US20220056450A1 (en) * 2017-08-30 2022-02-24 Arizona Board Of Regents On Behalf Of Arizona State University Rna nanostructures and methods of making and using rna nanostructures
US20230181761A1 (en) * 2021-12-09 2023-06-15 Nicholas Stephanopoulos Cellular uptake of functionalized dna nanostructures

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020034520A1 (en) * 1998-07-29 2002-03-21 Massimo Porro Vaccine for prevention of gram-negative bacterial infections and endotoxin related diseases
US20130330335A1 (en) * 2010-03-23 2013-12-12 Iogenetics, Llc Bioinformatic processes for determination of peptide binding
US20220056450A1 (en) * 2017-08-30 2022-02-24 Arizona Board Of Regents On Behalf Of Arizona State University Rna nanostructures and methods of making and using rna nanostructures
US20230181761A1 (en) * 2021-12-09 2023-06-15 Nicholas Stephanopoulos Cellular uptake of functionalized dna nanostructures

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