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WO2022260678A1 - Compositions et procédés pour excipients d'administration biologiques - Google Patents

Compositions et procédés pour excipients d'administration biologiques Download PDF

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Publication number
WO2022260678A1
WO2022260678A1 PCT/US2021/037011 US2021037011W WO2022260678A1 WO 2022260678 A1 WO2022260678 A1 WO 2022260678A1 US 2021037011 W US2021037011 W US 2021037011W WO 2022260678 A1 WO2022260678 A1 WO 2022260678A1
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WO
WIPO (PCT)
Prior art keywords
lipid
mole
composition
cargo
nanoparticle
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2021/037011
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English (en)
Inventor
Mubhij AHMAD
Timothy Day
Ismail Hafez
John Merritt
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Particella Inc
Original Assignee
Dnalite Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dnalite Therapeutics Inc filed Critical Dnalite Therapeutics Inc
Priority to PCT/US2021/037011 priority Critical patent/WO2022260678A1/fr
Priority to EP21840333.5A priority patent/EP4259099A1/fr
Priority to CA3205059A priority patent/CA3205059A1/fr
Priority to US18/267,206 priority patent/US20240058454A1/en
Priority to TW110146743A priority patent/TW202241388A/zh
Priority to PCT/US2021/063182 priority patent/WO2022132678A1/fr
Publication of WO2022260678A1 publication Critical patent/WO2022260678A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0031—Rectum, anus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • 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
    • C07K16/2866—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 cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific

Definitions

  • the present disclosure provides a composition that includes a cargo and a nanoparticle, the nanoparticle including: at least one bile salt; at least one cationic lipid; at least one structural lipid; and at least one conjugated lipid, wherein the conjugated lipid is conjugated with a hydrophilic polymer.
  • the at least one bile salt may be selected from one or more of deoxycholate, lithocholate, isolithocholate, alloisolithocholate, dehydrolithocholate, ursodiol, 5beta-cholanic acid, chenodeoxycholate, cholate, taurodeoxycholate, taurochenodeoxy cholate, glycocholate, 3-oxy-cholenic acid, and hyodeoxycholate.
  • the at least one bile salt may be included in the nanoparticle at a level of from about 5 to about 40 mole % of total nanoparticle lipid.
  • the at least one bile salt may be included in the nanoparticle at a level of from about 20 to about 40 mole % of total nanoparticle lipid.
  • the at least one bile salt may be included in the nanoparticle at a level of from about 33 to about 37 mole % of total nanoparticle lipid.
  • the at least one bile salt may include deoxycholate.
  • the composition may include two bile salts. At least one of the two bile salts may include lithocholate.
  • the composition may include deoxycholate at a level of from about 20 to about 30 mole % of total nanoparticle lipid; and lithocholate at a level of from about 5 to about 10 mole % of total nanoparticle lipid.
  • the at least one cationic lipid may include Nl-[2-((lS)-l-[(3-aminopropyl)amino]-4-[di(3-amino- propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5).
  • the MVL5 may be present at a level of from about 5 to about 20 mole % of total nanoparticle lipid.
  • the at least one cationic lipid may include one or more of (6Z,9Z,28Z,31Z)-heptatriaconta- 6,9,28,3 l-tetraen-19-yl 3-(dimethylamino)propanoate (MC2); 7-(4-(dimethylamino)butyl)-7- hydroxytridecane-l,13-diyl dioleate (CL1H6); and 7-(4-(diisopropylamino)butyl)-7- hydroxytride-cane-l,13-diyl dioleate (CL4H6).
  • Each one of the at least one cationic lipid may be present at a level of from about 5 to about 20 mole % of total nanoparticle lipid.
  • the at least one structural lipid may be selected from one or more of l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC) and l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC).
  • the at least one structural lipid may be present at a level of from about 35 to about 45 mole % of total nanoparticle lipid.
  • the hydrophilic polymer may include polyethylene glycol (PEG).
  • the at least one conjugated lipid may include one or more of 1,2-dimyristoyl-rac-glycerol (DMG)-PEG and l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE)-PEG.
  • the at least one conjugated lipid may be present at a level of from about 0.5 to about 2.0 mole % of total nanoparticle lipid.
  • the molar ratio between components may be: from about 1 to about 5 of the at least one bile salt; from about 0.5 to about 3 of each one of the at least one cationic lipid; from about 2 to about 10 of the at least one structural lipid; and from about 0.02 to about 0.10 of the at least one conjugated lipid.
  • the at least one bile salt may be selected from one or more of deoxycholate, ursodiol, lithocholate, isolithocholate, alloisolithocholate, dehydrolithocholate, and 5beta-cholanic acid.
  • the at least one cationic lipid may include MVL5.
  • the at least one cationic lipid may include MC2.
  • the at least one structural lipid may include DSPC.
  • the at least one conjugated lipid may include DMG-PEG.
  • the composition may include at least one bile salt, MVL5, MC2, DSPC, and DMG-PEG at a molar ratio of about 2.592:0.96:0.96:3.168:0.768.
  • the at least one bile salt may be deoxycholate.
  • the nanoparticle may include: MVL5, MC2, DSPC, deoxycholate, and DMPE-PEG at a molar ratio of about 2.4:2.4:7.9:6.48:0.192; MVL5, CL1H6, DSPC, deoxycholate, and DMG-PEG at a molar ratio of about 2.4:2.4:7.9:6.48:0.192; MVE5, CL4H6, DSPC, deoxycholate, and DMG-PEG at a molar ratio of about 2.4:2.4:7.9:6.48:0.192; MVL5, MC2, DSPC, chenodeoxycholate, and DMG-PEG at a molar ratio of about 2.4:2.4:7.9:6.48:0.192; MVL5, MC2, DMPC, deoxycholate, and DMG-PEG at a molar ratio of about 2.4:2.4:7.9:6.4
  • the composition may include 12.4 mole % of MVL5, 12.4 mole % of MC2, 40.8 mole % of DSPC, 33.4 mole % of the at least one bile salt, and 1 mole % of the at least one conjugated lipid.
  • the at least one conjugated lipid may include DMG-PEG or DMPE-PEG.
  • the at least one bile salt may be selected from one or more of taurodeoxycholate, taurochenodeoxycholate, glycocholate, 3-oxy-cholenic acid, and deoxycholate.
  • the cargo may include one or more of a nucleic acid, a protein, an antibody, a peptide, a small molecule, a biologic, a peptidomimetic, a ribozyme, a chemical agent, a viral particle, a growth factor, a cytokine, an immunomodulating agent, and a fluorescent dye.
  • the cargo may include a nucleic acid.
  • the nucleic acid may include DNA.
  • the DNA may include plasmid DNA.
  • the present disclosure provides a composition that includes a cargo; and a nanoparticle, the nanoparticle including: a first cationic lipid that includes CL1H6 or CL4H6; an optional second cationic lipid; at least one bile salt; at least one structural lipid; and at least one conjugated lipid, wherein the at least one conjugated lipid is conjugated with a hydrophilic polymer.
  • the at least one bile salt may be selected from one or more of deoxycholate, lithocholate, isolithocholate, alloisolithocholate, dehydrolithocholate, ursodiol, 5beta-cholanic acid, chenodeoxycholate, cholate, taurodeoxycholate, taurochenodeoxycholate, glycocholate, 3-oxy-cholenic acid, and hyodeoxycholate.
  • the at least one bile salt may be included in the nanoparticle at a level of from about 5 to about 40 mole % of total nanoparticle lipid.
  • the at least one bile salt may be included in the nanoparticle at a level of from about 20 to about 40 mole % of total nanoparticle lipid.
  • the at least one bile salt may include deoxycholate.
  • the first cationic lipid may include from about 5 to about 40 mole % of the total nanoparticle lipid.
  • the nanoparticle may include a second cationic lipid, the second cationic lipid including MVL5, MC2, or DODMA.
  • the second cationic lipid may be present at a level of from about 5 to about 20 mole % of total nanoparticle lipid.
  • Each of the first cationic lipid and the second cationic lipid may be present at a level of from about 5 to about 20 mole % of total nanoparticle lipid and each may be present in an equal amount.
  • the at least one structural lipid may be selected from one or more of DSPC, DMPC, and dioleoylphosphatidylethanolamine (DOPE).
  • DOPE dioleoylphosphatidylethanolamine
  • the at least one structural lipid may be present at a level of from about 10 to about 70 mole % of total nanoparticle lipid.
  • the at least one structural lipid may be present at a level of from about 30 to about 50 mole % of total nanoparticle lipid.
  • the at least one structural lipid and the at least one bile salt may be present at a combined level of from about 50 to about 80 mole % of total nanoparticle lipid.
  • the hydrophilic polymer may include PEG.
  • the at least one conjugated lipid may include DMG-PEG.
  • the at least one conjugated lipid may be present at a level of from about 0.5 to about 2.0 mole % of total nanoparticle lipid.
  • the first cationic lipid may include CL1H6.
  • the nanoparticle may include a second cationic lipid that includes MVL5.
  • the at least one bile salt may include deoxycholate.
  • the at least one structural lipid may include DSPC.
  • the at least one conjugated lipid may include DMG-PEG.
  • CL1H6, MVL5, and DMG-PEG may be included at molar ratios of about 1 : 1 :0.08; and deoxycholate and DSPC may be included at molar ratios of from about 0.5 to about 5.0.
  • the molar ratios of deoxycholate and DSPC may be from about 2.0 to about 4.0.
  • CL1H6 may be included at a level of from about 10 to about 20 mole % of total nanoparticle lipid
  • MVL5 may be included at a level of from about 10 to about 20 mole % of total nanoparticle lipid
  • deoxycholate may be included at a level of from about 10 to about 40 mole % of total nanoparticle lipid
  • DSPC, DMPC, or DOPE may be included at a level of from about 30 to about 60 mole % of total nanoparticle lipid
  • DMG-PEG may be included at a level of from about 0.5 to about 2.0 mole % of total nanoparticle lipid.
  • the nanoparticle may include: CL1H6 and MVL5 at a level of from about 10 to about 15 mole % of total nanoparticle lipid; deoxycholate at a level of from about 20 to about 40 mole % of total nanoparticle lipid; DSPC at a level of from about 35 to about 50 mole % of total nanoparticle lipid; and DMG-PEG at a level of from about 0.75 to about 1.5 mole % of total nanoparticle lipid.
  • the nanoparticle may include: CL1H6 and MVL5 at a level of from about 12 to about 14 mole % of total nanoparticle lipid; deoxycholate at a level of from about 27 to about 38 mole % of total nanoparticle lipid;
  • the nanoparticle may include: CL1H6 and MVL5 at a level of about 12 mole % of total nanoparticle lipid; deoxycholate at a level of about 33 mole % of total nanoparticle lipid; DSPC at a level of about 41 mole % of total nanoparticle lipid; and DMG-PEG at a level of about 1 mole % of total nanoparticle lipid.
  • the hydrophilic polymer may be conjugated with a polypeptide.
  • the polypeptide may be a mucus penetrating polypeptide (MPP).
  • the MPP may include an amino acid sequence according to SEQ ID NO: 17.
  • the hydrophilic polymer may include PEG.
  • the at least one conjugated lipid may include DMG-PEG.
  • the nanoparticle may include: CL1H6 and MVL5 at a level of from about 12 to about 14 mole % of total nanoparticle lipid; deoxycholate at a level of from about 27 to about 38 mole % of total nanoparticle lipid; DSPC at a level of from about 38 to about 45 mole % of total nanoparticle lipid; and DMG-PEG at a level of from about 0.75 to about 1.5 mole % of total nanoparticle lipid.
  • the cargo may include one or more of a nucleic acid, a protein, an antibody, a peptide, a small molecule, a biologic, a peptidomimetic, a ribozyme, a chemical agent, a viral particle, a growth factor, a cytokine, an immunomodulating agent, and a fluorescent dye.
  • the cargo may include a nucleic acid.
  • the nucleic acid may include DNA.
  • the DNA may include plasmid DNA.
  • the molar ratio of total nanoparticle cationic lipids to the total number of nucleotides of the nucleic acid cargo may be from about 2 to about 20.
  • the molar ratio of total nanoparticle cationic lipids to the total number of nucleotides of the nucleic acid cargo may be from about 14 to about 18.
  • the nucleic acid may include RNA.
  • the molar ratio of total nanoparticle cationic lipids to the total number of nucleotides of the nucleic acid cargo may be from about 2 to about 20.
  • the molar ratio of total nanoparticle cationic lipids to the total number of nucleotides of the nucleic acid cargo may be from about 2 to about 4.
  • the present disclosure provides a method of delivering a cargo to a target cell, the method including contacting the target cell with a composition (e.g., a cargo and nanoparticle composition) described herein.
  • the target cell may include a human cell.
  • the target cell may include an epithelial cell.
  • the epithelial cell may include an intestinal epithelial cell.
  • the present disclosure provides a method of delivering a cargo to a target cell, wherein the target cell is part of a mucosal tissue, the method including contacting the mucosal tissue with a composition described above or herein.
  • the mucosal tissue may be part of a gastrointestinal tract.
  • the target cell may be a gastrointestinal cell.
  • the gastrointestinal cell may include one or more of an intestinal epithelial cell, a lamina basement cell, an intraepithelial lymphocyte, an intestinal muscle cell, and an enteric neuron.
  • the present disclosure provides a method of delivering a cargo to a subject, the method including introducing a composition described above or herein to the gastrointestinal tract of the subject.
  • the composition may be introduced to the subject gastrointestinal tract by administering the composition to the subject by an administration route selected from one or more of oral administration and intrarectal administration.
  • the nanoparticle may target a gastrointestinal cell.
  • the gastrointestinal cell may be selected from one or more of an intestinal epithelial cell, a lamina basement cell, an intraepithelial lymphocyte, an intestinal muscle cell, and an enteric neuron.
  • the cargo may be delivered to the gastrointestinal cell.
  • the cargo may be delivered to the intracellular space of the gastrointestinal cell.
  • the cargo, a cargo component, or an expression product of the cargo may be secreted from the gastrointestinal cell.
  • Secretion of the cargo, cargo component, or expression product of the cargo may include apical secretion or basal secretion.
  • the cargo, cargo component, or expression product of the cargo may remain in an area proximal to the cell after secretion.
  • the cargo, cargo component, or expression product of the cargo may be secreted basally from the gastrointestinal cell and enter the circulation.
  • the cargo, cargo component, or expression product of the cargo may be distributed systemically after entering the circulation.
  • the cargo may include a therapeutic agent.
  • the therapeutic agent may include one or more of a nucleic acid, a polypeptide, a protein, a biologic, an antibody, an enzyme, a hormone, a cytokine, an immunogen, and a genetic or epigenetic editing system component.
  • the therapeutic agent may include a nucleic acid.
  • the nucleic acid may encode at least one polypeptide.
  • the nucleic acid may include DNA.
  • the nucleic acid may include plasmid DNA.
  • the nanoparticle may target a gastrointestinal cell and the gastrointestinal cell may be transfected with the nucleic acid.
  • the gastrointestinal cell may express a polypeptide encoded by the nucleic acid.
  • the nucleic acid may encode a cell signaling factor.
  • the cell signaling factor may be selected from one or more of interleukin (IL)-2, IL-2 mutein Fc-fusion, IL-10, IL-10 mutein, IL-22, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), adrenomedullin, glucagon-like peptide 1 (GLP-1), glucagon-like peptide 2 (GLP-2), GLP-2 analog teduglutide, peroxisome proliferator-activated receptor gamma (PPARy), human growth hormone (HGH), parathyroid hormone (PTH), fibroblast growth factor 21 (FGF21), and relaxin.
  • IL interleukin
  • IL-2 mutein Fc-fusion IL-10
  • IL-10 mutein IL-22
  • GM-CSF granulocyte-macrophag
  • the nucleic acid may encode an antibody.
  • the antibody may bind a target selected from one or more of IL-18, IL- 18 receptor 1 (IL18R1), IL-23, tumor necrosis factor a (TNFa), proprotein convertase subtilisin kexin 9 (PCSK9), and protein 19 (PI 9).
  • the antibody may be a bispecific antibody.
  • the bispecific antibody may bind to cluster of differentiation 3 (CD3).
  • the nucleic acid may encode an antimicrobial agent.
  • the antimicrobial agent may be selected from one or more of intestinal alkaline phosphatase (LAP) and a defensin.
  • the nucleic acid may encode a genetic editing system component.
  • the nucleic acid may encode an antigen as an immunogen for promotion of an immune response to the antigen by the subject.
  • the antigen may be derived from one or more of influenza virus, SARS-CoV-2 virus, Ebola virus, and polio virus.
  • the antigen may include a tumor cell neoantigen.
  • the immune response may include development of tolerance to the antigen by the subject.
  • the antigen may be associated with one or more of peanut allergies, celiac disease, rheumatoid arthritis, and IBD.
  • the nucleic acid may encode a clotting factor.
  • the clotting factor may include Factor VFQ.
  • the nucleic acid may encode an enzyme.
  • the enzyme may include b-glucocerebrosidase (GBA).
  • the nucleic acid may be a non-coding RNA.
  • the non-coding RNA may include one or more of short interfering RNA (siRNA), microRNA (miRNA), long non-coding RNA, piwi- interacting RNA (piRNA), small nucleolar RNA (snoRNA), small Cajal body-specific RNA (scaRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and small nuclear RNA (snRNA).
  • siRNA short interfering RNA
  • miRNA microRNA
  • piRNA piwi- interacting RNA
  • small nucleolar RNA small nucleolar RNA
  • scaRNA small Cajal body-specific RNA
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • small nuclear RNA small nuclear RNA
  • the cargo may include a nucleic acid encoding a therapeutic agent.
  • the therapeutic agent may be selected from the group consisting of IL-2, IL-2 mutein Fc-fusion, IL-10, IL-10 mutein, IL-22, adrenomedullin, an anti -microbial, and an anti-inflammatory antibody.
  • the cargo may be delivered to a gastrointestinal cell.
  • the gastrointestinal cell may express the therapeutic agent.
  • the gastrointestinal cell may secrete the therapeutic agent locally.
  • the immune-related indication may include a gastrointestinal indication.
  • the gastrointestinal indication may include one or more of gastrointestinal infection, inflammatory bowel disease (IBD), ulcerative colitis, and Crohn’s disease.
  • the gastrointestinal cell may secrete the therapeutic agent into circulation.
  • the immune-related indication may include a non-gastrointestinal-specific indication and/or a systemic indication.
  • the immune-related indication may include one or more of graft versus host disease (GVHD), systemic lupus erythematosus (SLE), type I diabetes, rheumatoid arthritis, an infection, a wound, and an allergy.
  • the therapeutic indication may include a cancer-related indication.
  • the cargo may include a nucleic acid encoding a therapeutic agent.
  • the therapeutic agent may include GM-CSF.
  • the cancer-related indication may include one or more of Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, acute lymphoblastic leukemia, and acute myelogenous leukemia.
  • the subject may have received or may be undergoing chemotherapy and/or stem cell transplantation.
  • the cargo may be delivered to gastrointestinal cells and the gastrointestinal cells may secrete the GM-CSF into circulation at a level sufficient to provide a circulating GM-CSF concentration of about 250 pg/rrr/day.
  • the therapeutic indication may include neutropenia.
  • the cargo may include a nucleic acid encoding G-CSF.
  • the cargo may be delivered to gastrointestinal cells and the gastrointestinal cells may secrete the G-CSF into circulation at a level sufficient to provide about 5 pg/kg/day of the G-CSF.
  • the subject may be treated until subject neutrophil blood levels reach 1000/m1.
  • the therapeutic indication may be microvillus inclusion disease (MVID) and the cargo may include a nucleic acid encoding MY05B gene product.
  • the therapeutic indication may include cystic fibrosis and the cargo may include a nucleic acid encoding cystic fibrosis transmembrane regulator protein (CFTR).
  • the therapeutic indication may include hemophilia and the cargo may include a nucleic acid encoding a clotting factor.
  • the clotting factor may include Factor VIIT
  • the hemophilia may include hemophilia A.
  • the therapeutic indication may include Gaucher’s disease and the cargo may include a nucleic acid encoding GBA.
  • the cargo may be delivered to gastrointestinal cells and the gastrointestinal cells may secrete GBA into circulation at a level sufficient to provide steady-state GBA plasma levels of about 6 ng/mL.
  • the therapeutic indication may include short bowel syndrome (SBS) and the cargo may include a nucleic acid encoding GLP-2.
  • the cargo may be delivered to gastrointestinal cells and the gastrointestinal cells may secrete GLP-2 into circulation at a level sufficient to provide a circulating GLP-2 concentration of about 36 ng/mL.
  • the therapeutic indication may include a hormone deficiency and the cargo may include a nucleic acid encoding the deficient hormone.
  • the deficient hormone may be selected from the group consisting of HGH and PTH.
  • the deficient hormone may be HGH, the cargo may be delivered to gastrointestinal cells, and the gastrointestinal cells may secrete the HGH into circulation at a level sufficient to provide a circulating HGH concentration of from about 1 to about 10 ng/mL in adults or from about 10 to about 50 ng/mL in children.
  • the deficient hormone may be PTH, the cargo may be delivered to gastrointestinal cells, and the gastrointestinal cells may secrete the PTH into circulation at a level sufficient to provide a circulating PTH concentration of about 150 pg/mL.
  • the therapeutic indication may include non-alcoholic steatohepatitis (NASH) and the cargo may include a nucleic acid encoding GLP-1 or FGF21.
  • NASH non-alcoholic steatohepatitis
  • the therapeutic indication may include elevated circulating low density lipoprotein (LDL) level and the cargo may include a nucleic acid encoding an anti-PCSK9 antibody.
  • LDL low density lipoprotein
  • the cargo may be delivered to gastrointestinal cells and the gastrointestinal cells may secrete the anti-PCSK9 antibody into circulation at a level sufficient to provide a circulating anti-PCSK9 antibody concentration of from about 18 to about 19 pg/mL.
  • FIG. 1 shows the results of an exemplary assay to measure transfection efficiency of exemplary delivery vehicles of this disclosure, carrying DNA as cargo, in HEK cells.
  • FIG. 2 shows results of an exemplary assay for measuring stability of exemplary delivery vehicles of this disclosure.
  • FIG. 3 shows results of an exemplary assay for measuring stability of exemplary delivery vehicles of this disclosure.
  • FIG. 4 shows results of an exemplary assay for measuring stability of exemplary delivery vehicles of this disclosure.
  • FIG. 5 shows an agarose gel electrophoresis with an exemplary delivery vehicle of this disclosure (Formulation No. 5 in Table 1).
  • the lanes from left are as follows: lane one shows the ladder; lane 2 shows untreated delivery vehicle; lane three shows delivery vehicle treated with 7% Triton-X 100; lane four shows delivery vehicle treated with 7% Triton-X plus heat (70° C for 30 mins).
  • FIG. 6 shows a mouse colon section of a mouse dosed with 30 micrograms of DNA encapsulated in a delivery vehicle that was Dil and DiO labelled.
  • FIG. 7 shows a mouse colon section of a mouse dosed with 30 micrograms of DNA encapsulated in a delivery vehicle that was Dil and DiO labelled. Observed is the distribution of 2% PEG containing vehicle (particle 6 of Table 3) labelled with Dil and DiO as shown by fluorescence imaging from Dil overlaid onto brightfield.
  • FIG. 8 shows a mouse colon section of a mouse dosed with 30 micrograms of DNA encapsulated in a delivery vehicle (particle 7 of Table 3) that was Dil and DiO labelled. Observed is the distribution of 3% PEG containing vehicle labelled with Dil and DiO as shown by fluorescence imaging from Dil overlaid onto brightfield.
  • FIG. 9 shows a mouse colon section of a mouse dosed with 30 micrograms of DNA encapsulated in a delivery vehicle that was Dil and DiO labelled. Observed is the distribution of 5% PEG containing vehicle (particle 8 of Table 3) labelled with Dil and DiO as shown by fluorescence imaging from Dil overlaid onto brightfield.
  • FIG. 10 shows a mouse colon section of a mouse dosed with 30 micrograms of DNA encapsulated in a delivery vehicle that was Dil and DiO labelled. Observed is the distribution of 10% PEG containing vehicle (particle 9 of Table 3) labelled with Dil and DiO as shown by fluorescence imaging from Dil overlaid onto brightfield.
  • FIG. 11A shows distribution of delivery vehicles in representative colon sections from mice administered particles at a ratio of 0%/25% MVL5/DODMA percent mols (particle 1 of Table 3).
  • FIG. 11B shows distribution of delivery vehicles in representative colon sections from mice administered particles at a ratio of 0%/25% MVL5/DODMA percent mols (particle 1 of Table 3).
  • FIG. 12A shows distribution of delivery vehicles in representative colon sections from mice administered particles at a ratio of 6.25%/l 8.75% (MVL5/DODMA) percent mols (particle 2 of Table 3).
  • FIG. 12B shows distribution of delivery vehicles in representative colon sections from mice administered particles at a ratio of 6.25%/l 8.75% (MVL5/DODMA) percent mols (particle 2 of Table 3).
  • FIG. 13A shows distribution of delivery vehicles in representative colon sections from mice administered particles at a ratio of 12.5%/12.5% (MVL5/DODMA) percent mols (particle 3 of Table 3).
  • FIG. 13B shows distribution of delivery vehicles in representative colon sections from mice administered particles at a ratio of 12.5%/12.5% (MVL5/DODMA) percent mols (particle 3 of Table 3).
  • FIG. 14A shows distribution of delivery vehicles in representative colon sections from mice administered particles at a ratio of 18.75%/6.25% (MVL5/DODMA) % mols (particle 4 of Table 3).
  • FIG. 14B shows distribution of delivery vehicles in representative colon sections from mice administered particles at a ratio of 18.75%/6.25% (MVL5/DODMA) % mols (particle 4 of Table 3).
  • FIG. 15A shows distribution of delivery vehicles in representative colon sections from mice administered particles at a ratio of 25%/0% MVL5/DODMA % mols (particle 10 of Table 3).
  • FIG. 15B shows distribution of delivery vehicles in representative colon sections from mice administered particles at a ratio of 25%/0% MVL5/DODMA % mols (particle 10 of Table 3).
  • FIG. 16A shows swiss roll images of colons of a section of a first mouse administered MVL5/DODMA/DOPC/Deoxycholate/DMG-PEG (particle 11 of Table 3) with Dil and DiO using the BioTek Cytation software. The figure shows the Dil channel.
  • FIG. 16B show swiss roll images of colons of a section of a first mouse administered MVL5/DODMA/DOPC/Deoxycholate/DMG-PEG (particle 11 of Table 3) with Dil and DiO using the BioTek Cytation software. The figure shows the Dil channel overlaid onto brightfield.
  • FIG. 16C shows swiss roll images of colons of a section of a second mouse administered MVL5/DODMA/DOPC/Deoxycholate/DMG-PEG (particle 11 of Table 3) with Dil and DiO using the BioTek Cytation software. The figure shows the Dil channel.
  • FIG. 16D show swiss roll images of colons of a section of a second mouse administered MVL5/DODMA/DOPC/Deoxycholate/DMG-PEG (particle 11 of Table 3) with Dil and DiO using the BioTek Cytation software. The figure shows the Dil channel overlaid onto brightfield.
  • FIG. 17A shows swiss roll images of colons of a section of a first mouse administered MVL5/DODMA/GMO/Deoxycholate/DMG-PEG (particle 12 of Table 3) with Dil and DiO using the BioTek Cytation software. The figure shows the Dil channel.
  • FIG. 17B show swiss roll images of colons of a section of a first mouse administered MVL5/DODMA/GMO/Deoxycholate/DMG-PEG (particle 12 of Table 3) with Dil and DiO using the BioTek Cytation software.
  • the figure shows the Dil channel overlaid onto brightfield.
  • FIG. 17C shows swiss roll images of colons of a section of a second mouse administered MVL5/DODMA/GMO/Deoxycholate/DMG-PEG (particle 12 of Table 3) with Dil and DiO using the BioTek Cytation software. The figure shows the Dil channel.
  • FIG. 17D show swiss roll images of colons of a section of a second mouse administered MVL5/DODMA/GMO/Deoxycholate/DMG-PEG (particle 12 of Table 3) with Dil and DiO using the BioTek Cytation software.
  • the figure shows the Dil channel overlaid onto brightfield.
  • FIG. 18A shows swiss roll images of colons of a section of a first mouse administered MVL5/DODMA/DSPC/Deoxycholate/DMG-PEG (particle 5 of Table 3) with Dil and DiO using the BioTek Cytation software. The figure shows the Dil channel.
  • FIG. 18B show swiss roll images of colons of a section of a first mouse administered MVL5/DODMA/DSPC/Deoxycholate/DMG-PEG (particle 5 of Table 3) with Dil and DiO using the BioTek Cytation software. The figure shows the Dil channel overlaid onto brightfield.
  • FIG. 18C shows swiss roll images of colons of a section of a second mouse administered MVL5/DODMA/DSPC/Deoxycholate/DMG-PEG (particle 5 of Table 3) with Dil and DiO using the BioTek Cytation software. The figure shows the Dil channel.
  • FIG. 18D show swiss roll images of colons of a section of a second mouse administered MVL5/DODMA/DSPC/Deoxycholate/DMG-PEG (particle 5 of Table 3) with Dil and DiO using the BioTek Cytation software. The figure shows the Dil channel overlaid onto brightfield.
  • FIG. 19A shows swiss roll images of colons of a section of a first mouse administered PBS with Dil and DiO using the BioTek Cytation software. The figure shows the Dil channel.
  • FIG. 19B show swiss roll images of colons of a section of a first mouse administered PBS with Dil and DiO using the BioTek Cytation software. The figure shows the Dil channel overlaid onto brightfield.
  • FIG. 19C shows swiss roll images of colons of a section of a second mouse administered PBS with Dil and DiO using the BioTek Cytation software. The figure shows the Dil channel.
  • FIG. 19D show swiss roll images of colons of a section of a second mouse administered PBS with Dil and DiO using the BioTek Cytation software. The figure shows the Dil channel overlaid onto brightfield.
  • FIG. 20 shows a bar graph comparing the stability of different bile salts incorporating lipid structures in lOg/L of bile salts (cholate: deoxycholate mixture) by measuring perturbations in the lipid structures using FRET between Dil and DiO. FRET values are normalized to no treatment.
  • agents such as therapeutic agents to epithelial tissues and cells, such as in the gastrointestinal (GI) tract, vagina and lung, present certain challenges.
  • epithelial cells are covered with a mucosal layer and thus therapeutic agents must penetrate and move through the mucus to reach the epithelial cells.
  • therapeutic agents once within or through the layer of mucus, must come into proximity to the intended target cells and in some cases, interact with the cell membrane and/or enter the cells.
  • delivery of an agent also referred to herein as “cargo” is improved with a delivery vehicle that not only penetrate and cross through the mucus layer but also come within reach of the intended epithelial cell target.
  • harsh environments such as naturally present bile acids of the GI, can present a challenge for the stability of delivery and for successful delivery of cargo to the intended target cells.
  • compositions for and methods of delivering a cargo using delivery vehicles provided herein.
  • delivery vehicles may be further modified to provide stability and/or to reach target epithelial cells in challenging environments.
  • the delivery vehicles provided herein also referred to herein as “mucosal epithelial reaching” and charge-separated” delivery vehicles
  • the charge-separated delivery vehicles herein provide both penetration through mucus thereby reducing or preventing entrapment of the delivery vehicle in the epithelial mucus as well as the epithelial reaching functionality which brings the delivery vehicle in proximity of the epithelial cells, such as within a distance of 20 microns or less.
  • lipid based delivery vehicles comprising lipid structures, such as lipid nanoparticles and a cargo, that have improved stability in high bile salt environments, such as in the gastrointestinal tract.
  • the delivery vehicle in some embodiments, can provide stability in harsh environments of the GI tract and can be further be suited for mucus environments.
  • the delivery vehicle can be suitable for delivering a cargo (e.g ., a nucleic acid) to mucosal epithelial cells such as intestinal epithelial cells, lung epithelial cells, cervical epithelial cells, rectal epithelial cells, endometrial cells and the likes.
  • the delivery vehicle can also be suitable for delivery to organs, such as the skin.
  • the delivery vehicles provided herein can include additional mucus- penetrating features that may assist in the penetration and movement of the delivery vehicle through the mucus surrounding the epithelial cells.
  • additional features include incorporating a polymer such as Polyethylene glycol (PEG), Polyoxazoline polymer with methyl (PMOZ), Polyoxazoline polymer with ethyl (PEOZ) into the delivery vehicle surface and/or by including a mucus penetrating peptide (MPP) linked to the surface of the delivery vehicle.
  • the vehicles contain no PEG coating or a low density PEG coating (or a low density coating of another polymer).
  • the term “about” and its grammatical equivalents in relation to a reference numerical value and its grammatical equivalents as used herein can include a range of values plus or minus 10% from that value.
  • the amount “about 10” includes amounts from 9 to 11.
  • the term “about” in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.
  • administering can refer to any method of providing a structure described herein to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent.
  • a structure disclosed herein can be administered therapeutically. In some instances, a structure can be administered to treat an existing disease or condition. In further various aspects, a structure can be administered prophylactically to prevent a disease or condition.
  • biodegradable and its grammatical equivalents can refer to polymers, compositions and formulations, such as those described herein that are intended to degrade during use.
  • biodegradable is intended to cover materials and processes also termed “bioerodible.”
  • cancer and its grammatical equivalents as used herein can refer to a hyperproliferation of cells whose unique trait — loss of normal controls — results in unregulated growth, lack of differentiation, local tissue invasion, and metastasis.
  • the cancer can be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, rectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, leukemia, liquid tumors, liver cancer, lung cancer, lymphoma, malignant mesothelioma, mastocytoma, melanoma, multiple myelom
  • cargo can refer to one or more molecules or structures encompassed in a delivery vehicle for delivery to or into a cell or tissue.
  • Non-limiting examples of cargo can include a nucleic acid, a dye, a drug, a protein, a liposome, a small chemical molecule, a large biological molecule, and any combinations thereof.
  • cell and its grammatical equivalents as used herein can refer to a structural and functional unit of an organism.
  • a cell can be microscopic in size and can consist of a cytoplasm and a nucleus enclosed in a membrane.
  • a cell can refer to an intestinal crypt cell.
  • a crypt cell can refer to the crypts of Lieberkiihn which are pit-like structures that surround the base of the villi in the intestine.
  • a cell can be of human or non human origin.
  • Conjugate can refer to the association, covalently or non- covalently of two or more molecules or structures, including without limitation, the association of a peptide, such as a mucus-penetrating peptide (MPP) with a delivery vehicle, a polymer, a surface modification, or any combinations thereof.
  • MPP mucus-penetrating peptide
  • the term “function” and its grammatical equivalents as used herein can refer to the capability of operating, having, or serving an intended purpose.
  • Functional can comprise any percent from baseline to 100% of an intended purpose.
  • functional can comprise or comprise about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or up to about 100% of an intended purpose.
  • the term functional can mean over or over about 100% of normal function, for example, 125, 150, 175, 200, 250, 300%, 400%, 500%, 600%, 700% or up to about 1000% of an intended purpose.
  • gastrointestinal disease can refer to diseases involving the gastrointestinal tract, including but not limited to esophagus, stomach, small intestine, large intestine and rectum, and the accessory organs of digestion, the liver, gallbladder, and pancreas, and any combinations thereof.
  • hydrophilic and its grammatical equivalents as used herein refers to substances or structures that have polar groups that readily interact with water.
  • hydrophobic and its grammatical equivalents as used herein refers to substances or structures that have polar groups that do not readily interact with water.
  • mucus can refer to a viscoelastic natural substance containing primarily mucin glycoproteins and other materials, which protects epithelial surface of various organs/tissues, including but not limited to respiratory, nasal, cervicovaginal, gastrointestinal, rectal, visual and auditory systems.
  • lipid structure refers to a lipid composition for delivery to a cell or tissue, such as to deliver a therapeutic product, such as a nucleic acid.
  • lipid structure and its grammatical equivalents as used herein can refer to a nanoparticle or delivery vehicle.
  • a structure can be a liposomal structure.
  • a lipid structure can also refer to a particle.
  • a lipid structure or particle can be a nanoparticle or delivery vehicle.
  • a lipid particle or lipid structure can be of any shape having a diameter from about 1 nm up to about 1 micron.
  • a nanoparticle or nanostructure can be or can be about 100 to 200 nm.
  • a nanoparticle or nanostructure can also be up to 500 nm. Nanoparticles or nanostructures having a spherical shape can be referred to as "nanospheres”.
  • structure and its grammatical equivalents as used herein can refer to a nanoparticle or delivery vehicle.
  • a structure can be a liposomal structure.
  • a structure can also refer to a particle.
  • a structure or particle can be a nanoparticle or delivery vehicle.
  • a particle or structure can be of any shape having a diameter from about 1 nm up to about 1 micron.
  • a nanoparticle or nanostructure can be or can be about 100 to 200 nm.
  • a nanoparticle or nanostructure can also be up to 500 nm.
  • Nanoparticles or nanostructures having a spherical shape can be referred to as "nanospheres”.
  • nucleic acid can refer to a deoxyribonucleotide and/or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form.
  • these terms should not to be construed as limiting with respect to length.
  • the terms can also encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties ( e.g ., phosphorothioate backbones).
  • an analogue of a particular nucleotide can have the same base-pairing specificity, i.e., an analogue of adenine “A” can base-pair with thymine “T”.
  • pharmaceutically acceptable carrier can refer to sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • These solutions, dispersions, suspensions or emulsions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly (orthoesters) and poly (anhydrides).
  • predisposed can be understood to mean an increased probability (e.g, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or more increase in probability) that a subject will suffer from a disease or condition.
  • a subject can be a mammal.
  • a subject can be a human male or a human female.
  • a subject can be of any age.
  • a subject can be an embryo.
  • a subject can be a newborn or up to about 100 years of age.
  • a subject can be in need thereof.
  • a subject can have a disease such as cancer.
  • sequence and its grammatical equivalents as used herein can refer to a nucleotide sequence, which can be DNA and/or RNA; can be linear, circular or branched; and can be either single-stranded or double stranded.
  • a sequence can be of any length, for example, between 2 and 1,000,000 or more nucleotides in length (or any integer value there between or there above), e.g. , between about 100 and about 10,000 nucleotides or between about 200 and about 500 nucleotides.
  • sequence as used herein can refer to an amino acid sequence, such as a sequence of a protein, polypeptide and/or peptide.
  • stem cell can refer to an undifferentiated cell of a multicellular organism that is capable of giving rise to indefinitely more cells of the same type.
  • a stem cell can also give rise to other kinds of cells by differentiation.
  • Stem cells can be found in crypts.
  • Stem cells can be progenitors of epithelial cells found on intestinal villi surface.
  • Stem cells can be cancerous.
  • a stem cell can be totipotent, unipotent or pluripotent.
  • a stem cell can be an induced stem cell.
  • treatment can refer to the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, condition, or disorder.
  • Treatment can include active treatment, that is, treatment directed specifically toward the improvement of a disease, condition, or disorder.
  • Treatment can include causal treatment, that is, treatment directed toward removal of the cause of the associated disease, condition, or disorder.
  • this treatment can include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, condition, or disorder.
  • Treatment can include preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of a disease, condition, or disorder.
  • Treatment can include supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the disease, condition, or disorder.
  • a condition can be pathological.
  • a treatment may not completely cure, ameliorate, stabilize or prevent a disease, condition, or disorder.
  • hydrogen means — H
  • hydroxy means — OH
  • halogen means independently — F, — Cl, — Br or — I;
  • (C n ) defines the exact number (n) of carbon atoms in the group.
  • (C2-10) alkyl designates those alkyl groups having from 2 to 10 carbon atoms (e.g, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any range derivable therein (e.g, 3 to 10 carbon atoms).
  • alkyl can refer to an aliphatic hydrocarbon group.
  • the alkyl moiety may be a “saturated alkyl” group, which means that it does not contain any alkene or alkyne moieties.
  • the alkyl moiety may also be an “unsaturated alkyl” moiety, which means that it contains at least one alkene or alkyne moiety.
  • An “alkene” moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon double bond
  • an “alkyne” moiety refers to a group consisting of at least two carbon atoms and at least one carbon- carbon triple bond.
  • the alkyl moiety, whether saturated or unsaturated, may be branched, straight chain, or cyclic. Furthermore, the alkyl moiety, whether saturated or unsaturated, may comprise branched, straight chain, and/or cyclic portions.
  • an alkyl group can be a monoradical or a diradical (i.e., an alkylene group).
  • a “heteroalkyl” group is as described for “alkyl” with at least one of the C atoms thereof substituted with an N, S, or O atom.
  • the “heteroalkyl” group may comprise linear, branched, and/or cyclic portions.
  • a “lower alkyl” is an alkyl group with 1-6 carbon atoms (i.e., a C1-C6 alkyl group). In specific instances, the “lower alkyl” may be straight chained or branched.
  • Aryl refers to a radical derived from an aromatic monocyclic or aromatic multicyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom.
  • the aromatic monocyclic or aromatic multicyclic hydrocarbon ring system contains only hydrogen and carbon and from five to eighteen carbon atoms, where at least one of the rings in the ring system is aromatic, i.e., it contains a cyclic, delocalized (4n+2) p-electron system in accordance with the Hiickel theory.
  • the ring system from which aryl groups are derived include, but are not limited to, groups such as benzene, fluorene, indane, indene, tetralin and naphthalene.
  • aryl can refer to an aromatic ring wherein each of the atoms forming the ring is a carbon atom.
  • Aryl rings can be formed by five, six, seven, eight, nine, or more than nine carbon atoms.
  • Aryl groups can be optionally substituted. Examples of aryl groups include, but are not limited to phenyl, naphthalenyl, phenanthrenyl, anthracenyl, fluorenyl, and indenyl.
  • an aryl group can be a monoradical or a diradical (i.e., an arylene group).
  • Heteroaryl refers to a radical derived from a 3- to 12-membered aromatic ring radical that comprises two to eleven carbon atoms and at least one heteroatom wherein each heteroatom may be selected from N, O, and S.
  • the heteroaryl ring may be selected from monocyclic or bicyclic and fused or bridged ring systems rings wherein at least one of the rings in the ring system is aromatic, i.e., it contains a cyclic, delocalized (4n+2) p- electron system in accordance with the Hiickel theory.
  • the heteroatom(s) in the heteroaryl radical may be optionally oxidized.
  • One or more nitrogen atoms, if present, are optionally quaternized.
  • heteroaryl may be attached to the rest of the molecule through any atom of the heteroaryl, valence permitting, such as a carbon or nitrogen atom of the heteroaryl.
  • heteroaryls include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxolyl, benzofuranyl, benzooxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[Z>][l,4]dioxepinyl, benzo[b][l,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (
  • X-membered heteroaryl refers to the number of endocylic atoms, i.e., X, in the ring.
  • a 5-membered heteroaryl ring or 5-membered aromatic heterocycle has 5 endocyclic atoms, e.g., triazole, oxazole, thiophene, etc.
  • heteroaryl when used without the “substituted” modifier refers to a monovalent group with an aromatic carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of an aromatic ring structure wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the monovalent group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur.
  • Non-limiting examples of heteraryl groups include acridinyl, furanyl, imidazoimidazolyl, imidazopyrazolyl, imidazopyridinyl, imidazopyrimidinyl, indolyl, indazolinyl, methylpyridyl, oxazolyl, phenylimidazolyl, pyridyl, pyrrolyl, pyrimidyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, tetrahydroquinolinyl, thienyl, triazinyl, pyrrolopyridinyl, pyrrolopyrimidinyl, pyrrolopyrazinyl, pyrrolotriazinyl, pyrroloimidazolyl, chromenyl (where the point of attachment is one of the aromatic atoms), and chromanyl (where the point of attachment is one of the aromatic atom
  • Substituted heteroaryl refers to a monovalent group with an aromatic carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of an aromatic ring structure wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the monovalent group further has at least one atom independently selected from the group consisting of non-aromatic nitrogen, non-aromatic oxygen, non-aromatic sulfur F, Cl, Br, I,
  • substituted refers to moieties having substituents replacing a hydrogen on one or more carbons or substitutable heteroatoms, e.g., NH, of the structure. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • substituted refers to moieties having substituents replacing two hydrogen atoms on the same carbon atom, such as substituting the two hydrogen atoms on a single carbon with an oxo, imino or thioxo group.
  • substituted is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.
  • delivery vehicles provided herein contain positive and negative charges separated into different loci within the particle, where each locus is comprised of a different polymer (conferring the charge to the locus).
  • delivery vehicles provided herein contain positively-charged and negatively-charged lipids, where the loci are separated by phase, such as into a liquid phase and a gel phase.
  • the delivery vehicle can comprise a positively charged liquid phase and a negatively charged gel phase; or, a positively charged gel phase and a negatively charged liquid phase.
  • Delivery vehicles provided herein can efficiently deliver cargo, such as nucleic acids, proteins, peptides, and/or small molecules to epithelial cells within mucosal tissues. Delivery vehicles herein are useful to treat diseases and conditions that effect and/or originate in mucosal tissues, such as in the mucosal tissues in the gastrointestinal tract. Non-limiting examples include familial adenomatous polyposis (FAP), attenuated FAP, colorectal cancer, chronic inflammatory bowel disease, chronic inflammatory bowel disease, microvillus inclusion disease and congenital diarrheal diseases.
  • FAP familial adenomatous polyposis
  • FAP familial adenomatous polyposis
  • colorectal cancer chronic inflammatory bowel disease
  • chronic inflammatory bowel disease chronic inflammatory bowel disease
  • microvillus inclusion disease and congenital diarrheal diseases.
  • Delivery vehicles herein also are useful to provide therapeutic agents and/or nucleic acids to express therapeutic agents in mucosal tissues and such agents may remain in the targeted epithelial cells and/or be transported to other disease-affected cells and tissues within a subject.
  • the delivery vehicle provides a proximity distance to an epithelial cell. In some aspects, such proximity distance is less than about 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 microns.
  • the delivery vehicles herein come in contact with the epithelial cell.
  • the delivery vehicle is internalized into the cell and a cargo carried by the delivery vehicle is released within the cell.
  • the delivery vehicle contacts the epithelial cell and a cargo from the delivery vehicle is released outside of the cell.
  • the delivery vehicle provided herein can be a lipid structure.
  • a lipid structure can be utilized for delivery of cargo to a cell or tissue.
  • cargo can encompass a therapeutic product, such as a nucleic acid.
  • Lipid structures include, but not are limited to, lipid particles, lipid nanoparticles, liposomes or vesicles, such as vesicles wherein an aqueous volume is encapsulated by amphipathic lipid bilayers (e.g., single; unilamellar or multiple; multilamellar), or wherein the lipids at least partially coat an interior comprising a therapeutic product, or lipid aggregates or micelles, wherein the lipid encapsulated therapeutic product is contained within a relatively disordered lipid mixture.
  • the delivery vehicles herein (such as lipid nanoparticles, liposomes and micelle like structures) have at least two loci, and comprise a positive charge and a negative charge that are not interspersed but instead located in separated loci.
  • a negative charge and a positive charge may be present on opposite loci on a lipid structure provided herein at a pH between about 5.5 and 8.0, such as at a pH of about 7.4.
  • a positive charge and a negative charge are in two separate loci where each locus is a different phase of a lipid structure, for example a liquid or solid (gel) phase.
  • a positive charge may be on a liquid phase and a negative charge may be on a solid phase, for example a gel phase or vice versa.
  • Charge separation can allow for both an attraction and repulsion force.
  • a positive lipid can be attracted towards a target cell due to its high negative potential.
  • a repulsive force on a negative face can prevent a positive face from being kinetically trapped in mucus.
  • a cationic charge for instance on a lipid on a delivery vehicle, maybe attracted to mucus, en route to a target cell, and may get kinetically trapped in the mucus thereby trapping the delivery vehicle. The mucus will eventually slough off clearing the delivery vehicle.
  • an anionic delivery vehicle can be repulsed by mucus and may not make its way through the mucus.
  • a zwitterionic particle can act like a neutral particle absent a net force. Zwitterionic particles may follow the flow of water similar to PEGylated systems and may not become trapped in the mucus, but may not reach the epithelial cells.
  • a lipid structure can include one or more of an anionic lipid or cationic lipid, a neutral lipid, a sterol, and a lipid selected to reduce aggregation of lipid particles during formation. Aggregation may result from steric stabilization of lipid structures which may prevent charge-induced aggregation during formation.
  • Lipid structures can include two or more cationic lipids.
  • a cationic lipid may be on a first phase and an anionic lipid on a second phase such that the lipid structure contains two phases with differentially charged lipids. The lipids can be selected to contribute different advantageous properties.
  • cationic lipids that differ in properties such as amine pK a , chemical stability, half-life in circulation, half-life in tissue, net accumulation in tissue, or toxicity can be used in a lipid structure.
  • cationic lipids can be chosen so that the properties of the mixed-lipid lipid structure are more desirable than the properties of a single-lipid structure of individual lipids.
  • Net tissue accumulation and long-term toxicity (if any) from cationic lipids can be modulated in a favorable way by choosing mixtures of cationic lipids instead of selecting a single cationic lipid in a given formulation.
  • Such mixtures can also provide better encapsulation and/or release of a cargo, such as a nucleic acid.
  • a combination of cationic lipids also can affect the systemic stability when compared to single entity in a formulation.
  • a cationic lipid may attain a positive charge through one or more amines present in a polar head group.
  • a lipid structure can be a cationic liposome.
  • a liposome may be a cationic liposome used to carry negatively charged polynucleic acid, such as DNA. The presence of positively charged amines may facilitate binding with anions such as those found in DNA. A liposome thus formed may be a result of energetic contributions by Van der Waals forces and electrostatic binding to a DNA cargo which may partially contribute to liposome shape.
  • a cationic (and neutral) lipid may be used for gene delivery.
  • an anionic liposome may be used to deliver other therapeutic agents.
  • the delivery vehicles provided herein further comprise a cargo.
  • the cargo comprises a therapeutic agent.
  • the cargo comprises a nucleic acid, a protein, an antibody, a peptide, a small molecule, a biologic or a combination of any thereof.
  • the delivery vehicles herein include a component for cell internalization.
  • the component is a peptide, a carbohydrate or ligand.
  • the delivery vehicles provided herein also include a stability component.
  • the stability component is a polyethylene glycol (PEG).
  • the first locus comprises an unsaturated or short-tail lipid.
  • the unsaturated lipid comprises a cationic or ionizable cationic lipid.
  • the cationic lipid comprises a multivalent cationic lipid or a monovalent cationic lipid.
  • charge separation may result in superior and/or unexpected performance of subject delivery vehicles.
  • utilizing PEG is thought to increase trafficking to target cells, for example intestinal epithelial cells as provided in Maisel K et al., Effect of surface chemistry on nanoparticle interaction with gastrointestinal mucus and distribution in the gastrointestinal tract following oral and rectal administration in the mouse. J Control Release, herein incorporated by reference.
  • increasing PEGylation results in decreased distribution within or at the intestinal tissue thereby providing support for utilizing delivery vehicles with reduced PEGylation as compared to conventional vehicles.
  • One mechanism by which reducing PEGylation may improve trafficking and/or distribution to and in proximity to a target cell is by increasing the exposure of positive charge at the surface of a subject vehicle by reducing the shielding properties of PEGylation.
  • a delivery vehicle that comprises charge separation as provided herein can have improved trafficking, transfection of target cells, epithelial reach, or a combination thereof as compared to a comparable delivery vehicle that lacks the charge separation.
  • the improvement is from about 1 fold, 50 fold, 99 fold, 148 fold, 197 fold, 246 fold, 295 fold, 344 fold, 393 fold, 442 fold, 491 fold, 540 fold, 589 fold, 638 fold, 687 fold, 736 fold, 785 fold, 834 fold, 883 fold, 932 fold, 981 fold, or up to about 1000 fold as compared to a comparable delivery vehicle that lacks the charge separation.
  • a delivery vehicle can comprise any one of: Nl-[2-((lS)-l-[(3- aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]- benzamide (MVL5)/ (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 3- (dimethylamino)propanoate (MC2)/ l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/Deoxycholate/l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG- PEG); MVL5/MC2/DSPC/Deoxycholate/l,2-dimyristoyl-sn-glycero-3-phosphoethanol
  • a delivery vehicle can be generated using a variety of molar ratios.
  • a pharmaceutical formulation comprises MVL5, MC2, Deoxycholate, DSPC, and DMG-PEG at a molar ratio of about 0.96:0.96:2.592:3.168:0.0768:0.0384:0.0384.
  • the ratio of a cationic charge in a first locus to an anionic charge in a second locus at pH 7.4 is from about 0.25, 0.45, 0.65, 0.85, 1.05, 1.25, 1.45, 1.65, 1.85, 2.05, 2.25, 2.45, 2.65, or 2.85.
  • the ratio of a cationic charge in a first locus to an anionic charge in a second locus at pH 7.4 is from about 0.25 to about 1.05, 0.75 to about 1.25, 1.05 to about 1.45, or 0.85 to about 1.85.
  • a ratio of a multivalent lipid to an ionizable cationic lipid in a delivery vehicle is from about (6%, 6.25%, 6.5%, 6.75%, 7%, 7.25%, 7.5%, 7.75%, or 8%) to (8%, 8.25%, 8.5%, 8.75%, 9%, 9.25%, 9.5%, 9.75%, 10%), (12%, 12.25%, 12.5%, 12.75%, or 13%) to (12%, 12.25%, 12.5%, 12.75%, or 13%), or (18%, 18.25%, 18.5%, 18.75%, 19%, 19.25%, 19.5%, 19.75%, 20%) to (6%, 6.25%, 6.5%, 6.75%, 7%, 7.25%,
  • a bile salt is at a concentration from about 10 mole %, 15 mole %, 20 mole %, 25 mole %, 30 mole %, 35 mole %, 40 mole %, 45 mole %, 50 mole %, 55 mole %, 60 mole %, 65 mole %, 70 mole %, 75 mole %, or about 80 mole %. In some cases, a bile salt is from about 10 mole % to 30 mole %, 20 mole % to 50 mole %, 30 mole % to 60 mole %, or 40 mole % to 80 mole %.
  • Suitable alternate formulations can comprise multivalent lipid, ionizable cationic lipid, bile salt, structural lipid, and/or lipid-PEG at molar ratios from about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% more or less to those provided herein.
  • the delivery vehicle stability can be increased with the incorporation of a bile acid or bile salt.
  • bile acid bile salt
  • bile acid/salt are, unless otherwise indicated, utilized interchangeably herein. Any reference to a bile acid used herein can include reference to a bile acid or a salt thereof.
  • bile acid can include steroid acids (and an anion thereof), and salts thereof, found in the bile of an animal (e.g ., a human), including, by way of non limiting example, cholic acid, cholate, deoxycholic acid, deoxycholate, hyodeoxycholic acid, hyodeoxycholate, glycocholic acid, glycocholate, taurocholic acid, taurocholate, chenodeoxycholic acid, chenodeoxycholate, lithocholic acid, lithocolate, and the like, or salts thereof.
  • a bile acid is ursodiol, isolithocholate, alloisolithocholate, dehydrolithochlate, or 5-beta-cholanic acid.
  • Taurocholic acid and taurocholate are referred to herein as TCA.
  • Any reference to a bile acid used herein can include reference to a bile acid, one and only one bile acid, one or more bile acids, or to at least one bile acid.
  • pharmaceutically acceptable bile acid esters can be utilized as the “bile acids” described herein, e.g., bile acids conjugated to an amino acid (e.g, glycine or taurine).
  • bile acid esters can include, e.g, substituted or unsubstituted alkyl ester, substituted or unsubstituted heteroalkyl esters, substituted or unsubstituted aryl esters, substituted or unsubstituted heteroaryl esters, or the like.
  • bile acid can include cholic acid conjugated with either glycine or taurine: glycocholate and taurocholate, respectively (and salts thereof). Any reference to a bile acid used herein can include reference to an identical compound naturally or synthetically prepared.
  • any singular reference to a component (bile acid or otherwise) used herein can include reference to one and only one, one or more, or at least one of such components.
  • any plural reference to a component used herein can include reference to one and only one, one or more, or at least one of such components, unless otherwise noted.
  • a bile salt can be cholic acid.
  • a bile salt can be deoxycholate.
  • the incorporation of bile salts can be cholic acid and deoxycholate.
  • the bile salt can comprise cholate, deoxycholate, their conjugates and derivatives, or combination thereof.
  • bile salts can be chenodeoxycholic, lithocholic, taurodeoxycholic, or combination thereof.
  • the bile salt concentration in the lipid nanoparticles of a delivery vehicle can comprise from about 80 mole % to about 10 mole %, such as from about 80 mole % to about 70 mole %, from about 65 mole % to about 55 mole %, from about 60 mole % to about 50%, from about 55 mole % to about 45 mole %, from about 50 mole % to about 40 mole %, from about 45 mole % to about 35 mole %, from about 40 mole % to about 30 mole%, from about 35 mole% to about 25 mole %, from about 30 mole % to about 20 mole %, from about 25 mole % to about 15 mole %, from about 20 mole % to about 10 mole%, from about 15 mole % to about 10 mole %, from about 60 mole % to about 20 mole %, from about 25.9
  • the bile salt concentration in a lipid nanoparticle of a delivery vehicle can comprise about 5 mole %, 10 mole %, 15 mole %, 20 mole %, 25 mole %, 30 mole %, 35 mole %, 40 mole %, 45 mole %, 50 mole %, 55 mole %, 60 mole %, 65 mole %, 70 mole %, 75 mole %, 80 mole %, or 85 mole %.
  • An efficiency of cellular uptake with a structure, such as the compositions described herein having a bile salt included in the lipid nanoparticle of a delivery vehicle can permit efficient penetration and transit through the mucus layer to the target cells and thereby have an efficient uptake by the target cell(s), for example, uptake can be or can be about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more than 99.9% of the total number of cells that are contacted.
  • the compositions can have a higher percent of cellular uptake as compared to a comparable delivery vehicle that does include a bile salt.
  • the improvement can be from about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or up to about 80% better.
  • an efficiency of transfection or integration of a polynucleic acid cargo delivered to a cell by a delivery vehicle composition as described herein can be from about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or up to 65% better than a comparable delivery vehicle that does not include the bile salt, and additional features, such as an MPP and/or a particular composition of lipids.
  • an efficiency of transfection or integration of a polynucleic acid cargo delivered to a cell by a delivery vehicle composition as described herein can be from about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or up to 65% better than a comparable delivery vehicle that does not include a bile salt.
  • stability of the delivery vehicle can be measured by a bile salt stability assay, in a high bile salt mimicking environment.
  • bile salt stability can be measured by fluorescence spectroscopy, such as relative fluorescence of delivery vehicles containing varying concentrations of bile salts, in a Forster resonance energy transfer (FRET) assay.
  • FRET Forster resonance energy transfer
  • the incorporated bile salt (s) can increase the stability of the delivery vehicle from about 80 % to about 10 %, such as about 80 % to about 70 %, about 65 % to about 55 %, about 60 % to about 50%, about 55 % to about 45 %, about 50 % to about 40 %, about 45 % to about 35 %, about 40 % to about 30 %, about 35 % to about 25 %, about 30 % to about 20 %, about 25 % to about 15 %, about 20 % to about 10 %, about 15 % to about 10, about 60 % to about 20 %, about 25.9 %, about 30.4 %, about 34.9 %, about 39.4 %, about 37.1 %, about 43.9 %, or about 45 %.
  • the incorporated bile salt (s) can increase the stability of the delivery vehicle as compared to a comparable delivery vehicle that lacks the bile salt.
  • a delivery vehicle that comprises a bile salt as provided herein can have improved trafficking, transfection of target cells, epithelial reach, or a combination thereof as compared to a comparable delivery vehicle that lacks the bile salt.
  • the improvement is from about 1 fold, 50 fold, 99 fold, 148 fold, 197 fold, 246 fold, 295 fold, 344 fold, 393 fold, 442 fold, 491 fold, 540 fold, 589 fold, 638 fold, 687 fold, 736 fold, 785 fold, 834 fold, 883 fold, 932 fold, 981 fold, or up to about 1000 fold as compared to a comparable delivery vehicle that lacks the bile salt.
  • the percent increase in stability can be measured by increased relative fluorescence units or relative luminescence units in an assay, such as FRET in vivo or ex vivo.
  • a delivery vehicle of this disclosure can comprise a cationic lipid and a bile salt, wherein the lipid can be a saturated cationic lipid or an unsaturated cationic lipid, wherein the saturated cationic lipid may have a phase transition temperature that is at least about 20 °C.
  • a delivery vehicle of this disclosure can comprise at least one saturated cationic lipid and at least a bile salt, wherein the at least one saturated cationic lipid can have a phase transition temperature of at least about 37 °C.
  • the saturated cationic lipid has a phase transition temperature of at least about 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, and/or up to about 60°C.
  • the saturated cationic lipid can have a phase transition temperature of 30 °C-60 °C, 35 °C-60 °C, 37 °C-60 °C, 37 °C-55 °C, 37 °C-50 °C, 37 °C-45 °C, or 37 °C-40 °C.
  • a delivery vehicle of this disclosure can comprise at least one saturated cationic lipid and at least a bile salt, wherein the at least one saturated cationic lipid can have a phase transition temperature of at least about 37 °C.
  • the lipid delivery vehicle can further comprise a saturated non- cationic lipid.
  • the saturated non-cationic lipid may have a phase transition temperature of at least about 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, and/or up to about 60°C.
  • the saturated non-cationic lipid may have phase transition temperatures of about 30 °C-60 °C, 35 °C-60 °C, 37 °C-60 °C, 37 °C-55 °C, 37 °C-50 °C, 37 °C-45 °C, or 37 °C-40 °C.
  • the lipid delivery vehicle in some cases, can further comprise a lipid conjugated to a hydrophilic polymer, such a polyethylene glycol (PEG).
  • the delivery vehicle in some cases, may be conjugated to at least one of: a cell penetrating peptide, a ligand, a mucus penetrating polymer, a peptide that enables mucus penetration, a cell penetrating peptide that is not substantially mucus adhesive, or any combinations thereof.
  • a delivery vehicle comprising a cargo in a lipid structure, for example a lipid nanoparticle, and wherein the lipid nanoparticle comprises a bile salt and at least one of: (a) a saturated cationic lipid that has a phase transition temperature of at least about 37 °C, and a non-cationic lipid; (b) a saturated cationic lipid, an unsaturated cationic lipid, a non-cationic lipid, wherein the unsaturated cationic lipid, the non-cationic lipid, or the unsaturated cationic lipid and the non-cationic lipid, have a phase transition temperature of at least about 37 °C; or (c) a multivalent cationic lipid, a non- cationic lipid, wherein the multivalent cationic lipid, the non-cationic lipid, or the multivalent cationic lipid and the non-cationic lipid have a phase transition temperature of at least about 37 °C,
  • the saturated cationic lipid, unsaturated cationic lipid, non-cationic lipid, and/or multivalent cationic lipid may have a phase transition temperature of at least about 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, and/or up to about 60°C.
  • the saturated cationic lipid, unsaturated cationic lipid, non-cationic lipid, and/or multivalent cationic lipid may have phase transition temperatures of about 30 °C-60 °C, 35 °C-60 °C, 37 °C-60 °C, 37 °C-55 °C, 37 °C-50 °C, 37 °C-45 °C, or 37 °C-40 °C.
  • a delivery vehicle comprising a cargo and a lipid structure, such as a lipid nanoparticle, wherein the lipid nanoparticle comprises a bile salt and at least one of: (a) a saturated cationic lipid that has a phase transition temperature of at least about 37 °C; (b) a saturated cationic lipid, an unsaturated cationic lipid and a non- cationic lipid, wherein the unsaturated cationic lipid, the non-cationic lipid, or the unsaturated cationic lipid and the non-cationic lipid, have a phase transition temperature of at least about 37 °C; or (c) a multivalent cationic lipid and a non-cationic lipid, wherein the multivalent cationic lipid, the non-cationic lipid, or the multivalent cationic lipid and the non-cationic lipid have a phase transition temperature of at least about 37 °C, wherein the delivery vehicle demonstrates an increased stability in
  • the delivery vehicle demonstrates an increased stability in a solution containing at least about 0.5 g/L, 1 g/L, 5 g/L, 7g/L, 9g/L, llg/L, 13g/L, 15g/L, 17g/L, 19g/L, 21 g/L, 23g/L, or up to about 25g/L of bile acid, for example, a mixture of about 40%, 45%, 50%, or up to about 55% cholic acid and about 40%, 45%, 50%, 55%, or up to about 60% deoxycholate, compared to an otherwise identical delivery vehicle that (i) does not comprise the bile salt, wherein the stability is measured by relative fluorescence intensity of a fluorescent lipid incorporated into the lipid nanoparticle, in a Forster resonance energy transfer (FRET) assay.
  • FRET Forster resonance energy transfer
  • a delivery vehicle comprising (i) a cargo and (ii) a lipid structure, such as a lipid nanoparticle, wherein the lipid nanoparticle comprises at least one saturated cationic lipid and a bile salt, wherein the at least one saturated cationic lipid has a phase transition temperature of at least about 37 °C.
  • a delivery vehicle comprising (i) a cargo and a (ii) lipid nanoparticle, wherein the lipid nanoparticle comprises at least one saturated lipid, at least one unsaturated cationic lipid, and a bile salt, wherein the concentration of the at least one unsaturated cationic lipid in the lipid nanoparticle is less than 50 mole %.
  • Exemplary delivery vehicles are described herein and provided for example at Table 1, Table 2, Table 3, Table 4, and Table 8. Any one of the delivery vehicles exemplified in Table 1 -Table 4 and Table 8 can be further modified. For example, additional lipids, cargo, modifications to, additions to, subtractions to, can be made. In some cases, any one of the delivery vehicles in Table 1 can further comprise lipid-PEG.
  • Table 1 Exemplary delivery vehicles for delivering cargo _ _
  • BS bile salt
  • SC saturated cationic
  • UC unsaturated cationic
  • SN saturated non-cationic
  • UN unsaturated non-cationic
  • MV multivalent cationic
  • SMV Multivalent cationic saturated
  • UMV Multivalent cationic unsaturated.
  • Lipids for use in delivery vehicles Lipids for use in delivery vehicles
  • the delivery vehicles herein, including those with a cargo include one or more lipids such as in a lipid nanoparticle.
  • the lipid nanoparticle includes at least one saturated lipid, at least one of an unsaturated cationic lipid or an unsaturated non- cationic lipid, and a bile salt.
  • the lipid nanoparticle includes at least one saturated lipid, where the saturated lipid comprises a saturated cationic lipid that has a phase transition temperature of at least about 37 °C or a saturated non-cationic lipid that has a phase transition temperature of at least about 37 °C.
  • the lipid nanoparticle further includes at least one of: a non-cationic lipid, a multivalent cationic lipid, a permanently charged cationic lipid, or any combinations thereof.
  • the lipid nanoparticle comprises a bile salt and a multivalent cationic lipid and a non-cationic lipid, where the multivalent cationic lipid, the non-cationic lipid, or the multivalent cationic lipid and the non-cationic lipid have a phase transition temperature of at least about 37 °C.
  • the lipid nanoparticle comprises a bile salt and a saturated cationic lipid that has a phase transition temperature of at least about 37 °C, and a non-cationic lipid.
  • the lipid nanoparticle comprises a bile salt and a saturated cationic lipid, an unsaturated cationic lipid, and a non-cationic lipid, wherein the unsaturated cationic lipid, the non-cationic lipid, or the unsaturated cationic lipid and the non-cationic lipid have a phase transition temperature of at least about 37 °C.
  • the delivery vehicle has a first locus that is positively charged at a pH between about 5.5 and 8.0, and a second locus that is negatively charged at a pH between about 5.5 and 8.0, wherein the first and second loci are separated such that the positive and negative charges are not interspersed, and wherein one or both loci contain a lipid.
  • the first locus comprises an unsaturated or short-tail lipid, such as a cationic or ionizable cationic lipid, for example, a multivalent cationic lipid or a monovalent cationic lipid.
  • a cationic lipid for use in the lipid nanoparticles of the delivery vehicles herein can include N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), [l,2-bis(oleoyloxy)-3-(trimethylammonio)propane] (DOTAP), dimethyldioctadecylammonium (DDA), 3b[N-(N', N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), and dioctadecylamidoglycylspermine (DOGS).
  • DOTMA N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
  • DOTAP [l,2-bis(oleoyloxy)-3-(trimethylammonio)propane]
  • DDA dimethyldioctade
  • Dioleoylphosphatidylethanolamine DOPE
  • PEI polyethyleneimines
  • a neutral lipid may often be used in conjunction with cationic lipids because of its membrane destabilizing effects at low pH, which can aide in endolysosomal escape.
  • a saturated cationic lipid can be employed in a delivery vehicle provided herein.
  • a saturated cationic lipid can have a positive charge at pH 4, or at a pH greater than pH 4.
  • the saturated cationic lipid can comprise at least one of: 1,2-dialkyl-sn- glycero-3-ethylphosphocholine, l,2-dialkyl-3-dimethylammonium -propane, l,2-dialkyl-3- trimethylammonium-propane, l,2-di-0-alkyl-3-trimethylammonium propane, 1,2-dialkyloxy- 3-dimethylaminopropane, N,N-dialkyl-N,N-dimethylammonium, N-(4-carboxybenzyl)-N,N- dimethyl-2,3-bis(alkyloxy)propan-l-aminium, l,2-dialkyl-sn-glycero-3-[(N-(5-amino-l- carboxypentyl)iminodiacetic acid)succinyl], Nl-[2-((lS)-l-[(3-aminopropyl)amin
  • the alkyl can be a conjugated derivative of at least one of: myristoyl, pentadecanoyl, palmitoyl, heptadecanoyl, stearoyl, lauroyl, tridecanoyl, nonadecanoyl, arachidoyl, heneicasnoyl, behenoyl, tricosanoyl, lignoceroyl, or any combinations thereof.
  • the saturated cationic lipid can comprise at least one of: saturated cationic lipid that has a phase transition temperature of at least about 37 °C comprises at least one of: l,2-stearoyl-3-trimethylammonium -propane (DSTAP), l,2-dipalmitoyl-3-trimethylammonium -propane (DPTAP), 1, 2-Distearoyl-3- Dimethylammonium-Propane (DSDAP), or any combinations thereof.
  • DSTAP l,2-stearoyl-3-trimethylammonium -propane
  • DPTAP 1, 2-Distearoyl-3- Dimethylammonium-Propane
  • a cationic lipid may be in a gel phase of a lipid structure and an anionic lipid may be in a liquid phase.
  • a lipid nanoparticle of a delivery vehicle can comprise at least one unsaturated cationic lipid.
  • the unsaturated cationic lipid can have a positive charge at about pH 4, or at a pH greater than about pH 4 and less than about pH 8.
  • the unsaturated cationic lipid can comprise at least one of: 1,2- dialkyl-sn-glycero-3 -ethylphosphocholine, 1 ,2-dialkyl-3 -dimethylammonium -propane, 1 ,2- dialkyl-3-trimethylammonium -propane, l,2-di-0-alkyl-3-trimethylammonium propane, 1,2- dialkyloxy-3-dimethylaminopropane, N,N-dialkyl-N,N-dimethylammonium, N-(4- carboxybenzyl)-N,N-dimethyl-2,3-bis(alkyloxy)propan-l-aminium, l,2-dialkyl-sn-glycero-3- [(N-(5-amino-l-carboxypentyl)iminodiacetic acid)succinyl], Nl-[2-((lS)-l-[(3- aminopropyl), Nl-
  • the alkyl can be a conjugated derivative of at least one of: oleic acid, elaidic acid, gondoic acid, erucic acid, nervonic acid, mead acid, paullinic acid, vaccenic acid, palmitoleic acid, Docosatetraenoic acid, Arachidonic acid, Dihomo-Y-linolenic acid, g- Linolenic acid, linolelaidic acid, linoleic acid, Docosahexaenoic acid, Eicosapentaenoic acid, Stearidonic acid, a-Linolenic acid, or any combinations thereof.
  • the unsaturated cationic lipid can comprise at least one of: l,2-Dialkyloxy-N,N- dimethylaminopropane, 4-(2,2-diocta-9,12-dienyl-[l,3]dioxolan-4-ylmethyl)-dimethylamine, O-alkyl ethylphosphocholines, MC3, MC2, MC4, 3B-[N-(N',N'-dimethylaminoethane)- carbamoyl jchol esterol , N4-Cholesteryl-Spermine, or salts thereof, or any combinations thereof.
  • a lipid can comprise or can be 7-(4-(dimethylamino)butyl)-7- hydroxytridecane-l,13-diyl dioleate (CL1H6), CL1A6, CL1A6, CL3A6, CL4A6, CL5A6, CL6A6, CL7A6, CL8A6, CL9A6, CL10A6, CL11A6, CL12A6, CL13A6, CL14A6,
  • a cationic lipid may be in a liquid phase of a lipid structure and an anionic lipid may be in a gel phase or solid phase of a lipid structure.
  • a lipid nanoparticle of a delivery vehicle may comprise a multivalent cationic lipid.
  • a multivalent cationic lipid can be selected from: N 1 - [2-((l S)- 1 - [(3 -aminopropyljamino] -4- [di(3 - amino-propyl)amino]butylcarboxamido)ethyl]-3,4- di[oleyloxy]-benzamide (MVL5) a salt thereof, and any combination thereof.
  • a delivery vehicle provided herein can be generated using MVL5.
  • MVL5, GL67, or a combination thereof are in a liquid phase of a delivery vehicle.
  • any of the multivalent cationic lipids provided herein can be incorporated in a provided vehicle or particle at less than about 50 mole %, 48 mole %, 46 mole %, 44 mole %, 42 mole %, 40 mole %, 38 mole %, 36 mole %, 34 mole %, 32 mole %, 30 mole %, 28 mole %, 26 mole %, 24 mole %, 22 mole %, 20 mole %, 18 mole %, 16 mole %, 14 mole %, 12 mole %, 10 mole %, 8 mole %, 6 mole %, 4 mole %, 2 mole %, or 0 mole %.
  • any of the multivalent cationic lipids provided herein can be incorporated in a provided vehicle or particle at about 50 mole %, 48 mole %, 46 mole %, 44 mole %, 42 mole %, 40 mole %, 38 mole %, 36 mole %, 34 mole %, 32 mole %, 30 mole %, 28 mole %, 26 mole %, 24 mole %, 22 mole %, 20 mole %, 18 mole %, 16 mole %, 14 mole %, 12 mole %, 10 mole %, 8 mole %, 6 mole %, 4 mole %, 2 mole %, or 0 mole %.
  • the multivalent cationic lipids provided herein can be incorporated in a provided vehicle or particle in a concentration of 5-50 mole %, 5-40 mole %, 5-30 mole %, 5-25 mole %, 5-20 mole %, 5-15 mole %, 10-50 mole %, 10-40 mole %, 10-30 mole %, 10-25 mole %, 15-50 mole %, 15-40 mole %, 15-30 mole % and 15-25 mole %.
  • a lipid nanoparticle of a delivery vehicle provided herein can also comprise an anionic lipid.
  • An anionic lipid can contain any of a wide range of fatty acid chains in the hydrophobic region. The specific fatty acids incorporated are responsible for the fluidic characteristics of the lipid structure in terms of phase behavior and elasticity.
  • divalent cations can be incorporated into an anionic lipid structure to enable the condensation of nucleic acids prior to envelopment by anionic lipids.
  • divalent cations can be used in anionic lipoplexes such as Ca 2+ , Mg 2+ , Mn 2+ , and Ba 2+ .
  • Ca 2+ can be utilized in an anionic lipid structure.
  • Suitable anionic lipids include but are not limited to: phosphatidylglycerol, a cardiolipin, a diacylphosphatidylserine, a diacylphosphatidic acid, a N-dodecanoyl phosphatidylethanolamine, a N-succinyl phosphatidylethanolamine, a N- glutarylphosphatidylethanolamine, a lysylphosphatidylglycerol, a palmitoyloleyolphosphatidylglyeerol (POPG), or any combinations thereof.
  • phosphatidylglycerol a cardiolipin
  • a diacylphosphatidylserine a diacylphosphatidic acid
  • a N-dodecanoyl phosphatidylethanolamine a N-succinyl phosphatidylethanolamine
  • a N- glutarylphosphatidylethanolamine a ly
  • the anionic lipid in the lipid nanoparticles comprises at least one of phosphatidylglycerol, cardiolipin, dialkylphosphatidylserine, dialkylphosphatidic acid, N-dodecanoyl phosphatidylethanolamine, N-succinyl phosphatidylethanolamine, N- glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleyolphosphatidylglycerol (POPG), glycerophosphoinositol monophosphate, glycerophosphoinositol bisphosphate, glycerophosphoinositol trisphosphate, glycerophosphate, a glyceropyrophosphate, glycerophosphoglycerophosphoglycerol, cytidine-5'-diphosphate-glycerols, glycosylglycero
  • the alkyl is a conjugated derivative of at least one of oleic acid, elaidic acid, gondoic acid, erucic acid, nervonic acid, mead acid, paullinic acid, vaccenic acid, palmitoleic acid, Docosatetraenoic acid, Arachidonic acid, Dihomo-Y-linolenic acid, g-Linolenic acid, linolelaidic acid, linoleic acid, Docosahexaenoic acid, Eicosapentaenoic acid, Stearidonic acid, a-Linolenic acid, or salts thereof, or any combinations thereof.
  • the alkyl is a conjugated derivative of at least one myristic acid, pentadecylic acid, palmitic acid, heptadecanoic acid, stearic acid, lauric acid, tridecylic acid, nonadecylic acid, arachidic acid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid, and/or salts thereof, or any combinations thereof.
  • the alkyl has a phase transition temperature of >37C it is considered to be in the gel phase otherwise it is present in the liquid phase.
  • an anionic lipid can be a saturated lipid with a phase transition temperature above 37C, such a lipid can be used in a solid phase and a cationic lipid in a liquid phase.
  • an anionic lipid is unsaturated or a short chain lipid with a transition temperature below 37C then it may be employed in a liquid phase and a cationic lipid can be used in a gel or solid phase.
  • the concentration of at least one unsaturated cationic lipid and/or unsaturated non-cationic lipid in a lipid nanoparticle can be less than 50 mole %, 45 mole %, 40 mole %, 35 mole %, 30 mole %, 25 mole %, 20 mole %, 15 mole %, 10 mole %, 5 mole %, or 2 mole % of the total lipid concentration of the lipid nanoparticle.
  • the concentration of at least one unsaturated cationic lipid and/or unsaturated non-cationic lipid in a lipid nanoparticle can be about 50 mole %, 45 mole %, 40 mole %, 35 mole %, 30 mole %, 25 mole %, 20 mole %, 15 mole %, 10 mole %, 5 mole %, or 2 mole % of the total lipid concentration of the lipid nanoparticle.
  • the concentration of at least one unsaturated cationic lipid and/or unsaturated non-cationic lipid in a lipid nanoparticle can be 5-50 mole %, 5-40 mole %, 5-30 mole %, 5-25 mole %, 5-20 mole %, 5-15 mole %, 10-50 mole %, 10-40 mole %, 10-30 mole %, 10-25 mole %, 15-50 mole %, 15-40 mole %, 15-30 mole % and 15-25 mole %.
  • a delivery vehicle may comprise a high temperature phase transition lipid, for example, a high temperature phase transition neutral lipid such as DSPC, and a bile salt such as deoxycholate, cholic acid or a conjugate thereof.
  • a high temperature phase transition neutral lipid such as DSPC
  • a bile salt such as deoxycholate, cholic acid or a conjugate thereof.
  • Deoxycholate can serve as a solid phase (gel phase) where deoxycholate provides the negative charge.
  • a cationic lipid can be present as unsaturated or a short tail lipid and can be present in the liquid phase.
  • Multivalent cationic lipids like MVL5, can be used to create enough positive to negative charge ratio to provide the system with a balance of attraction and repulsion thereby generating a delivery vehicle containing a charge separation.
  • a delivery vehicle can further comprise a conjugated lipid, wherein the conjugated lipid can comprise a lipid conjugated to a stabilizing component.
  • the stabilizing component can comprise a hydrophilic polymer.
  • the hydrophilic polymer can comprise polyethylene glycol, a poly (2-alkyl-2- oxazoline), a polyvinyl alcohol, or any combinations thereof.
  • the hydrophilic polymer can comprise a molecular weight from at least about 500Da to about 500kDa, from at least about.
  • the hydrophilic polymer can include polyethyleneglycol (PEG), and the conjugated lipid may be a pegylated lipid.
  • the pegylated lipid can comprise l,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE)-PEG, 1,2-distearoyl-rac-glycerol (DSG)-PEG, 1,2- dipalmitoyl-rac-glycerol (DPG)-PEG, diacylglycerol (DAG)-PEG, 1,2-dimyristoyl-rac- glycerol (DMG)-PEG, l,2-dipalmitoryl-sn-glycero-3-phosphoethanolamine (DPPE)-PEG, l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE)-PEG, or any combinations thereof.
  • DSPE disistearoyl-sn-glycero-3- phosphoethanolamine
  • DPG 1,2-dipalmitoyl-rac-glycerol
  • DMG 1,2-dimyristoyl-rac-gly
  • the concentration of the conjugated lipid can be less than about or more than about: 0 mole %, 0.5 mole %, l mole %, 1.5 mole %, 2 mole %, 2.5 mole %, 3 mole %, 3.5 mole %, 4 mole %, 4.5 mole %, 5 mole %, 5.5 mole %, 6 mole %, 6.5 mole %, 7 mole %, 7.5 mole %, 8 mole %, 8.5 mole %, 9 mole %, 9.5 mole %, 10 mole %,
  • a concentration of the conjugated lipid is from about 0.5 mole % to about 20 mole %, 0.5 mole % to about 5 mole %,0.5 mole % to about 10 mole %, 5 mole % to about 10 mole %, or 10 mole % to about 20 mole %.
  • bile salts can be used as an anionic component in a delivery vehicle.
  • non-bile salts can be used as an anionic component.
  • the delivery vehicle stability can be increased with the incorporation of the bile salts (also referred to herein as bile acids), such as cholic acid, cholate, deoxycholic acid, deoxycholate, hyodeoxycholic acid, hyodeoxycholate, glycocholic acid, glycocholate, taurocholic acid, taurocholate, chenodeoxycholic acid, chenodeoxycholate, lithocholic acid, and lithocolate.
  • a bile salt can be cholic acid.
  • a bile salt can be deoxycholate.
  • the incorporation of bile salts can be cholic acid and deoxycholate.
  • stability of the delivery vehicle can be measured by a bile salt stability assay, in a high bile salt mimicking environment.
  • bile salt stability can be measured by fluorescence spectroscopy, such as relative fluorescence of delivery vehicles containing varying concentrations of bile salts, in a Forster resonance energy transfer (FRET) assay.
  • FRET forster resonance energy transfer
  • the incorporated bile salt (s) can increase the stability of the delivery vehicle from about 80 % to about 10 %, such as about 80 % to about 70 %, about 65 % to about 55 %, about 60 % to about 50%, about 55 % to about 45 %, about 50 % to about 40 %, about 45 % to about 35 %, about 40 % to about 30 %, about 35 % to about 25 %, about 30 % to about 20 %, about 25 % to about 15 %, about 20 % to about 10 %, about 15 % to about 10, about 60 % to about 20 %, about 25.9 %, about 30.4 %, about 34.9 %, about 39.4 %, about 37.1 %, about 43.9 %, or about 45 %.
  • the percent increase in stability can be measured by increased relative fluorescence units or relative luminescence units in an assay, such as FRET.
  • a delivery vehicle provided herein can comprise at least one of a multivalent lipid, cationic lipid, structural lipid, bile salt, or lipid-PEG. Any or all of the lipids provided herein can be formulated at any mole% for example, including but not limited to: 0 mole %, 0.5 mole %, 1 mole %, 1.5 mole %, 2 mole %, 2.5 mole %, 3 mole %, 3.5 mole %, 4 mole %, 4.5 mole %, 5 mole %, 5.5 mole %, 6 mole %, 6.5 mole %, 7 mole %, 7.5 mole %, 8 mole %, 8.5 mole %, 9 mole %, 9.5 mole %, 10 mole %, 10.5 mole %, 11 mole %, 11.5 mole %, 12 mole %, 12.5 mole %, 13 mole %, 13.5
  • a lipid structure for a delivery vehicle can include a lipid bilayer.
  • a lipid bilayer can be generated of one or more compositions selected from the group consisting of a phospholipid, a phosphatidyl-choline, a phosphatidyl- serine, a phosphatidyl-diethanolamine, a phosphatidylinosite, a sphingolipid, and an ethoxylated sterol, or mixtures thereof.
  • the phospholipid can be a lecithin; the phosphatidylinosite can be derived from soy, rape, cotton seed, egg and mixtures thereof; the sphingolipid can be ceramide, a cerebroside, a sphingosine, and a sphingomyelin, and a mixture thereof; the ethoxylated sterol can be phytosterol, PEG- (polyethyleneglycol)-5 rapeseed sterol. In certain embodiments, the phytosterol comprises a mixture of at least two of the following compositions: sistosterol, camposterol and stigmasterol.
  • a lipid layer can be comprised of one or more phosphatidyl groups selected from the group comprising phosphatidyl choline, phosphatidyl- ethanolamine, phosphatidyl-serine, phosphatidyl- inositol, lyso- phosphatidyl-choline, lyso- phosphatidyl-ethanolamnine, lyso-phosphatidyl-inositol or lyso- phosphatidyl-inositol.
  • a lipid bilayer can be comprised of phospholipid selected from a monoacyl or diacylphosphoglyceride.
  • a lipid bilayer can be comprised of one or more phosphoinositides selected from the group comprising phosphatidyl-inositol-3 -phosphate (PI-3-P), phosphatidyl-inositol -4-phosphate (PI-4-P), phosphatidyl-inositol-5 -phosphate (PI- 5-P), phosphatidyl-inositol-3, 4-diphosphate (PI-3, 4-P2), phosphatidyl-inositol-3, 5- diphosphate (PI-3, 5-P2), phosphatidyl-inositol-4, 5-diphosphate (PI-4, 5-P2), phosphatidyl- inositol-3, 4, 5-triphosphate (PI-3,4,5-P3), lysophosphatidyl-inositol-3-phosphate (LPI-3-P), lysophosphatidyl-inositol -4-phosphate
  • Lipid structures used as delivery vehicles may be modified.
  • a modification can be a surface modification.
  • a surface modification can enhance an average rate at which a lipid structure moves in mucus compared to a comparable lipid structure.
  • a comparable lipid structure may not be surface modified, or a comparable lipid structure may be modified with a polyethylene glycol (PEG) polymer.
  • PEG polyethylene glycol
  • a modification can facilitate protection from degradation in vivo.
  • a modification may also assist in trafficking of a lipid structure. For example, a modification may allow a lipid structure to traffic within a gastrointestinal (GI) track with an acidic pH due to pH sensitive modifications.
  • GI gastrointestinal
  • a surface modification can also improve an average rate at which a lipid structure moves in mucous.
  • a modification may enhance a rate by IX, 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 20X, 30X, 40X, 5 OX, 60X, 70X, 80X, 90X, 100X, 300X, 500X, 700X, 900X, or up to about 1000X when compared to a comparable lipid structure without a modification or a lipid structure with a modification comprising PEG.
  • a modification to a lipid structure occurs via a bond.
  • a bond can be covalent, noncovalent, polar, ionic, hydrogen, or any combination thereof.
  • a bond can be considered an association of two groups or portions of groups.
  • a lipid structure can be bonded to a PEG via a linker comprising a covalent bond.
  • a bond can occur between two adjacent groups. Bonds can be dynamic. A dynamic bond can occur when one group temporarily associates with a second group.
  • a polynucleic acid in suspension within a liposome may bond with portions of a lipid bilayer during its suspension.
  • a modification can be a polyethylene glycol (PEG) addition.
  • PEG polyethylene glycol
  • Methods of modifying lipid structure surfaces with PEG can include its physical adsorption onto a lipid structure surface, its covalent attachment onto a lipid structure, its coating onto a lipid structure, or any combination thereof.
  • PEG can be covalently attached to a lipid particle before a lipid structure is formed.
  • a variety of molecular weights of PEG may be used.
  • PEG can range from about 10 to about 100 units of an ethylene PEG component which may be conjugated to phospholipid through an amine group comprising or comprising about 1% to about 20%, preferably about 5% to about 15%, about 10% by weight of the lipids which are included in a lipid structure.
  • a lipid structure can comprise a phosphatidylcholine.
  • exemplary phosphatidylcholines include but are not limited to dilauroyl phophatidylcholine, dimyristoylphophatidylcholine, dipalmitoylphophatidylcholine, distearoylphophatidyl- choline, diarachidoylphophatidylcholine, dioleoylphophatidylcholine, dilinoleoyl- phophatidylcholine, dierucoylphophatidylcholine, palmitoyl-oleoyl- phophatidylcholine, egg phosphatidylcholine, myristoyl- palmitoylphosphatidylcholine, palmitoyl-myristoyl- phdsphatidylcholine, myristoy
  • Asymmetric phosphatidylcholines can be referred to as 1-acyl, 2-acyl- sn-glycero-3-phosphocholines, wherein the acyl groups are different from each other.
  • Symmetric phosphatidylcholines can be referred to as l,2-diacyl-sn-glycero-3- phosphocholines.
  • PC refers to phosphatidylcholine.
  • the phosphatidylcholine l,2-dimyristoyl-sn-glycero-3-phosphocholine can be abbreviated herein as "DMPC.”
  • the phosphatidylcholine l,2-dioleoyl-sn-glycero-3- phosphocholine can be abbreviated herein as "DOPC.”
  • the phosphatidylcholine l,2-dipalmitoyl-sn-glycero-3- phosphocholine can be abbreviated herein as "DPPC.”
  • saturated acyl groups found in various lipids include groups having the names propionyl, butanoyl, pentanoyl, caproyl, heptanoyl, capryloyl, nonanoyl, capryl, undecanoyl, lauroyl, tridecanoyl, myristoyl, pentadecanoyl, palmitoyl, phytanoyl, hept
  • the corresponding IUPAC names for saturated acyl groups are trianoic, tetranoic, pentanoic, hexanoic, heptanoic, octanoic, nonanoic, decanoic, undecanoic, dodecanoic, tridecanoic, tetradecanoic, pentadecanoic, hexadecanoic, 3,7,11,15-tetramethylhexadecanoic, heptadecanoic, octadecanoic, nonadecanoic, eicosanoic, heneicosanoic, docosanoic, trocosanoic and tetracosanoic.
  • Unsaturated acyl groups found in both symmetric and asymmetric phosphatidylcholines include myristoleoyl, palmitoleyl, oleoyl, elaidoyl, linoleoyl, linolenoyl, eicosenoyl and arachidonoyl.
  • the corresponding IUPAC names for unsaturated acyl groups are 9-cis- tetradecanoic, 9-cis-hexadecanoic, 9-cis-octadecanoic, 9-trans- octadecanoic, 9-cis-12-cis- octadecadienoic, 9-cis-12-cis-15-cisoctadecatrienoic, 11-cis-eicosenoic and 5 ⁇ ci s-8 -ci s-11 -ci s- 14-cis- eicosatetraenoic.
  • Exemplary phosphatidylethanolamines include dimyristoyl- phosphatidylethanolamine, dipalmitoyl-phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dioleoyl-phosphatidylethanolamine and egg phosphatidylethanolamine.
  • Phosphatidylethanolamines may also be referred to under IUPAC naming systems as l,2-diacyl-sn-glycero-3- phosphoethanolamines or 1 -acyl-2-acyl-sn- glycero-3 -phosphoethanolamine, depending on whether they are symmetric or asymmetric lipids.
  • Exemplary phosphatidic acids include dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid and dioleoyl phosphatidic acid.
  • Phosphatidic acids may also be referred to under IUPAC naming systems as 1,2-diacyl-sn- glycero-3 -phosphate or l-acyl-2-acyl-sn- glycero-3 -phosphate, depending on whether they are symmetric or asymmetric lipids.
  • Exemplary phosphatidylserines include dimyristoyl phosphatidylserine, dipalmitoyl phosphatidylserine, dioleoylphosphatidylserine, distearoyl phosphatidylserine, palmitoyl- oleylphosphatidylserine and brain phosphatidylserine.
  • Phosphatidylserines may also be referred to under EJPAC naming systems as 1,2-diacyl-sn- glycero-3 -[phospho-L-serine] or l-acyl-2-acyl-sn-glycero-3- [phospho-L- serine], depending on whether they are symmetric or asymmetric lipids.
  • PS refers to phosphatidylserine.
  • Exemplary phosphatidylglycerols include dilauryloylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoyl- phosphatidylglycerol, dimyristoylphosphatidylglycerol, palmitoyl-oleoyl- phosphatidylglycerol and egg phosphatidylglycerol.
  • Phosphatidylglycerols may also be referred to under EJPAC naming systems as 1,2-diacyl-sn -glycero-3- [phospho-rac-(l- glycerol)] or 1 -acyl-2-acyl-sn-glycero-3- [phospho-rac- (1 -glycerol)], depending on whether they are symmetric or asymmetric lipids.
  • the phosphatidylglycerol 1,2-dimyristoyl-sn- glycero-3- [phospho-rac- (1- glycerol)] is abbreviated herein as "DMPG".
  • DPPG phosphatidylglycerol 1,2- dipalmitoyl-sn-glycero-3- (phospho-rac-1 -glycerol) (sodium salt)
  • Suitable sphingomyelins might include brain sphingomyelin, egg sphingomyelin, dipalmitoyl sphingomyelin, and distearoyl sphingomyelin.
  • Other suitable lipids include glycolipids, sphingolipids, ether lipids, glycolipids such as the cerebrosides and gangliosides, and sterols, such as cholesterol or ergosterol.
  • a lipid structure can comprise cholesterol or a derivative thereof, a phospholipid, a mixture of a phospholipid and cholesterol or a derivative thereof, or a combination.
  • cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'- hydroxybutyl ether, and mixtures thereof.
  • the lipid structure may comprise up to about 40, 50, or 60 mol % of the total lipid present in the lipid structure.
  • One or more phospholipids and/or cholesterol may comprise from about 10 mol % to about 60 mol %, from about 15 mol % to about 60 mol %, from about 20 mol % to about 60 mol %, from about 25 mol % to about 60 mol %, from about 30 mol % to about 60 mol %, from about 10 mol % to about 55 mol %, from about 15 mol % to about 55 mol %, from about 20 mol % to about 55 mol %, from about 25 mol % to about 55 mol %, from about 30 mol % to about 55 mol %, from about 13 mol % to about 50 mol %, from about 15 mol % to about 50 mol % or from about 20 mol % to about 50 mol % of the total lipid present in the lipid structure.
  • a delivery vehicle herein is designed to be internalized in an epithelial cell, such as an epithelial cell within the gastrointestinal tract.
  • Peptides in particular, cell penetrating peptides (CPPs) and cell penetrating peptides having mucus- penetrating functionality (MPPs) provide for internalization into the cell.
  • the delivery vehicles herein such as the lipid structures described herein for such purposes further comprise mucus-penetrating peptide (MPPs), cell- penetrating peptides (CPP), or both.
  • cell penetrating peptides can be short polypeptides that can allow for increased uptake of delivery vehicles and/or cargo into cells.
  • Cell-penetrating peptides can be peptide sequences that facilitate crossing the cytoplasmic membrane efficiently.
  • Exemplary CPPs and MPPs include those disclosed in PCT/US17/61111 and PCT/US2019/032484 (e.g., any of those listed in Table 3 thereof), each of which are herein incorporated by reference.
  • MPPs mucus-penetrating cell-penetrating peptides
  • MPPs have cell-penetrating properties and in addition, permit penetration through a layer of mucus such as the naturally- occurring layers of mucus in the colon, lung, eye and cervix.
  • MPP can further be used to target structures to intracellular components of cells. They can also be designed to specifically target certain cell types.
  • MPPs can be conjugated to delivery vehicles to allow penetration of the particles through the mucus layer and also for interaction with cells so as to result in increased penetration or targeting of cells.
  • a lipid structure that has an MPP can be internalized into a cell with an efficacy of at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to about 100% as compared to a comparable particles that does not contain an MPP.
  • a delivery vehicle can comprise a mucus-penetrating peptide (MPP).
  • the MPP may be conjugated to the lipid structure, such as conjugated to a lipid nanoparticle, a surface modification of the lipid nanoparticle or the cargo, such that the MPP is exposed such that it may come into contact, in whole or in part, with a mucus layer, mucus-containing tissue, organ or extracellular surface.
  • the presence of the MPP can confer improved penetration of the delivery vehicle through the mucus (diffusion and/or movement through).
  • an MPP can have an amino acid sequence having from about 3 to 100 amino acids, including without limitation from about 3 to 5, 5 to 10, 10 to 20, 20 to 40, 30 to 60, or 80 to 100 amino acids.
  • An MPP can have from about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
  • an MPP may have the ability to penetrate a mucus-layer that overlays or surrounds a target cell or tissue.
  • An MPP can be employed to penetrate the mucus layer of a target tissue such as the intestinal epithelium, colon, lung, eye or cervix of a mammal.
  • MPPs can be conjugated to delivery vehicles, including nanoparticles, to allow penetration of the delivery vehicle through the mucus layer and also for interaction with cells so as to result in increased penetration or targeting of cells.
  • a particle that has an MPP permeates a mucus layer with an efficacy of at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to about 100% as compared to a comparable particles that does not contain an MPP.
  • Numerous methods of determining the penetration of a mucus layer can be used to assess the penetration by an MPP or an MPP conjugated directly or indirectly with a delivery vehicle.
  • a lipid structure can be a mucus-penetrating particle or MPP as used herein, can refer to particles which have been coated with a mucosal penetration enhancing coating.
  • a particle can be or can deliver a particle of an active agent, such as a therapeutic, diagnostic, prophylactic, and/or nutraceutical agent (i.e., drug particle) that can be coated with a mucosal penetrating enhancing coating.
  • particles can be formed of a matrix material, such as a polymeric material, in which a therapeutic, diagnostic, prophylactic, and/or nutraceutical agent can be encapsulated, dispersed, and/or associated.
  • a delivery vehicle can further comprise at least one targeting agent.
  • the term targeting agent can refer to a moiety, compound, antibody, etc. that specifically binds a particular type or category of cell and/or other particular type compounds, ( e.g ., a moiety that targets a specific cell or type of cell).
  • a targeting agent can be specific ( e.g ., have an affinity) for the surface of certain target cells, a target cell surface antigen, a target cell receptor, or a combination thereof.
  • a targeting agent can refer to an agent that has a particular action (e.g., cleaves) when exposed to a particular type or category of substances and/or cells, and this action can drive the delivery vehicle to target a particular type or category of cell.
  • the term targeting agent can refer to an agent that can be part of a delivery vehicle and plays a role in the delivery vehicle’s targeting mechanism, although the agent itself may or may not be specific for the particular type or category of cell itself.
  • the efficiency of the cellular uptake of a polynucleic acid delivered by a delivery vehicle can be enhanced and/or made more specific by incorporation of targeting agents into the present delivery vehicles.
  • delivery vehicles described herein can comprise one or more small molecule targeting agents (e.g ., carbohydrate moieties).
  • Suitable targeting agents also include, by way of non-limiting example, antibodies, antibody-like molecules, or peptides, such as an integrin-binding peptides such as RGD- containing peptides, or small molecules, such as vitamins, e.g., folate, sugars such as lactose and galactose, or other small molecules.
  • Cell surface antigens include a cell surface molecule such as a protein, sugar, lipid or other antigen on the cell surface. In specific embodiments, the cell surface antigen undergoes internalization.
  • Examples of cell surface antigens targeted by the targeting agents of embodiments of the present delivery vehicles include, but are not limited, to the transferrin receptor type 1 and 2, the EGF receptor, HER2/Neu, VEGF receptors, integrins, NGF, CD2, CD3, CD4, CDS, CDI9, CD20, CD22, CD33, CD43).
  • a targeting agent can also comprise an artificial affinity molecule, e.g., a peptidomimetic or an aptamer.
  • Peptidomimetics can refer to compounds in which at least a portion of a peptide, such as a therapeutic peptide, is modified, and the three-dimensional structure of the peptidomimetic remains substantially the same as that of the peptide.
  • Peptidomimetics both peptide and non-peptidyl analogues
  • Peptidomimetics may have improved properties (e.g., decreased proteolysis, increased retention or increased bioavailability).
  • Peptidomimetics generally have improved oral availability, which makes them especially suited to treatment of disorders in a human or animal. It should be noted that peptidomimetics may or may not have similar two- dimensional chemical structures but share common three-dimensional structural features and geometry.
  • the targeting agent can be a proteinaceous targeting agent (e.g., a peptide, and antibody, an antibody fragment).
  • a delivery vehicle can comprise a plurality of different targeting agents.
  • a lipid structure modification can provide biocompatibility and can be modified to possess targeting species including, for example, targeting peptides including antibodies, aptamers, polyethylene, or combinations thereof.
  • a targeting agent be a receptor.
  • a T cell receptor (TCR), B cell receptor (BCR), single chain variable fragment (scFv), chimeric antigen receptor (CAR), or combinations thereof are used as targeting agents.
  • one or more targeting agents can be coupled to the polymers that form the delivery vehicle.
  • the targeting agents can be bound to a polymer that coats a delivery vehicle.
  • a targeting agent can be covalently coupled to a polymer.
  • a targeting agent can be bound to a polymer such that a targeting agent can be substantially at or near the surface of the resulting delivery vehicle.
  • a monomer comprising a targeting agent residue e.g ., a polymerizable derivative of a targeting agent such as an (alkyl) acrylic acid derivative of a peptide
  • a targeting agent residue e.g ., a polymerizable derivative of a targeting agent such as an (alkyl) acrylic acid derivative of a peptide
  • one or more targeting agents can be coupled to the polymer of the present delivery vehicles through a linking moiety.
  • the linking moiety coupling the targeting agent to the membrane -destabilizing polymer can be a cleavable linking moiety (e.g., comprises a cleavable bond).
  • the linking moiety can be cleavable and/or comprises a bond that can be cleavable in endosomal conditions.
  • the linking moiety can be cleavable and/or comprise a bond that can be cleaved by a specific enzyme (e.g., a phosphatase, or a protease).
  • the linking moiety can be cleavable and/or comprise a bond that may be cleavable upon a change in an intracellular parameter (e.g., pH, redox potential), in some embodiments, a linking moiety can be cleavable and/or comprise a bond that can be cleaved upon exposure to a matrix metalloproteinase (MMP) (e.g., MMP -cleavable peptide linking moiety).
  • MMP matrix metalloproteinase
  • a targeting mechanism of a delivery vehicle can depend on a cleavage of a cleavable segment in a polymer.
  • the present polymers can comprise a cleavable segment that, when cleaved, exposes the delivery vehicle and/or the core of a delivery vehicle.
  • the cleavable segment can be located at either or both terminal ends of the present polymers in some embodiments.
  • the cleavable segment is located along a length of a polymer, and optionally can be located between blocks of a polymer.
  • the cleavable segment can be located between a first block and a second block of a polymer, and when a delivery vehicle can be exposed to a particular cleaving substance the first block can be cleaved from a second block.
  • a cleavable segment can be an MMP-cleavable peptide that can be cleaved upon exposure to MMP.
  • Attachment of a targeting agent, such as an antibody or a peptide, to a polymer or a lipid can be achieved in any suitable manner, e.g., by any one of a number of conjugation chemistry approaches including but not limited to amine-carboxyl linkers, amine-sulfhydryl linkers, amine-carbohydrate linkers, amine-hydroxyl linkers, amine-amine linkers, carboxyl - sulfhydryl linkers, carboxyl-carbohydrate linkers, carboxyl-hydroxyl linkers, carboxyl- carboxyl linkers, sulfhydryl-carbohydrate linkers, sulfhydryl -hydroxyl tinkers, sulfhydryl- sulfhydryl linkers, carbohydrate-hydroxyl linkers, carbohydrate-carbohydrate linkers, and hydroxyl-hydroxyl linkers.
  • "click" chemistry can be used to attach the targeting agent to the polymers of the delivery vehicles provided herein.
  • targeting agents can be attached to a monomer and the resulting compound can then be used in a polymerization synthesis of a polymer ( e.g ., copolymer) utilized in a delivery vehicle described herein.
  • a targeting agent can be attached to the sense or antisense strand of siRNA bound to a polymer of a delivery vehicle.
  • a targeting agent can be attached to a 5' or a 3' end of the sense or the antisense strand.
  • Methods for linking compounds can include but are not limited to proteins, labels, and other chemical entities, to nucleotides.
  • Cross-linking reagents such as n- maleimidobutyryloxy-succinimide ester (GMBS) and sulfo-GMBS, have reduced immunogenicity.
  • Substituents have been attached to the 5' end of preconstructed oligonucleotides using amidite or H-phosphonate chemistry. Substituents can also be attached to the 3' end of oligomers.
  • an oligonucleotide may include one or more modified nucleotides having a group attached via a linker arm to the base.
  • the attachment of biotin to the C-5 position of dUTP by an allylamine linker arm may be utilized.
  • the attachment of biotin and other groups to the 5-position of pyrimidines via a linker arm may also be performed.
  • Chemical cross-linking may include the use of spacer arms, i.e., linkers or tethers.
  • Spacer arms provide intramolecular flexibility or adjust intramolecular distances between conjugated moieties and thereby may help preserve biological activity.
  • a spacer arm may be in the form of a peptide moiety comprising spacer amino acids.
  • a spacer arm may be part of the cross-linking reagent, such as in “long-chain SPDP”.
  • a variety of coupling or crosslinking agents such as protein A, carbodiimide, dimaleimide, dithio-bis-nitrobenzoic acid (DTNB), N-succinimidyl-5-acetyl-thioacetate (SATA), and N-succinimidyl-3-(2-pyrid-yldithio)propionate (SPDP), 6-hydrazinonicotimide (HYNIC), N3S and N2S2 can be used in well-known procedures to synthesize targeted constructs.
  • biotin can be conjugated to an oligonucleotide via DTPA using a bicyclic anhydride method.
  • biotin a lysine conjugate of biotin
  • NHS- LC-biotin which can be purchased from Pierce Chemical Co. Rockford, Ill.
  • biocytin a lysine conjugate of biotin
  • biotin acid chloride or acid precursors can be coupled with an amino derivative of the therapeutic agent by known methods.
  • a biotin moiety By coupling a biotin moiety to the surface of a particle, another moiety may be coupled to avidin and then coupled to the particle by the strong avidin-biotin affinity, or vice versa.
  • the free hydroxyl group of PEG may be used for linkage or attachment (e.g ., covalent attachment) of additional molecules or moieties to the particle.
  • the lipid structures herein (delivery vehicles) have sizes can fall into the nanometer to micrometer range, such as 20-200 nm, 200 nm-1 pm.
  • a polynucleic acid may be condensed to be properly encapsulated by a lipid structure.
  • Condensation of DNA may be performed by divalent metal ions such as Mn 2+ , Ni 2+ , Co 2+ , and Cu 2+ that can condense DNA through neutralization of phosphate groups of the DNA backbone and distortion of the B-DNA structure through hydrogen bonding with bases, permitting both local bending of the DNA and inter-helical associations.
  • the concentration of metal ions utilized for condensation can be dependent on the dielectric constant of a medium used in the condensation.
  • the addition of ethanol or methanol may also reduce the concentration of metal ion required for condensation.
  • ethanol can be used to condense DNA at concentrations from about 0.5% up to about 60% by volume.
  • ethanol can be used to condense DNA at concentrations from about 0.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or up to 60% by volume.
  • calcium may also be used for condensation. Calcium not only binds to DNA phosphates but can also form a complex with the nitrogen and oxygen of guanine, disrupting base pairing.
  • a polynucleic acid can be fully encapsulated in a lipid structure.
  • Full encapsulation can indicate that a polynucleic acid in a lipid structures may not be significantly degraded after exposure to serum or a nuclease or protease assay that would significantly degrade free DNA, RNA, or protein.
  • a fully encapsulated system preferably less than about 25% of a polynucleic acid in a lipid structure can be degraded in a treatment that would normally degrade 100% of free polynucleic acid, more preferably less than about 10%, and most preferably less than about 5% of a polynucleic acid in a lipid structure can be degraded.
  • full encapsulation may be determined by an Oligreen® assay.
  • Oligreen® is an ultra-sensitive fluorescent nucleic acid stain for quantitating oligonucleotides and single-stranded DNA or RNA in solution (available from Invitrogen Corporation; Carlsbad, Calif.).
  • Fluly encapsulated can also indicate that a lipid structure may be serum-stable, that is, that they do not rapidly decompose into their component parts upon in vivo administration.
  • a moiety can be utilized to identify a number of cells that have received a polynucleic acid.
  • a moiety can be an antibody, dye, scFv, peptide, glycoprotein, carbohydrate, ligand, polymer, to name a few.
  • a moiety can be in contact with a linker.
  • a linker can be non-cleavable. Accordingly, in some cases, a linker can be a cleavable linker. This may enable a moiety to be released from a lipid structure once contact to a target cell has been made.
  • a moiety may have a better ability to be absorbed by an intracellular component of a cell, such as an intestinal crypt cell or intestinal crypt stem cell, when separated from a lipid structure.
  • a linker may comprise a disulfide bond, acyl hydrazone, vinyl ether, orthoester, or aN-P03.
  • Cleavage of a linker releasing a moiety may be as a result of a change in conditions within a cell as compared to outside cells, for example, due to a change in pH within a cell. Cleavage of a linker may occur due to the presence of an enzyme within a cell which cleaves a linker once a drug, such as a polynucleic acid, enters a cell. Alternatively, cleavage of a linker may occur in response to energy or a chemical being applied to the cell.
  • a linker may be a photolabile linker.
  • a linker used to link a complex may also be an acid labile linker.
  • acid labile linkers include linkers formed by using cis-aconitic acid, cis-carboxylic alkatriene, polymaleic anhydride, and other acid labile linkers.
  • a lipid structure such as a liposome can be biocompatible and biodegradable.
  • a liposome may biodegrade after introduction into a subject. Biodegradation can begin immediately after introduction in some cases. Biodegradation can occur within a mucosal tract of a subject that has received an administration of a liposome or liposomal structure. Biodegradation can result release of a liposomal cargo such as a polynucleic acid.
  • biodegradation can comprise decomposition of a component of a liposomal structure such as a polymer. Biodegradation can occur under standard bodily conditions such as from about 97.6 °F to about 99 °F.
  • biodegradation can occur under a temperature from about 95 °F to about 106 °F.
  • Biodegradation can occur from about 95 °F, 96 °F, 97 °F, 98 °F, 99 °F, 100 °F, 101 °F, 102 °F, 103°F, 104 °F, 105 °F, or up to 106 °F.
  • biodegradation can occur from about 50 °F to about 150 °F.
  • biodegradation may not occur.
  • biodegradation can take from about 1 minute to about 100 years after administration of a liposome or a structure to a subject.
  • Biodegradation can take from about 1 minute, 5 minutes, 30 minutes, 1 hour, 3 hours, 7 hours, 10 hours, 15 hours, 20 hours, 25 hours, 2 days, 4 days, 8 days, 12 days, 20 days, 30 days, 1.5 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1.5 yrs., 3 years, 5 years, 8 years, 10 years, 15 years, 20 years, 30 years, 40 years, 50 years, 60 years, 70 years, 80 years, 90 years, or at least about 100 years.
  • Lipid of a structure such as a liposome may be or may comprise: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits); sterol lipids prenol lipids (derived from condensation of isoprene subunits) or any combination thereof.
  • a cargo can comprise a therapeutic agent.
  • therapeutic agents can comprise: a nucleic acid, a protein, an antibody, a peptide, a small molecule, a biologic, an antisense oligonucleotide, peptidomimetics, ribozymes, a chemical agent such as a chemotherapeutic molecule, or any large molecule including, but not limited to, viral particles, growth factors cytokines, immunomodulating agents, small molecule drugs, fluorescent dyes, including fluorescent dye peptides which may be expressed by a DNA incorporated within a liposome, or any combination thereof.
  • a cargo can be a nucleic acid.
  • a nucleic acid can be DNA- or RNA- based.
  • a nucleic acid can be a vector.
  • DNA-based vectors can be non-viral, and can include molecules such as plasmids, minicircles, nanoplasmid, closed linear DNA (doggybone), linear DNA, and single-stranded DNA.
  • a nucleic acid that can be present in a lipid-nucleic acid particle includes any form of nucleic acid that is known.
  • the nucleic acids used herein can be single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrids.
  • double-stranded DNA examples include structural genes, genes including control and termination regions, and self-replicating systems such as viral or plasmid DNA.
  • double-stranded RNA examples include siRNA and other RNA interference reagents.
  • Single-stranded nucleic acids include antisense oligonucleotides, ribozymes, microRNA, and triplex-forming oligonucleotides.
  • the nucleic acid that is present in a lipid-nucleic acid particle may include one or more of the oligonucleotide modifications described below. Nucleic acids may be of various lengths, generally dependent upon the particular form of nucleic acid.
  • plasmids or genes may be from about 1,000 to 100,000 nucleotide residues in length.
  • oligonucleotides may range from about 10 to 100 nucleotides in length.
  • oligonucleotides, single-stranded, double-stranded, and triple-stranded may range in length from about 10 to about 50 nucleotides, from about 20 to about 50 nucleotides, from about 15 to about 30 nucleotides, from about 20 to about 30 nucleotides in length.
  • oligonucleotides may range from about 2 nucleotides to 10 nucleotides in length.
  • DNA-based vectors can also be viral, and include adeno-associated virus, lentivirus, adenovirus, and others.
  • Vectors can also be RNA.
  • RNA vectors can be linear or circular forms of unmodified RNA. They can also include various nucleotide modifications designed to increase half-life, decrease immunogenicity, and/or increase level of translation.
  • a vector as used herein can be composed of either DNA or RNA.
  • a vector can be composed of DNA.
  • Vectors can be capable of autonomous replication in a prokaryote such as E. coli , used for growth.
  • a vector may be stably integrated into a genome of an organism.
  • a vector can remain separate, either in a cytoplasm or a nucleus.
  • a vector can contain a targeting sequence.
  • a vector can contain an antibiotic resistance gene.
  • a vector can contain regulatory elements for regulating gene expression.
  • a mini-circle can be enclosed within a delivery vehicle.
  • Minicircle (MC) DNA can be delivered as cargo by a vehicle provided herein.
  • MC can be similar to plasmid DNA as both may contain expression cassettes that may permit transgene products to be made at high levels shortly after delivery.
  • a MC can differ in that MC DNA can be devoid of prokaryotic sequence elements (e.g ., bacterial origin of replication and antibiotic-resistance genes). Removal of prokaryotic sequence elements from a backbone plasmid DNA can be achieved via site- specific recombination in Escherichia coli before episomal DNA isolation.
  • a mini circle DNA can be free of a bacterial origin of replication.
  • a mini circle DNA or closed linear DNA can be free of a bacterial origin of replication from about 50% of a bacterial origin of replication sequence or up to 100% of a bacterial origin of replication.
  • a bacterial origin of replication is truncated or inactive.
  • a polynucleic acid can be derived from a vector that initially encoded a bacterial origin of replication.
  • a method can be utilized to remove the entirety of a bacterial origin of replication or a portion thereof, leaving a polynucleic acid free of a bacterial origin of replication.
  • a bacterial origin of replication can be identified by its high adenine and thymine content.
  • Minicircle DNA vectors can be supercoiled minimal expression cassettes, derived from conventional plasmid DNA by site-specific recombination in vivo in Escherichia coli for the use in non- viral gene therapy and vaccination.
  • Minicircle DNA may lack or have reduced bacterial backbone sequences such as an antibiotic resistance gene, an origin of replication, and/or inflammatory sequences intrinsic to bacterial DNA. In addition to their improved safety profile, minicircles can greatly increase efficiency of transgene expression.
  • a portion of a gene can be delivered by a polynucleic acid cargo.
  • a portion of a gene can be from three nucleotides up to the entire whole genomic sequence.
  • a portion of a gene can be from about 1% up to about 100% of an endogenous genomic sequence.
  • a portion of a gene can be from about 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to about 100% of a whole genomic sequence of a gene.
  • a variety of protein and polypeptides can be delivered as cargo by vehicles described herein including but not limited to proteins for treating metabolic disorders and endocrine disorders.
  • proteins are phenylalanine hydroxylase, insulin, anti diuretic hormone and growth hormone.
  • Disorders include phenylketonuria, diabetes, organic acidurias, tyrosinemia, urea cycle disorders, familial hypercholesteremia.
  • Genes for any of the proteins or peptides which can correct the defects in phenylketonuria, diabetes, organic acidurias, tyrosinemia, urea cycle disorders, familial hypercholesteremia can be introduced into stem cells such that the protein or peptide products are expressed by the intestinal epithelium.
  • Coagulation factors such as antihemophilic factor (factor 8), Christmas factor (factor 9) and factor 7 can likewise be produced in the intestinal epithelium.
  • Proteins which can be used to treat deficiency of a circulatory protein can also be expressed in the intestinal epithelium. Proteins which can be used to treat deficiency of a circulatory protein can be, for example, albumin for the treatment of an albuminemia, alpha- 1 -antitrypsin, hormone binding protein.
  • the intestinal symptoms of cystic fibrosis can be treated by inserting the gene for the normal cystic fibrosis transmembrane conductance regulator into the stem cells of intestinal epithelium.
  • Abetalipoproteinemia can be treated by the insertion of the apolipoprotein B.
  • Disaccharidase intolerance can be treated by the insertion of sucrase- isomaltose, lactase-phlorizin hydrolase and maltase-glucoamylase.
  • the insertion of the intrinsic factor for the absorption of vitamin B12 or the receptor for the intrinsic factor/cobalamin complex for absorption of vitamin B12, as well as the transporter for bile acids can be inserted into the intestinal epithelium.
  • any drug which can be encoded by nucleic acid can be inserted into the stem cell of the intestinal epithelium to be secreted in localized, high concentrations for the treatment of cancer.
  • antisense RNA can be encoded into the stem cells after production of antisense it can incorporate into the cancerous cells for the treatment of cancer.
  • a therapeutic agent or drug can be a small molecule, protein, polysaccharide or saccharide, nucleic acid molecule, lipid, peptidomimetic, or a combination thereof.
  • a delivery vehicle can include any molecule or compound capable of exerting a desired effect on a cell, tissue, organ, or subject. Such effects may be biological, physiological, or cosmetic, for example.
  • Molecules or compounds may include e.g., nucleic acids, peptides and polypeptides, including, e.g., antibodies, such as, e.g., polyclonal antibodies, monoclonal antibodies, antibody fragments; humanized antibodies, recombinant antibodies, recombinant human antibodies, and PrimatizedTM antibodies, cytokines, growth factors, apoptotic factors, differentiation-inducing factors, cell surface receptors and their ligands; hormones; and small molecules, including small organic molecules or compounds.
  • a molecules or compound can be a therapeutic agent, or a salt or derivative thereof.
  • Therapeutic agent derivatives may be therapeutically active themselves or they may be prodrugs, which become active upon further modification.
  • a molecules or compound derivative may retain some or all of the therapeutic activity as compared to the unmodified agent, while in another embodiment, a therapeutic derivative lacks therapeutic activity.
  • therapeutic agents include any therapeutically effective agent or drug, such as anti-inflammatory compounds, anti-depressants, stimulants, analgesics, antibiotics, birth control medication, antipyretics, vasodilators, anti-angiogenics, cytovascular agents, signal transduction inhibitors, cardiovascular drugs, e.g, anti -arrhythmic agents, vasoconstrictors, hormones, and steroids.
  • a molecule or compound can be an oncology drug, which may also be referred to as an anti-tumor drug, an anti-cancer drug, a tumor drug, an antineoplastic agent, or the like.
  • oncology drugs examples include, but are not limited to, adriamycin, alkeran, allopurinal, altretamine, amifostine, anastrozole, araC, arsenic trioxide, azathioprine, bexarotene, biCNU, bleomycin, busulfan intravenous, busulfan oral, capecitabine (Xeloda), carboplatin, carmustine, CCNU, celecoxib, chlorambucil, cisplatin, cladribine, cyclosporin A, cytarabine, cytosine arabinoside, daunorubicin, cytoxan, daunorubicin, dexamethasone, dexrazoxane, dodetaxel, doxorubicin, doxorubicin, DTIC, epirubicin, estramustine, etoposide phosphate, etoposide and VP-16,
  • a polynucleic acid for use as a cargo with the delivery vehicles herein include nucleic acids encoding for a tumor-suppressor gene.
  • a tumor-suppressor gene can generally encode for a protein that in one way or another can inhibit cell proliferation. Loss of one or more of these “brakes” may contribute to the development of a cancer.
  • Intracellular proteins such as the pl6 cyclin-kinase inhibitor, that can regulate or inhibit progression through a specific stage of the cell cycle, receptors for secreted hormones (e.g ., tumor derived growth factor b) that may function to inhibit cell proliferation, checkpoint-control proteins that arrest the cell cycle if DNA may be damaged or chromosomes are abnormal, proteins that can promote apoptosis, enzymes that participate in DNA repair, or a combination thereof.
  • DNA-repair enzymes may not directly function to inhibit cell proliferation, cells that have lost the ability to repair errors, gaps, or broken ends in DNA accumulate mutations in many genes, including those that are critical in controlling cell growth and proliferation.
  • loss-of-function mutations in the genes encoding DNA-repair enzymes may promote inactivation of other tumor-suppressor genes as well as activation of oncogenes. Since generally one copy of a tumor-suppressor gene suffices to control cell proliferation, both alleles of a tumor-suppressor gene must be lost or inactivated in order to promote tumor development.
  • oncogenic loss-of-function mutations in tumor-suppressor genes act recessively.
  • Tumor-suppressor genes in many cancers have deletions or point mutations that prevent production of any protein or lead to production of a nonfunctional protein.
  • introducing a tumor suppressor gene encoding for a protein may ameliorate disease, prevent disease, or treat disease in a subject.
  • a tumor suppressor gene that can be delivered by a delivery vehicle herein includes, for example, APC, ARHGEF12, ATM, BCL11B, BLM, BMPR1A, BRCA1, BRCA2, CARS, CBFA2T3, CDH1, CDH11, CDK6, CDKN2C, CEBPA, CHEK2, CREBl, CREBBP, CYLD, DDX5, EXT1, EXT2, FBXW7, FH, FLT3, FOXP1, GPC3, IDH1, IL2, JAK2, MAP2K4, MDM4, MENl, MLHl, MSH2, NF1, NF2, NOTCH1, NPM1, NR4A3, NUP98, PALB2, PML, PTEN, RBI, RUNX1, SDHB, SDHD, SMARCA4, SMARCBl, SOCS1, STK11, SUFU, SUZ12, SYK, TCF3, TNFAIP3, TP53, TSC1, TSC2, VHL,
  • a vehicle can comprise an imaging agent that may be further attached to a detectable label (e.g ., the label can be a radioisotope, fluorescent compound, enzyme or enzyme co-factor).
  • the active moiety may be a radioactive agent, such as: radioactive heavy metals such as iron chelates, radioactive chelates of gadolinium or manganese, positron emitters of oxygen, nitrogen, iron, carbon, or gallium, 43 K, 52 Fe, 57 Co, 67 Cu, 67 Ga, 68 Ga, 123 I, 125 I, 131 I, 132 I, or "Tc.
  • a delivery vehicle including such a moiety may be used as an imaging agent and be administered in an amount effective for diagnostic use in a mammal such as a human.
  • the localization and accumulation of the imaging agent can be detected.
  • the localization and accumulation of the imaging agent may be detected by radioscintiography, nuclear magnetic resonance imaging, computed tomography, or positron emission tomography.
  • the amount of radioisotope to be administered is dependent upon the radioisotope.
  • Those having ordinary skill in the art can readily formulate the amount of the imaging agent to be administered based upon the specific activity and energy of a given radionuclide used as the active moiety.
  • compositions useful as imaging agents can comprise a targeting moiety conjugated to a radioactive moiety that can comprise 0.1-100 millicuries, in some embodiments preferably 1-10 millicuries, in some embodiments preferably 2-5 millicuries, in some embodiments more preferably 1-5 millicuries.
  • the means of detection used to detect the label is dependent of the nature of the label used and the nature of the biological sample used, and may also include fluorescence polarization, high performance liquid chromatography, antibody capture, gel electrophoresis, differential precipitation, organic extraction, size exclusion chromatography, fluorescence microscopy, or fluorescence activated cell sorting (FACS) assay.
  • a targeting moiety can also refer to a protein, nucleic acid, nucleic acid analog, carbohydrate, or small molecule.
  • the entity may be, for example, a therapeutic compound such as a small molecule, or a diagnostic entity such as a detectable label.
  • a locale may be a tissue, a particular cell type, or a subcellular compartment.
  • the targeting moiety can direct the localization of an active entity.
  • the active entity may be a small molecule, protein, polymer, or metal.
  • the active entity such as a liposome comprising a nucleic acid, may be useful for therapeutic, prophylactic, or diagnostic purposes.
  • a moiety may allow a delivery vehicle to penetrate a blood brain barrier.
  • a cargo can be a drug.
  • a drug can be a substance that when administered can cause a physiological change in a subject.
  • a drug can be a medication used to treat a disease, such as cancer.
  • drugs can be entrapped completely in a liposomal lipid bilayer, in an aqueous compartment, or in both a liposomal lipid bilayer and an aqueous compartment. Strongly lipophilic drugs can be entrapped almost completely in a lipid bilayer. Strongly hydrophilic drugs can be located exclusively in an aqueous compartment.
  • Drugs with intermediate logP can easily partition between a lipid and aqueous phases, both in a bilayer and in an aqueous core.
  • Exemplary drugs can comprise drugs such as adalimumab, anti-TNF, insulin-like growth factor, interleukin, Mesalamine, GLP-1 analogs, GLP-2 analogs, and combinations thereof.
  • a polynucleic acid can encode for a heterologous sequence.
  • a heterologous sequence can provide for subcellular localization (e.g ., a nuclear localization signal (NLS) for targeting to a nucleus; a mitochondrial localization signal for targeting to a mitochondria; a chloroplast localization signal for targeting to a chloroplast; an ER retention signal; and the like).
  • a polynucleic acid such as minicircle DNA or closed linear DNA, can comprise a nuclear localization sequence (NLS).
  • a cargo can comprise one or more nuclear localization sequences (NLSs).
  • NLS sequences can be from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs.
  • a vector comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy -terminus, or a combination of these (e.g. one or more NLS at the amino-terminus and one or more NLS at the carboxy terminus).
  • NLSs can include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 1); the NLS from nucleoplasmin (e.g.
  • nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 2)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 3) or RQRRNELKRSP (SEQ ID NO: 4); the hRNPAl M9 NLS having the sequence NQ S SNF GPMKGGNF GGRS S GP Y GGGGQ YF AKPRNQGGY (SEQ ID NO: 5); the sequence RMRIZFKNKGKDT AELRRRRVE V S VELRKAKKDEQILKRRNV (SEQ ID NO: 6) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 7) and PPKKARED (SEQ ID NO: 8) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 9) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 10) of mouse
  • the one or more NLSs can be of sufficient strength to drive accumulation of the minicircle DNA vector or short linear DNA vector in a detectable amount in the nucleus of a eukaryotic cell.
  • a eukaryotic cell can be a human intestinal crypt cell.
  • a particle may contain a DNAse inhibitor.
  • a DNAse inhibitor may be localized within a particle or on a particle.
  • a polynucleic acid encoding for an inhibitor can be enclosed within a particle.
  • an inhibitor can be a DNA methyltransferase inhibitor such as DNA methyltransferase inhibitors-2 (. DMI-2 '). DMI-2 can be produced by Streptomyces sp. strain No. 560.
  • a structure of DMI-2 can be 4"'R, 6aR,l OS, 10aS-8-acetyl-6a,10a-dihydroxy-2-methoxy-12-mefhyl-10-[4'-[3 ''-hydroxy s'', 5 ''-dimethyl-4" (Z- 2'",4'''-dimethyl-2'"-heptenoyloxy) tetrahydropyran-l"-yloxy]-5'- methylcyclohexan-l'-yloxy] -l,4,6,7,9-pentaoxo-l,4,6,6a,7,8,9,10,10a,ll- decahydronaphthacen
  • Other inhibitors, such as chloroquine can also be enclosed within a particle or on a particle, such as on a surface of a particle.
  • Detection of accumulation in the nucleus may be performed by any suitable technique.
  • a detectable marker may be fused to a vector, such that location within a cell may be visualized, such as in combination with a means for detecting the location of the nucleus (e.g . a stain specific for the nucleus such as DAPI).
  • Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay.
  • An embodiment herein can exhibit time dependent pH triggered release of a cargo into a target site.
  • An embodiment herein can contain and provide cellular delivery of complex multiple cargoes.
  • An additional cargo can be a small molecule, an antibody, an inhibitor such as a DNAse inhibitor or RNAse inhibitor.
  • a lipid structure can carry to a capacity up to over 100 % weight: defined as (cargo weight/weight of the lipid structure) x 100.
  • the optimal loading of cargo can be or can be from about 1 % to 100% weight of a lipid structure.
  • a lipid structure can contain a polynucleic acid cargo from about 1% weight of a structure to about 10%, from about 10% to about 20%, from about 20% to about 30%, from about 30% to about 40%, from about 40% to about 50%, from about 50%, to about 60%, from about 60% to about 70%, from about 70% to about 80%, from about 80% to about 90%, from about 90% to about 100%, from about 100% to about 200%, from about 200% to about 300%, from about 300% to about 400%, from about 400% to about 500% or greater weight of a structure.
  • Polynucleic acids can be delivered to cells of the intestinal tract.
  • a polynucleic acid can be delivered by the delivery vehicles herein to an intestinal crypt stem cell.
  • a delivered polynucleic acid can be: (1) not normally found in intestinal epithelial stem cells; (2) normally found in intestinal epithelial stem cells, but not expressed at physiological significant levels; (3) normally found in intestinal epithelial stem cells and normally expressed at physiological desired levels in the stem cells or their progeny; (4) any other DNA which can be modified for expression in intestinal epithelial stem cells; and (5) any combination of the above.
  • a protein that is encoded by a polynucleic acid comprised within a lipid structure can be measured and quantified.
  • modified cells can be isolated, and a western blot performed on modified cells to determine a presence and a relative amount of protein production as compared to unmodified cells.
  • intracellular staining of a protein utilizing flow cytometry can be performed to determine a presence and a relative amount of protein production. Additional assays can also be performed to determine if a protein, such as APC, is functional. For example, modified cells expressing an APC transgene, can be measured for cytosolic b-catenin expression and compared to unmodified cells.
  • a murine model of FAP can be utilized to determine functionality of a transgene encoding an APC protein.
  • mice with FAP can be treated with modified cells, encoding for APC, and a reduction of FAP disease measured versus untreated mice.
  • Subjects can receive procedures such as blood transfusions, blood draws, computerized tomography scan (CT) can, magnetic resonance imaging (MRI), X rays, radiation therapy, organ transplants, and any combination thereof.
  • CT computerized tomography scan
  • MRI magnetic resonance imaging
  • X rays radiation therapy
  • organ transplants organ transplants
  • an evaluation of a lesion such as a cancerous lesion, can be performed.
  • non-target lesions can be evaluated.
  • a complete response of a non target lesion can be a disappearance and normalization of tumor marker level. All lymph nodes must be non-pathological in size (less than 10 mm short axis). If tumor markers are initially above the upper normal limit, they must normalize for a patient to be considered a complete clinical response.
  • Non-CR/Non-PD is persistence of one or more non-target lesions and or maintenance of tumor marker level above the normal limit.
  • Progressive disease can be appearance of one or more new lesions and or unequivocal progression of existing non-target lesions. Unequivocal progression should not normally trump target lesion status.
  • a best overall response can be the best response recorded from the start of treatment until disease progression/recurrence.
  • Delivery vehicles provided herein can be utilized to deliver cargo to a target cell.
  • a target cell is found in a gastrointestinal tract, reproductive tract, circulatory system, respiratory system, musculoskeletal system, excretory system, nervous system, oculatory system, and combinations thereof.
  • suitable target cells can be found in any major organ of the body including but not limited to the skin, lungs, heart, liver, stomach, urinary system, reproductive system, intestine, pancreas, kidneys, thymus gland, thyroid, and/or brain.
  • a target cell is part of the gastrointestinal tract and is in the anus, rectum, large intestine, small intestine, liver, stomach, esophagus, or mouth.
  • a target cell is an enteroendocrine cell, mast cell, enterocyte, brush cell, Paneth cell, or goblet cell.
  • a target cell is an enteroendocrine cell and is an EC cell, D cell, CCK cell, L cell, P/Dl cell, or G cell.
  • a target cell is in the intestinal epithelium and is selected from an intestinal stem cell, Paneth cell, goblet cell, enterocyte, transit amplifying cell, enteroendocrine cell, or any combination thereof.
  • a target cell is an intestinal stem cell.
  • a target cell is a crypt cell.
  • Delivery vehicles can be utilized to introduce cargo to target cells.
  • introduction comprises contacting the target cell with the cargo.
  • introduction comprises transfecting or transducing the target cell with the cargo.
  • the cargo can modify the genome of the cell or exist within the cell extragenomically.
  • a delivery vehicle employed may contain a cargo that is delivered to a target cell, for example for expression in the cell and/or to genetically modify a target cell.
  • An efficiency of such delivery, e.g., transfection, with a cargo, such as a polynucleic acid described herein, for example, can be or can be about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more than 99.9% of the total number of cells that are contacted (in vivo or ex vivo) and/or are present in a tissue or location.
  • An efficiency of such delivery, e.g., transfection, with a cargo, such as a polynucleic acid described herein, for example, can be or can be about 1 fold, 10 fold, 20 fold, 40 fold, 60 fold, 80 fold, 100 fold, 120 fold, 140 fold, 160 fold, 180 fold, 200 fold, 300 fold, 400 fold, 500 fold, or over 1000 fold of the total number of cells that are contacted (in vivo or ex vivo) and/or are present in a tissue or location.
  • Efficiency of cellular uptake with subject delivery vehicles can permit efficient penetration and transit (such as through the mucus layer) to the target cells and thereby have an efficient uptake by the target cell(s), for example, uptake can be or can be about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more than 99.9% of the total number of cells that are contacted.
  • the compositions can have a higher percent of cellular uptake as compared to a comparable delivery vehicle that does not include a bile salt and/or charge separation or compared to a delivery vehicle which lacks one or more components.
  • the improvement can be from about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or up to about 80% better.
  • an efficiency of transfection or delivery of a cargo (such as integration of or expression of protein from a polynucleic acid) delivered to a cell by a delivery vehicle composition as described herein can be from about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or up to 65% better than a comparable delivery vehicle that does not include a bile salt and/or charge separation or compared to a delivery vehicle which lacks one or more components.
  • an efficiency of transfection or integration of or expression from a polynucleic acid cargo delivered to a cell by a delivery vehicle composition as described herein can be from about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or up to 65% better than a comparable delivery vehicle that does not include a bile salt and/or charge separation or compared to a delivery vehicle which lacks one or more components.
  • compositions provided herein for delivering a cargo can be functional for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 6, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or 100 days after introduction to a subject in need thereof.
  • Structures can be functional for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after introduction into a subject.
  • a delivery vehicle as provided herein can be functional for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 years after introduction to a subject.
  • a delivery vehicle can be functional for up to the lifetime of a recipient. Further, a delivery vehicle can function at 100% of its normal intended operation. Delivery vehicles can also function 1, 2,
  • Function of a delivery vehicle may refer to the efficiency of delivery, persistence of a lipid nanoparticles, stability of a lipid nanoparticles, or any combination thereof.
  • the delivery vehicle provided herein can deliver a cargo, such as a nucleic acid to a target cell (such as RNA, DNA (for example, minicircle DNA)).
  • a target cell such as RNA, DNA (for example, minicircle DNA)
  • function can include a percent of cells that received a nucleic acid from the delivery vehicle composition.
  • function can refer to a frequency or efficiency of protein generation from a nucleic acid.
  • a delivery vehicle composition may deliver a nucleic acid to a cell that encodes for at least a portion of a gene, such as APC, and a frequency of efficiency may describe a functionality complete gene as restored or created by the delivery of the cargo.
  • a nucleic acid cargo concentration in a delivery vehicle composition can be from 0.5 nanograms to 50 micrograms. Such concentration can be from about 0.5 ng, 1 ng, 2 ng, 5 ng, 10 ng, 50 ng, 100 ng, 150 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, lOOOng, lpg, 2 pg, 5 pg, 10 gg, 20 gg, 30 gg, 40 gg, 50 gg, 60 gg, or up to 50 gg or greater.
  • the amount of nucleic acid (e.g ., ssDNA, dsDNA, RNA) that may be introduced to a cell by a delivery vehicle may be varied to optimize transfection efficiency and/or cell viability. In some cases, less than about 100 picograms of nucleic acid may be introduced to a subject.
  • an effective amount of a structure can mean an amount sufficient to increase the expression level of at least one gene which can be decreased in a subject prior to the treatment or an amount sufficient to alleviate one or more symptoms of cancer.
  • an effective amount can be an amount sufficient to increase the expression level of at least one gene selected from the group consisting of gastrointestinal differentiation genes, cell cycle inhibition genes, and tumor suppressor genes by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 1000%, 1500%, or more compared to a reference value or the expression level without the treatment of any compound.
  • an effective amount can mean an amount sufficient to decrease the expression level of at least one gene which may be increased in the subject prior to the treatment or an amount sufficient to alleviate one or more symptoms of cancer.
  • an effective amount can be an amount sufficient to decrease the expression level of a gene by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 1000%, 1500%, or more compared to a reference value or the expression level without the treatment of any compound.
  • treating comprises reduction of the disease in the subject in need thereof by at least about 1 fold, 5 fold, 10 fold, 20 fold, 40 fold, 80 fold, 100 fold, 300 fold, 600 fold, or 1000 fold as measured by an in vitro or in vivo assay as compared to a comparable subject that does not undergo the administering.
  • reduction of the disease can be the result of an increase or decreases in the expression level of at least one gene in the subject.
  • Various gene expression assays can be utilized and include but are not limited to sequencing, PCR, RT-PCR, western blot, northern blot, ELISA, protein quantification, mRNA quantification, FISH, RNA-Seq, SAGE, or a combination thereof. Additional assays that can be utilized include microscopy, histology, in vivo animal experiments, human experiments, or any combination thereof.
  • the present disclosure provides compositions that include nanoparticles and cargo.
  • the nanoparticles may include at least one bile salt, at least one cationic lipid, at least one structural lipid, and at least one conjugated lipid.
  • Bile salts may be selected from one or more of deoxycholate, lithocholate, isolithocholate, alloisolithocholate, dehydrolithocholate, ursodiol, 5beta-cholanic acid, chenodeoxycholate, cholate, taurodeoxycholate, taurochenodeoxy cholate, glycocholate, 3-oxy-cholenic acid, and hyodeoxycholate.
  • Bile salts may be included in nanoparticles at levels of from about 5 to about 40 mol % of total nanoparticle lipid (e.g., from about 20 to about 40 or from about 33 to about 37 mol % of total nanoparticle lipid).
  • Nanoparticle bile salt may include deoxycholate and/or lithocholate.
  • Nanoparticles may include two bile salts.
  • Nanoparticles may include deoxycholate at a level of from about 20 to about 30 mol % of total nanoparticle lipid and lithocholate at a level of from about 5 to about 10 mol % of total nanoparticle lipid.
  • Nanoparticle cationic lipid may include MVL5.
  • Nanoparticle cationic lipid may include one or more of MC2, CL1H6, and CL4H6, each of which may be present at a level of from about 5 to about 20 mol % of total nanoparticle lipid.
  • Nanoparticle structural lipids may include one or more of DSPC and DMPC and may be present at a level of from about 35 to about 45 mol % of total nanoparticle lipid.
  • Nanoparticle conjugated lipids may be conjugated with hydrophilic polymers. Hydrophilic polymers may include PEG.
  • Conjugated lipids may include one or more of DMG-PEG and DMPE-PEG and may be present at a level of from about 0.5 to about 2.0 mol % of total nanoparticle lipid.
  • composition nanoparticles include a molar ratio between components of from about 1 to about 5 of at least one bile salt, from about 0.5 to about 3 of each one of at least one cationic lipid, from about 2 to about 10 of at least one structural lipid, and from about 0.02 to about 0.10 of at least one conjugated lipid.
  • Nanoparticle bile salts may include one or more of deoxycholate, ursodiol, lithocholate, isolithocholate, alloisolithocholate, dehydrolithocholate, and 5beta-cholanic acid.
  • Nanoparticle cationic lipids may include MVL5.
  • Nanoparticle cationic lipids may include MC2.
  • Nanoparticle structural lipids may include DSPC.
  • Nanoparticle conjugated lipids may include DMG-PEG.
  • Molar ratios of bile salt:MVL5:MC2:DSPC:DMG-PEG may be about 2.592:0.96:0.96:3.168:0.768.
  • Nanoparticle bile salts may include deoxycholate.
  • Composition nanoparticles may include MVL5, MC2, DSPC, deoxycholate, and DMPE-PEG at a molar ratio of 2.4:2.4:7.9:6.48:0.192.
  • Nanoparticles may include MVL5, CL1H6, DSPC, deoxycholate, and DMG-PEG at a molar ratio of about 2.4, 2.4, 7.9, 6.48, and 0.192.
  • Nanoparticles may include MVL5, CL4H6, DSPC, deoxycholate, and DMG-PEG at a molar ratio of about 2.4, 2.4, 7.9, 6.48, and 0.192.
  • Nanoparticles may include MVL5, MC2, DSPC, chenodeoxycholate, and DMG-PEG at a molar ratio of about 2.4, 2.4, 7.9, 6.48, and 0.192. Nanoparticles may include MVL5, MC2, DMPC, deoxycholate, and DMG-PEG at a molar ratio of about 2.4, 2.4, 7.9, 6.48, and 0.192. Nanoparticles may include MVL5, MC2, DMPC, deoxycholate, and DMPE-PEG at a molar ratio of about 2.4, 2.4, 7.9, 6.48, and 0.192.
  • Nanoparticles may include MVL5, CL1H6, DMPC, deoxycholate, and DMG-PEG at a molar ratio of about 2.4, 2.4, 7.9, 6.48, and 0.192.
  • Nanoparticles may include MVL5, MC2, DSPC, deoxycholate, lithocholate, and DMG-PEG at a molar ratio of about 2.4, 2.4, 7.9, 5.2, 1.3, and 0.192.
  • Nanoparticles may include MVL5, CL1H6, DSPC, deoxycholate, lithocholate, and DMG-PEG at a molar ratio of about 2.4, 2.4, 7.9, 5.2, 1.3, and 0.192.
  • Nanoparticles may include MVL5, MC2, DSPC, alloisolithocholate, and DMG-PEG at a molar ratio of about 2.4, 2.4, 7.92, 6.48, and 0.192.
  • Nanoparticles may include MVL5, MC2, DSPC, dehydrolithocholate, and DMG-PEG at a molar ratio of about 2.4, 2.4, 7.92, 6.48, and 0.192.
  • Compositions may include 12.4 mole % of MVL5, 12.4 mole % of MC2, 40.8 mole % of DSPC, 33.4 mole % of at least one bile salt, and 1 mole % of at least one conjugated lipid.
  • the at least one conjugated lipid may include DMG-PEG or DMPE-PEG.
  • the at least one bile salt include one or more of taurodeoxycholate, taurochenodeoxycholate, glycocholate, 3- oxy-cholenic acid, and deoxycholate.
  • Cargo included in compositions may include one or more of a nucleic acid, a protein, an antibody, a peptide, a small molecule, a biologic, a peptidomimetic, a ribozyme, a chemical agent, a viral particle, a growth factor, a cytokine, an immunomodulating agent, and a fluorescent dye.
  • Cargo may include nucleic acids. Such nucleic acids may include DNA (e.g., plasmid DNA).
  • compositions that include a cargo; and a nanoparticle, the nanoparticle including: a first cationic lipid and an optional second cationic lipid; at least one bile salt; at least one structural lipid; and at least one conjugated lipid conjugated with a hydrophilic polymer.
  • the at least one bile salt may be selected from one or more of deoxycholate, lithocholate, isolithocholate, alloisolithocholate, dehydrolithocholate, ursodiol, 5beta-cholanic acid, chenodeoxycholate, cholate, taurodeoxycholate, taurochenodeoxy cholate, glycocholate, 3-oxy-cholenic acid, and hyodeoxycholate.
  • the at least one bile salt may be included in nanoparticles at levels of from about 5 to about 40 mole % of total nanoparticle lipids.
  • the at least one bile salt may be included in nanoparticles at levels of from about 20 to about 40 mole % of total nanoparticle lipid.
  • the at least one bile salt may include deoxycholate.
  • the first cationic lipid may include CL1H6 or CL4H6.
  • the first cationic lipid may be included at levels of from about 5 to about 40 mole % of the total nanoparticle lipid.
  • the second cationic lipid may include MVL5,
  • the second cationic lipid may be present at levels of from about 5 to about 20 mole % of total nanoparticle lipid. Each of the first cationic lipid and the second cationic lipid may be present at levels of from about 5 to about 20 mole % of total nanoparticle lipid and may be present in equal amounts.
  • the at least one structural lipid may be selected from one or more of DSPC, DMPC, and dioleoylphosphatidylethanolamine (DOPE).
  • DOPE dioleoylphosphatidylethanolamine
  • the at least one structural lipid may be present at levels of from about 10 to about 70 mole % of total nanoparticle lipid.
  • the at least one structural lipid may be present at levels of from about 30 to about 50 mole % of total nanoparticle lipid.
  • the at least one structural lipid and the at least one bile salt may be present at combined levels of from about 50 to about 80 mole % of total nanoparticle lipid.
  • the hydrophilic polymer may include PEG.
  • the at least one conjugated lipid may include DMG-PEG.
  • the at least one conjugated lipid may be present at levels of from about 0.5 to about 2.0 mole % of total nanoparticle lipid.
  • the first cationic lipid may be CL1H6.
  • the nanoparticle may include a second cationic lipid that includes MVL5.
  • the at least one bile salt may be deoxycholate.
  • the at least one structural lipid may be DSPC.
  • the at least one conjugated lipid may be DMG-PEG.
  • compositions that include a cargo and nanoparticles, wherein the nanoparticles include CL1H6, MVL5, and DMG-PEG at molar ratios of about 1 : 1 :0.08; and deoxycholate and DSPC at molar ratios of from about 0.5 to about 5.0.
  • the molar ratios of deoxycholate and DSPC may be from about 2.0 to about 4.0.
  • the nanoparticles may include CL1H6 at levels of from about 10 to about 20 mole % of total nanoparticle lipid; MVL5 at levels of from about 10 to about 20 mole % of total nanoparticle lipid; deoxycholate at levels of from about 10 to about 40 mole % of total nanoparticle lipid; DSPC, DMPC, or DOPE at levels of from about 30 to about 60 mole % of total nanoparticle lipid; and DMG-PEG at levels of from about 0.5 to about 2.0 mole % of total nanoparticle lipid.
  • the nanoparticle may include: CL1H6 and MVL5 at levels of from about 10 to about 15 mole % of total nanoparticle lipid; deoxycholate at levels of from about 20 to about 40 mole % of total nanoparticle lipid; DSPC at levels of from about 35 to about 50 mole % of total nanoparticle lipid; and DMG-PEG at levels of from about 0.75 to about 1.5 mole % of total nanoparticle lipid.
  • the nanoparticle may include: CL1H6 and MVL5 at levels of from about 12 to about 14 mole % of total nanoparticle lipid; deoxycholate at levels of from about 27 to about 38 mole % of total nanoparticle lipid; DSPC at levels of from about 38 to about 45 mole % of total nanoparticle lipid; and DMG-PEG at levels of from about 0.75 to about 1.5 mole % of total nanoparticle lipid.
  • the nanoparticles may include: CL1H6 and MVL5 at levels of about 12 mole % of total nanoparticle lipid; deoxycholate at levels of about 33 mole % of total nanoparticle lipid; DSPC at levels of about 41 mole % of total nanoparticle lipid; and DMG-PEG at levels of about 1 mole % of total nanoparticle lipid.
  • Hydrophilic polymers may be conjugated with polypeptides.
  • Polypeptides may include MPPs, for example, any of those described in Table 3 of International Publication Number WO20 19222400, the contents of which are herein incorporated by reference in their entirety.
  • MPPs may include amino acid sequences according to TVDNDAPTKRASKLFAV (SEQ ID NO: 17).
  • Hydrophilic polymers may include PEG.
  • the at least one conjugated lipid may include DMG-PEG.
  • Nanoparticles may include: CL1H6 and MVL5 at levels of from about 12 to about 14 mole % of total nanoparticle lipid; deoxycholate at levels of from about 27 to about 38 mole % of total nanoparticle lipid; DSPC at levels of from about 38 to about 45 mole % of total nanoparticle lipid; and DMG-PEG at levels of from about 0.75 to about 1.5 mole % of total nanoparticle lipid.
  • the cargo may include one or more of a nucleic acid, a protein, an antibody, a peptide, a small molecule, a biologic, a peptidomimetic, a ribozyme, a chemical agent, a viral particle, a growth factor, a cytokine, an immunomodulating agent, and a fluorescent dye.
  • the cargo may include nucleic acids.
  • the nucleic acids may include DNA.
  • the DNA may include plasmid DNA.
  • the molar ratio of nanoparticle cationic lipids to nanoparticle nucleotides may be from about 2 to about 20.
  • the molar ratio of nanoparticle cationic lipids to nanoparticle nucleotides may be from about 14 to about 18.
  • the nucleic acid may include RNA.
  • the molar ratio of nanoparticle cationic lipids to the total number cargo RNA nucleotides may be from about 2 to about 20.
  • the molar ratio of nanoparticle cationic lipids to the cargo RNA nucleotides may be from about 2 to about 4.
  • the delivery vehicle compositions herein provide delivery to epithelial cells within mucosal tissues, such as those of the gastrointestinal tract (GI tract), as well as find uses in other mucosal tissues such as lung, vagina and eye.
  • the delivery vehicles herein provide penetration through a mucus layer as well as reaching epithelial cells.
  • a delivery vehicle delivers a cargo to epithelial cells of the GI tract and delivers a cargo (such as those described herein) for purposes of a therapeutic, diagnostic or theranostic purpose.
  • Exemplary diseases that can be treated with subject delivery vehicles provided herein, particularly such delivery vehicles with a therapeutic cargo can be cancerous or non- cancerous.
  • Such disease can be cardiovascular disease, a neurodegenerative disease, an ocular disease, a reproductive disease, a gastrointestinal disease, a brain disease, a skin disease, a skeletal disease, a muscoskeletal disease, a pulmonary disease, a thoracic disease, to name a few.
  • a disease can be a genetic disease such as cystic fibrosis, tay-sachs, fragile X, Huntington’s, neurofibromatosis, sickle cell, thalassemias, Duchenne’s muscular dystrophy, or a combination thereof.
  • a disease is a gastrointestinal disease.
  • a gastrointestinal disease is a monogenic GI disease.
  • a gastrointestinal disease is inherited.
  • a gastrointestinal disease is of the epithelium.
  • Suitable gastrointestinal diseases can be: familial adenomatous polyposis (FAP), attenuated FAP, microvillus inclusion disease (MVID), chronic inflammatory bowel disease, chronic inflammatory bowel disease, ileal Crohn’s, juvenile polyposis, hereditary diffuse gastric cancer syndrome (HDGC), Koz-Jeghers syndrome, lynch syndrome, gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS), Li-Fraumeni syndrome, familial gastric cancer, or a combination thereof.
  • a GI disease can produce polyps in a gastrointestinal tract. In some cases, a disease is FAP. FAP can progress to cancer.
  • a gastrointestinal disease can be hereditary.
  • a hereditary gastrointestinal disease can be Gilbert’s syndrome, telangiectasia, mucopolysaccaride, Osler-Weber-Rendu syndrome, pancreatitis, keratoacanthoma, biliary atresia, Morquio’s syndrome, Hurler’s syndrome, Hunter’s syndrome, Crigler-Najjar, Rotor’s, Peutz-Jeghers’ syndrome, Dubin- Johnson, Osteochondroses, Osteochondrodysplasias, polyposis, or a combination thereof.
  • a subject can be screened for the presence of a disease. Screens can be utilized to identify suitable subjects.
  • a disease can be identified by genetic, phenotypic, molecular, or chromosomal screening.
  • a suitable subject is positive for a disease provided herein.
  • a genetic screen can identify a mutation in an APC gene that can result in FAP.
  • a screen can comprise analyzing a gene such as CDH1, STK11, SMAD4, MLHl, MSH2, EPCAM, MSH6, PMS2, MY05B, APC, TP53, portions thereof, promoters thereof, and combinations thereof.
  • the delivery vehicles herein carry a therapeutic cargo (such as a nucleic acid, a protein or a drug) are used to treat a disease affecting the GI tract such as familial polyposis (FAP), attenuated FAP, colorectal cancer, chronic inflammatory bowel disease, ileal Crohn’s, Microvillus Inclusion Disease and congenital diarrheas.
  • a disease affecting the GI tract such as familial polyposis (FAP), attenuated FAP, colorectal cancer, chronic inflammatory bowel disease, ileal Crohn’s, Microvillus Inclusion Disease and congenital diarrheas.
  • a gene for delivery by a liposome may be administered to a subject as a preventive measure.
  • a subject may not have diagnosed disease and may appear to be predisposed to a disease such as cancer.
  • a cancer can be a colon cancer.
  • the delivery vehicles herein carry a diagnostic cargo and are used to visualize or diagnose the state of cells or tissues or to diagnose or monitor a subject for a condition or a disease.
  • a subject is administered an effective amount of delivery vehicles and a diagnostic method for FAP includes determining a level of APC incorporated into a cell genome whereupon a difference in APC levels before the start of therapy in a patient and during and/or after therapy will evidence the effectiveness of therapy in a patient, including whether a patient has completed therapy or whether the disease state has been inhibited or eliminated.
  • a pharmaceutical composition containing a delivery vehicle with its cargo can be administered chronically in some cases. Administration can encompass hourly, daily, monthly, or yearly administration of a structure to a subject. For example, in some cases, a subject may be administered a pharmaceutical composition daily for the entirety of the subject’s life. In other cases, a pharmaceutical composition may be administered daily for the duration of the presence of disease in a subject.
  • a subject may be administered a pharmaceutical composition, such as with a delivery vehicle and a polynucleic acid cargo, to treat a disease or disorder until the disease or disorder is reduced, controlled, or eliminated. Disease control may encompass the stabilization of a disease. For example, a cancer that is controlled may have stopped growing or spreading as measured by CT scan. A cancer may be a colon cancer.
  • a pharmaceutical composition may be administered prophylactically.
  • a subject may have undergone a genetic screen that identifies the subject as being predisposed to a cancer, such as colon cancer.
  • a predisposed subject may begin prophylactic treatment by receiving a pharmaceutical composition comprising delivery vehicle and a polynucleic acid cargo.
  • a subject may begin prophylactic treatment with such pharmaceutical composition.
  • prophylactic treatment can prevent a disease, such as cancer.
  • prevention can be used in relation to a condition, such as a local recurrence (e.g ., pain)
  • a disease such as cancer
  • prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.
  • Prevention of an infection includes, for example, reducing the number of diagnoses of the infection in a treated population versus an untreated control population, and/or delaying the onset of symptoms of the infection in a treated population versus an untreated control population.
  • Prevention of pain includes, for example, reducing the magnitude of, or alternatively delaying, pain sensations experienced by subjects in a treated population versus an untreated control population.
  • Assays can be utilized to determine therapeutic effectiveness of delivery vehicles provided herein.
  • an assay can be performed before, during, and/or after administration of subject delivery vehicles.
  • An assay can be performed for example on days -
  • Suitable assays can be in vivo or ex vivo.
  • an assay comprises a scan. Suitable scans can comprise CT, PET, MRI, or combinations thereof.
  • an assay comprises an in vitro assay such as histology, serology, sequencing, ELISA, microscopy, and the like.
  • the present disclosure provides methods of delivering cargo to target cells by contacting target cells with compositions (e.g., compositions that include cargo and nanoparticles) described herein.
  • Target cells may include human cells.
  • Target cells may include epithelial cells.
  • Epithelial cells may include intestinal epithelial cells.
  • Target cells may be part of mucosal tissue.
  • Target cell mucosal tissue may be part of the gastrointestinal tract.
  • Target cells may include gastrointestinal cells.
  • Gastrointestinal cells may include, but are not limited to intestinal epithelial cells, lamina basement cells, intraepithelial lymphocytes, intestinal muscle cells, and enteric neurons.
  • the present disclosure provides methods of delivering cargo to subjects by introducing compositions to subject gastrointestinal tracts, wherein the compositions include the cargo and nanoparticles encapsulating the cargo.
  • Nanoparticles may include at least one cationic lipid; at least one structural lipid; at least one bile salt; and at least one conjugated lipid, conjugated with a hydrophilic polymer (e.g., PEG).
  • Bile salts may be selected from one or more of deoxycholate, lithocholate, isolithocholate, alloisolithocholate, dehydrolithocholate, ursodiol, 5beta-cholanic acid, chenodeoxycholate, cholate, taurodeoxycholate, taurochenodeoxycholate, glycocholate, 3-oxy-cholenic acid, and hyodeoxycholate.
  • Bile salts may be included in nanoparticles at levels of from about 5 to about 40 mol % of total nanoparticle lipid (e.g., from about 20 to about 40 or from about 33 to about 37 mol % of total nanoparticle lipid).
  • Nanoparticle bile salt may include deoxycholate and/or lithocholate. Nanoparticles may include two bile salts. Nanoparticles may include deoxycholate at a level of from about 20 to about 30 mol % of total nanoparticle lipid and lithocholate at a level of from about 5 to about 10 mol % of total nanoparticle lipid. Nanoparticle cationic lipids may include MVL5. MVL5 may be present at a level of from about 5 to about 20 mol % of total nanoparticle lipid.
  • Nanoparticle cationic lipids may include one or more of MC2, CL1H6, and CL4H6 and each may be present at a level of from about 5 to about 20 mol % of total nanoparticle lipid.
  • Nanoparticle structural lipids may include one or more of DSPC and DMPC and may be present at a level of from about 35 to about 45 mol % of total nanoparticle lipid.
  • Nanoparticle conjugated lipids may be conjugated with hydrophilic polymers. Hydrophilic polymers may include PEG.
  • Conjugated lipids may include one or more of DMG-PEG and DMPE-PEG and may be present at a level of from about 0.5 to about 2.0 mol % of total nanoparticle lipid.
  • Methods of delivering cargo to subjects by introducing compositions to subject gastrointestinal tracts may include administering compositions to subjects by oral administration and/or intrarectal administration.
  • Composition nanoparticles may target gastrointestinal cells.
  • Targeted gastrointestinal cells may include, but are not limited to, intestinal epithelial cells, lamina basement cells, intraepithelial lymphocytes, intestinal muscle cells, and enteric neurons.
  • Composition cargo may be delivered to the gastrointestinal cells.
  • Cargo may be delivered to the intracellular space of the gastrointestinal cells.
  • Cargo, cargo components, and/or cargo expression products may be secreted from gastrointestinal cells after delivery.
  • the term “expression product” refers to a nucleic acid, amino acid polymer, protein, biomolecule, or other structure synthesized or “expressed” from a coded template (e.g., DNA or RNA).
  • Cargo expression products may be expressed from nucleic acid cargo components directly or may be expressed by cells in response to some other cargo component or component activity (e.g., enzymatic activity, cell signaling activity, transcriptional/translational activation/repression, etc.).
  • Secretion of cargo, cargo components, and/or cargo expression products may be by apical secretion or basal secretion from gastrointestinal cells.
  • Cargo, cargo components, and/or cargo expression products may remain in an area proximal to the cell after secretion.
  • Cargo, cargo components, and/or cargo expression products may be secreted basally from gastrointestinal cells and enter the circulation.
  • Cargo, cargo components, and/or cargo expression products may be distributed systemically after entering the circulation.
  • Methods of delivering cargo to subjects by introducing compositions to subject gastrointestinal tracts may include introducing compositions with nanoparticle cargo that includes therapeutic agents that may include, but are not limited to, nucleic acids, polypeptides, proteins, biologies, antibodies, enzymes, hormones, cytokines, immunogens, and genetic or epigenetic editing system components.
  • Therapeutic agent nucleic acids may encode polypeptides or proteins that also act as therapeutic agents.
  • Nucleic acids may include DNA (e.g., plasmid DNA).
  • Nanoparticles may target gastrointestinal cells and transfect them with nucleic acid cargo. Transfected gastrointestinal cells may express polypeptides encoded by nucleic acid cargo.
  • Nucleic acids may encode cell signaling factors.
  • cell signaling factor refers to any molecule that elicits a cellular response, including, but not limited to, cytokines, growth factors, and receptor ligands.
  • Cell signaling factors encoded by nucleic acid nanoparticle cargos may include, but are not limited to, interleukin (IL)-2, IL-2 mutein Fc-fusion, IL-10, IL-10 mutein, IL-22, granulocyte-macrophage colony- stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), adrenomedullin, glucagon-like peptide 1 (GLP-1), glucagon-like peptide 2 (GLP-2), GLP-2 analog teduglutide, peroxisome proliferator-activated receptor gamma (PPARy), human growth hormone (HGH), parathyroid hormone (PTH), fibroblast growth factor 21 (FGF21), and relaxin.
  • IL interleukin
  • nucleic acids encode antibodies.
  • antibody is used in the broadest sense and specifically embraces various antibody formats including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies formed from at least two intact antibodies), and antibody fragments (e.g., diabodies) so long as they exhibit a desired biological activity (e.g., are "functional” fragments).
  • Encoded antibodies may bind targets that include one or more of IL-18, IL-18 receptor 1 (IL18R1), IL-23, tumor necrosis factor a (TNFa), proprotein convertase subtilisin kexin 9 (PCSK9), and protein 19 (PI 9).
  • Encoded antibodies may include bispecific antibodies. Bispecific antibodies may bind to cluster of differentiation 3 (CD3) for recruitment of immune cells to targets of a second bispecific antibody epitope.
  • CD3 cluster of differentiation 3
  • transfected gastrointestinal cells may express nucleic acid cargo encoding antimicrobial agents.
  • antimicrobial agent refers to any substance capable of killing or otherwise slowing or stopping the growth, spread, or reproduction of microbiological organisms or viruses.
  • Antimicrobial agents encoded by nucleic acid cargo may include, but are not limited to, intestinal alkaline phosphatase (LAP) and defensins.
  • transfected gastrointestinal cells may express nucleic acid cargo encoding genetic editing system components.
  • genetic editing system refers to any technological approach to modifying nucleic acids together with associated components for carrying out the approach.
  • Genetic editing systems may include, but are not limited to, systems utilizing clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein technology.
  • Genetic editing systems may include epigenetic editing systems.
  • Epigenetic editing systems are genetic editing systems that alter non-sequence-related nucleic acid characteristics, for example methylation and organization into chromatin.
  • Nucleic acid cargo encoding genetic editing system components may be used to correct mutations in epithelial cell genes, including, but not limited to, CFTR gene mutations, GPR35 gene mutations, RNF186 A64T germline mutations associated with increased ulcerative colitis risk (see Beaudoin, M. et al. PLoS Genetics. 2013. 9(9): el003723, the content of which is herein incorporated by reference in its entirety), mutations associated with very early onset IBD (see Leung, G. and Muise, A.M., Physiology. 2018.
  • Nucleic acid cargo encoding genetic editing system components may be used to delete or silence genes encoding IL-18 and/or IL-18R1 in gastrointestinal stem cells (in vivo or in vitro ) to treat or prevent IBD.
  • Nucleic acid cargo encoding genetic editing system components may be used to generate RNF186 (179X) mutations in gastrointestinal stem cells to confer protection against IBD.
  • Nucleic acid cargo encoding genetic editing system components may be used to insert transgenes into gastrointestinal cell DNA (e.g., via CRISPR or RNA-mediated retrotransposons) to provide permanent sources for expression of therapeutic proteins or other factors.
  • inserted transgenes encode anti- TNFa antibodies, anti-P19 antibodies, or anti-IL-23 antibodies to treat or prevent IBD.
  • inserted transgenes express GLP-1 or FGF21 for treatment or prevention of metabolic diseases.
  • transfected gastrointestinal cells may express nucleic acid cargo encoding antigens.
  • antigen refers to an entity or structure that can be specifically bound or “recognized” by an antibody binding partner.
  • An antigen which evokes an immune response in organisms is referred to herein as an “immunogen.”
  • Nucleic acid cargo encoding immunogens may be delivered to subjects to promote immune responses to the encoded immunogens.
  • Encoded immunogens may be derived from pathogenic organisms or viruses. Pathogens associated with encoded immunogens may include, but are not limited to, influenza virus, SARS-CoV-2 virus, Ebola virus, and polio virus.
  • nucleic acid cargo encodes tumor cell neoantigens.
  • tumor cell neoantigen refers to an antigen that is expressed by tumor cells (e.g., due to mutation or other mechanism), distinguishing them from non-tumor cells. Expression of neoantigens may be used to promote immune responses in subjects against tumor cells.
  • nucleic acid cargo encode antigens useful for development of tolerance to the antigens by the subject. Such antigens may include, but are not limited to, antigens associated with peanut allergies, celiac disease, rheumatoid arthritis, and IBD.
  • transfected gastrointestinal cells express nucleic acid cargo encoding clotting factors (e.g., Factor VIII).
  • transfected gastrointestinal cells express nucleic acid cargo encoding enzymes [e.g., b- glucocerebrosidase (GBA)].
  • GBA b- glucocerebrosidase
  • gastrointestinal cells may be transfected with nucleic acid cargo that includes non-coding RNA.
  • non-coding RNA refers to RNA molecules with sequences that do not encode proteins, but typically have significance in some other RNA function.
  • Non-coding RNA may include, but is not limited to, short interfering RNA (siRNA), microRNA (miRNA), long non-coding RNA, piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), small Cajal body-specific RNA (scaRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and small nuclear RNA (snRNA).
  • the present disclosure provides methods of treating therapeutic indications in subjects by providing compositions, nanoparticles, and/or cargo described herein, including methods of delivering cargo to subjects according to any of the methods described above involving composition introduction to subject gastrointestinal tracts.
  • therapeutic indication refers to any disease, condition, disorder, or symptom that may be improved, cured, stabilized, alleviated, or otherwise addressed by medical treatment or other intervention.
  • Composition cargo used in therapeutic indication treatment may include and/or encode therapeutic agents.
  • Therapeutic indications may include immune-related indications.
  • the term “immune-related indication” refers to any therapeutic indication relating to the immune system.
  • methods of the present disclosure may include the use of nucleic acid cargo encoding one or more of IL-2, IL-2 mutein Fc- fusion, IL-10, IL-10 mutein, IL-22, adrenomedullin, an anti -microbial, and an anti inflammatory antibody.
  • Nucleic acid cargo may be delivered to gastrointestinal cells. Gastrointestinal cells may express therapeutic agents from nucleic acid cargo.
  • Gastrointestinal cells may secrete therapeutic agents locally or systemically (e.g., via entry into circulation).
  • immune-related indications addressed by treatment methods of the present disclosure include gastrointestinal indications, which may include gastrointestinal diseases and any other disorders involving the gastrointestinal tract and related components.
  • Gastrointestinal indications may include, but are not limited to, gastrointestinal infections, inflammatory bowel disease (IBD), ulcerative colitis, and Crohn’s disease.
  • Gastrointestinal cells may express and locally secrete (e.g., into the intestinal lumen) therapeutic agents encoded by cargo nucleic acids for treatment of such gastrointestinal indications.
  • immune-related indications addressed by treatment methods of the present disclosure are systemic or are not specific to the gastrointestinal tract. Gastrointestinal cells may express and secrete therapeutic agents into circulation for treatment of such indications. Non-limiting examples include graft versus host disease (GVHD), systemic lupus erythematosus (SLE), type I diabetes, rheumatoid arthritis, infections, wounds, and allergies.
  • GVHD graft versus host disease
  • SLE systemic lupus erythe
  • therapeutic indications treated according to methods of the present disclosure include cancer and associated disorders, referred to herein as “cancer- related indications.”
  • Therapeutic agents encoded by nucleic acid cargo associated with methods of treating cancer-related indications may include GM-CSF.
  • Gastrointestinal cells may express and secrete nucleic acid cargo encoded GM-CSF locally or into circulation.
  • Cancer-related indications treated according to such methods may include, but are not limited to, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, acute lymphoblastic leukemia, and acute myelogenous leukemia.
  • subjects receiving treatment have previously received or are receiving concurrent chemotherapy treatment and/or stem cell transplantation treatment.
  • GM-CSF is secreted into circulation at a level sufficient to provide circulating GM-CSF concentrations of from about 10 to about 500 pg/nr/day (e.g., from about 50 to about 200, from about 100 to about 250, or from about 150 to about 400 pg/nr/day).
  • Therapeutic indications treated according to methods of the present disclosure may include neutropenia, a disorder characterized by low neutrophil blood levels.
  • Nucleic acid cargo associated with such methods may encode G-CSF, which promotes granulocyte production and neutrophil regulation.
  • Gastrointestinal cells may express and secrete G-CSF locally and/or systemically for neutropenia treatment.
  • G-CSF is secreted into circulation at a level sufficient to provide subjects with a dose of from about 1 to about 20 pg/kg/day of the G-CSF (e.g., from about 1 to about 10, from about 5 to about 15, or from about 10 to about 20 pg/kg/day).
  • subjects are treated until neutrophil blood levels reach about 1000/pl.
  • Therapeutic indications treated according to methods of the present disclosure may include microvillus inclusion disease (MVID). Nucleic acid cargo associated with such methods may encode MY05B gene product. In some embodiments, therapeutic indications treated according to methods of the present disclosure may include cystic fibrosis. Nucleic acid cargo associated with such methods may encode cystic fibrosis transmembrane regulator protein (CFTR). [0210] In some embodiments, therapeutic indications treated according to methods of the present disclosure may include hemophilia. Nucleic acid cargo associated with such methods may encode clotting factors. Clotting factors may include Factor VIII. In some embodiments, treated hemophilia may include hemophilia A.
  • MVID microvillus inclusion disease
  • Nucleic acid cargo associated with such methods may encode MY05B gene product.
  • therapeutic indications treated according to methods of the present disclosure may include cystic fibrosis. Nucleic acid cargo associated with such methods may encode cystic fibrosis transmembrane regulator protein (CFTR).
  • Therapeutic indications treated according to methods of the present disclosure may include Gaucher’s disease.
  • Nucleic acid cargo associated with such methods may encode GBA.
  • Nucleic acid cargo encoding GBA may be delivered to gastrointestinal cells. Gastrointestinal cells may secrete GBA into circulation at a level sufficient to provide steady- state subject GBA plasma levels of from about 1 ng/mL to about 10 ng/mL (e.g., about 6 ng/mL).
  • therapeutic indications treated according to methods of the present disclosure include short bowel syndrome (SBS).
  • Nucleic acid cargo associated with such methods may encode GLP-2.
  • Nucleic acid cargo encoding GLP-2 may be delivered to and expressed by gastrointestinal cells.
  • GLP-2 may be secreted into circulation at levels sufficient to provide circulating GLP-2 concentrations of from about 10 ng/mL to about 50 ng/mL (e.g., about 36 ng/mL).
  • Therapeutic indications treated according to methods of the present disclosure may include hormone deficiencies.
  • Nucleic acid cargo delivered according to such methods may encode deficient hormones.
  • Deficient hormones may include, but are not limited to, HGH and PTH.
  • Nucleic acid cargo may be delivered to and expressed by gastrointestinal cells. Expressed hormones may be secreted into circulation.
  • HGH may be secreted into circulation at a level sufficient to provide circulating HGH concentrations of from about 0.1 to about 100 ng/mL.
  • levels in adults are from about 1 to about 10 ng/mL.
  • levels in children are from about 10 to about 50 ng/mL.
  • PTH may be secreted into circulation at levels sufficient to provide circulating PTH concentrations of from about 50 to about 300 pg/mL (e.g., about 150 pg/mL).
  • therapeutic indications treated according to methods of the present disclosure include non-alcoholic steatohepatitis (NASH).
  • Nucleic acid cargo associated with such methods may encode GLP-1 or FGF21.
  • therapeutic indications treated according to methods of the present disclosure include elevated circulating low density lipoprotein (LDL) levels.
  • Nucleic acid cargo associated with such methods may encode anti-PCSK9 antibodies. Such cargo may be delivered to and expressed by gastrointestinal cells.
  • Anti-PCSK9 antibodies may be secreted into circulation at a level sufficient to provide circulating antibody concentrations of from about 1 to about 50 mg/mL (e.g., from about 1 to about 10, from about 6 to about 18, from about 12 to about 19, or from about 15 to about 45 pg/mL).
  • compositions described throughout can be formulation into a pharmaceutical medicament and be used to treat a human or mammal, in need thereof. Medicaments can be co-administered with any additional therapy.
  • an excipient may include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like.
  • a delivery vehicle composition may also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, or buffers.
  • a composition can be administered orally, by subcutaneous or other injection, intravenously, intracerebrally, intramuscularly, parenterally, transdermally, nasally or rectally.
  • the form in which the compound or composition is administered depends at least in part on the route by which the compound is administered.
  • a composition can be employed in the form of solid preparations for oral administration; preparations may be tablets, granules, powders, capsules or the like.
  • a composition is typically formulated with additives, e.g.
  • a composition to be administered may contain a quantity of a delivery vehicle in a pharmaceutically effective amount for therapeutic use in a biological system, including a patient or subject.
  • a pharmaceutical composition may be administered daily or administered on an as needed basis.
  • the delivery vehicles herein include those formulated as a pharmaceutical composition for administration.
  • Suitable formulations can include aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, bactericidal antibiotics and solutes that render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents.
  • Suitable inert carriers can include sugars such as lactose.
  • the compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g, peanut oil, corn oil, sesame oil, etc.), and combinations thereof.
  • polyols e.g., glycerol, propylene glycol, and liquid polyethylene glycol
  • oils such as vegetable oils (e.g, peanut oil, corn oil, sesame oil, etc.), and combinations thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants.
  • isotonic agents for example, sugars or sodium chloride.
  • Solutions and dispersions of the active compounds as the free acid or base or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, and combination thereof.
  • Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface active agents.
  • Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions.
  • anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2- ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate.
  • Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimoniuni bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine.
  • nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG- 1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide.
  • amphoteric surfactants include sodium N-dodecyl-beta-alanine, sodium N-lauryl-beta- iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.
  • the formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.
  • the formulation may also contain an antioxidant to prevent degradation of the active agent(s).
  • the formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution.
  • Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.
  • Water soluble polymers can be often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.
  • Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization.
  • dispersions can be prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above.
  • a method of preparation can be vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles.
  • a formulation can be an ocular formulation or a topical formation.
  • Pharmaceutical formulations for ocular administration can be in the form of a sterile aqueous solution or suspension of particles formed from one or more polymer-drug conjugates.
  • Acceptable solvents include, for example, water, Ringer's solution, phosphate buffered saline (PBS), and isotonic sodium chloride solution.
  • PBS phosphate buffered saline
  • the formulation may also be a sterile solution, suspension, or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as 1,3-butanediol.
  • the liposomes can be formulated for topical administration to mucosa.
  • Suitable dosage forms for topical administration include creams, ointments, salves, sprays, gels, lotions, emulsions, liquids, and transdermal patches.
  • the formulation may be formulated for transmucosal, transepithelial, transendothelial, or transdermal administration.
  • the compositions contain one or more chemical penetration enhancers, membrane permeability agents, membrane transport agents, emollients, surfactants, stabilizers, and combination thereof.
  • the liposomes can be administered as a liquid formulation, such as a solution or suspension, a semi-solid formulation, such as a lotion or ointment, or a solid formulation.
  • the liposomes can be formulated as liquids, including solutions and suspensions, such as eye drops or as a semi-solid formulation, such as ointment or lotion for topical application to mucosa, such as the eye or vaginally or rectally.
  • the formulation may contain one or more excipients, such as emollients, surfactants, emulsifiers, and penetration enhancers.
  • An appropriate dosage (“therapeutically effective amount”) of an active agent(s) in a composition may depend, for example, on the severity and course of a condition, a mode of administration, a bioavailability of a particular agent(s), the age and weight of a subject, a subject’s clinical history and response to an active agent(s), discretion of a physician, or any combination thereof.
  • a therapeutically effective amount of an active agent(s) in a composition to be administered to a subject can be in the range of about 100 pg/kg body weight/day to about 1000 mg/kg body weight/day whether by one or more administrations.
  • the range of each active agent administered daily can be from about 100 pg/kg body weight/day to about 50 mg/kg body weight/day, 100 pg/kg body weight/day to about 10 mg/kg body weight/day, 100 pg/kg body weight/day to about 1 mg/kg body weight/day, 100 pg/kg body weight/day to about 10 mg/kg body weight/day, 500 pg/kg body weight/day to about 100 mg/kg body weight/day, 500 pg/kg body weight/day to about 50 mg/kg body weight/day, 500 pg/kg body weight/day to about 5 mg/kg body weight/day, 1 mg/kg body weight/day to about 100 mg/kg body weight/day, 1 mg/kg body weight/day to about 50 mg/kg body weight/day, 1 mg/kg body weight/day to about 10 mg/kg body weight/day, 5 mg/kg body weight/dose to about 100 mg/kg body weight/day, 5 mg/kg body weight/dose to about 50 mg/kg body weight/day,
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, sweeteners, salts, buffers, and the like.
  • the pharmaceutically acceptable carriers may be prepared from a wide range of materials including, but not limited to, flavoring agents, sweetening agents and miscellaneous materials such as buffers and absorbents that may be needed in order to prepare a particular therapeutic composition.
  • a composition comprising a delivery vehicle can be formulated under sterile conditions within a reasonable time prior to administration.
  • a composition comprising a delivery vehicle can be formulated from about 1 month, 2 weeks, 1 week, 5 days, 3 days, 2 days, 1 day, 10 hours, 5 hours, or immediately prior to administration to a subject.
  • a delivery vehicle can be frozen and thawed prior to administration.
  • Provided delivery vehicles can be used in combination with secondary therapies.
  • a secondary therapy such as chemotherapy or radiation therapy may be administered before or subsequent to the administration of a delivery vehicle, for example within 12 hr. to 7 days.
  • a combination of therapies such as both chemotherapy and radiation therapy may be employed in addition to the administration of the delivery vehicles [0225]
  • provided delivery vehicles can comprise a coating.
  • a coating can be an enteric coating. Enteric coatings can be utilized to prevent or minimize dissolution in the stomach but allow dissolution in the small intestine.
  • a coating can include an enteric coating.
  • An enteric coating can be a barrier applied to oral medication that prevents release of medication before it reaches the small intestine. Delayed-release formulations, such as enteric coatings, can an irritant effect on the stomach from administration of a medicament from dissolving in the stomach.
  • Such coatings are also used to protect acid-unstable drugs from the stomach's acidic exposure, delivering them instead to a basic pH environment (intestine's pH 5.5 and above) where they may not degrade.
  • Dissolution can occur in an organ.
  • dissolution can occur within a duodenum, jejunum, ilium, and/or colon, or any combination thereof.
  • dissolution can occur in proximity to a duodenum, jejunum, ilium, and/or colon.
  • Some enteric coatings work by presenting a surface that is stable at a highly acidic pH found in the stomach, but break down rapidly at a less acidic (relatively more basic) pH. Therefore, an enteric coated pill may not dissolve in the acidic environment of the stomach but can dissolve in an alkaline environment present in a small intestine.
  • enteric coating materials include, but are not limited to, methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxy propyl methyl cellulose phthalate, hydroxy propyl methyl cellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, sodium alginate and stearic acid.
  • enteric coating materials include, but are not limited to, methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxy propyl methyl cellulose phthalate, hydroxy propyl methyl cellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, sodium alginate and stearic acid.
  • PVAP polyvinyl acetate phthalate
  • An enteric coating can be applied at a functional concentration.
  • An enteric coating can be cellulose acetate phthalate, Polyvinyl acetate phthalate, Hydroxypropylmethylcellulose acetate succinate, Poly(methacylic acid-co-ethyl acrylate) 1:1, Poly(methacrylic acid-co-ethyl acrylate) 1:1, Poly(methacylic acid-co-methyl methacrylate) 1:1, Poly(methacylic acid-co-methyl methacrylate) 1:1, Poly(methacylic acid- co-methyl methacrylate) 1:2, Poly(methacylic acid-co-methyl methacrylate) 1:2,
  • An enteric coating can be applied from about 6 mg/(cm 2 ) to about 12 mg/( cm 2 ).
  • An enteric coating can also be applied to a structure from about 1 mg/(cm 2 ) , 2 mg/(cm 2 ), 3 mg/(cm 2 ), 4 mg/(cm 2 ), 5 mg/(cm 2 ), 6 mg/(cm 2 ), 7 mg/(cm 2 ), 8 mg/(cm 2 ), 9 mg/(cm 2 ), 10 mg/(cm 2 ), ll mg/(cm 2 ), 12 mg/(cm 2 ), 13 mg/(cm 2 ), 14 mg/(cm 2 ), 15 mg/(cm 2 ), 16 mg/(cm 2 ), 17 mg/(cm 2 ), 18 mg/(cm 2 ), 19 mg/(cm 2 ), to about 20 mg/(
  • a pharmaceutical composition comprising a subject delivery vehicle can be orally administered from a variety of drug formulations designed to provide delayed-release.
  • Delayed oral dosage forms include, for example, tablets, capsules, caplets, and may also comprise a plurality of granules, beads, powders or pellets that may or may not be encapsulated. Tablets and capsules can represent oral dosage forms, in which case solid pharmaceutical carriers can be employed.
  • one or more barrier coatings may be applied to pellets, tablets, or capsules to facilitate slow dissolution and concomitant release of drugs into the intestine.
  • a barrier coating can contain one or more polymers encasing, surrounding, or forming a layer, or membrane around a therapeutic composition or active core.
  • active agents such as a polynucleic acid
  • the delay may be up to about 10 minutes, about 20 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, or up to 1 week in length.
  • an enteric coating may not be used to coat a particle.
  • Polymers or coatings that can be used to achieve enteric release can be anionic polymethacrylates (copoly-merisate of methacrylic acid and either methyl-methacrylate or ethylacrylate (Eudragit®), cellulose based polymers, e.g. cellulose acetatephthalate (Aquateric®) or polyvinyl derivatives, e.g. polyvinyl acetate phthalate (Coateric®) in some cases.
  • anionic polymethacrylates copoly-merisate of methacrylic acid and either methyl-methacrylate or ethylacrylate (Eudragit®)
  • cellulose based polymers e.g. cellulose acetatephthalate (Aquateric®)
  • polyvinyl derivatives e.g. polyvinyl acetate phthalate (Coateric®) in some cases.
  • formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a frozen or freeze-dried (lyophilized) condition requiring only the addition of sterile liquid carrier immediately prior to use.
  • compositions can take the form of, for example, tablets or capsules prepared by a conventional technique with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato star
  • Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations can be prepared by conventional techniques with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g. lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters, ethyl alcohol
  • compositions can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • Preparations for oral administration can be suitably formulated to give controlled release of the active compound.
  • buccal administration the compositions can take the form of tablets or lozenges formulated in conventional manner.
  • compositions can also be formulated as a preparation for implantation or injection.
  • a structure can be formulated with suitable polymeric, aqueous, and/or hydrophilic materials, or resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).
  • the compounds can also be formulated in rectal compositions, creams or lotions, or transdermal patches.
  • a pharmaceutical composition may include a salt.
  • a salt can be relatively non-toxic.
  • pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like.
  • suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts.
  • the class of such organic bases may include mono-, di-, and trialkylamines, such as methylamine, dimethylamine, and triethylamine; mono-, di- or trihydroxyalkylamines such as mono-, di-, and triethanolamine; amino acids, such as arginine and lysine; guanidine; N-methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; (trihydroxymethyl)aminoethane; and the like.
  • mono-, di-, and trialkylamines such as methylamine, dimethylamine, and triethylamine
  • mono-, di- or trihydroxyalkylamines such as mono-, di-, and triethanolamine
  • amino acids such as arginine and lysine
  • guanidine N-methylglucosamine
  • N-methylglucamine N-methylglucamine
  • articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the disclosure includes embodiments in which exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process.
  • the invention includes embodiments in which more than one, or all group members are present in, employed in, or otherwise relevant to a given product or process.
  • Example 1 Preparation of exemplary delivery vehicles of this disclosure
  • This example provides an exemplary method of preparing a delivery vehicle of this disclosure.
  • Lipid components of the delivery vehicle l,2-dioleyloxy-3-dimethylaminopropane (DODMA) (Sigma Aldrich), Deoxycholate (Sigma Aldrich), MVL5 (Avanti Polar Lipids), DSPC (Avanti Polar Lipids), DMG-PEG 2000 (Avanti Polar Lipids), DOPC (Avanti Polar Lipids), Dil (ThermoFisher Scientific), DiO (ThermoFisher Scientific) were dissolved in ethanol and heated above their phase transition temperature, for instances where the phase transition temperatures were higher than 37 °C.
  • the lipids and aqueous phase were heated to 70 °C.
  • DOPC DO-dimethylcellulose
  • the lipids and aqueous phase were not heated and used at room temperature.
  • Nucleic acids were dissolved in an aqueous buffer heated above the phase transition temperature of the lipids.
  • the aqueous buffer pH was set at below the pKa of the bile salt and the cationic lipids. In this way, the lipids were strongly cationic when formulated with the nucleic acids.
  • the lipids and nucleic acids were mixed using microfluidic channels followed by removal of ethanol via dialysis. Other suitable methods can also be used for this step.
  • lipid structures such as liposomes can be formed by thin film hydration where the lipids may be dissolved in organic phase and dried using a rotovap under rotation. The thin film that is formed can be hydrated in water.
  • the hydrated lipids can be heated to 70 °C for example, for DSPC or used at room temperature for example, for DOPC and extruded through the appropriate extruder pore size.
  • the nucleic acid cargo can be mixed with the lipids to form lipoplexes.
  • Another suitable alternate method for the preparation of exemplary delivery vehicles is using the thin film hydration.
  • Lipids are dissolved and mixed in an organic solvent. The solvent is removed, and the formed thin film is hydrated in an aqueous solution. The lipids are sized appropriately using sonication or extrusion. Nucleic acids can be complexed by mixing the lipid mixture and nucleic acids together.
  • exemplary delivery vehicles containing encapsulated nucleic acid
  • 300 pg of plasmid DNA encoding for Gaussia luciferase under a cytomegalovirus (CMV) promoter was dissolved in a final volume of 3 mL 50 mM Sodium Acetate buffer (pH 4.8).
  • Appropriate moles of MVL5, DODMA, Deoxycholate, MVL5, DSPC, DMG-PEG2000 and/or DOPC were mixed in ethanol according to their mole and cationic lipid: Nucleic acid ratio ( See Table 2 for mole % of the lipids in the various formulations prepared).
  • the cationic lipid: nucleotide molar ratio was maintained at about 16.
  • Fluorescently labelled lipids, such as Dil and DiO when used, were added to the mix at 0.5 % of the total lipid moles. Ethanol volume was raised to lmL.
  • the nucleic acid is in the aqueous sodium acetate buffer phase in a 3mL syringe.
  • the lipids are in ethanol in a lmL syringe.
  • the two syringes are mounted on to a NanoAssemblr (Precision Nanosystems) and then the two samples are mixed using the microfluidics chip on the NanoAssemblr.
  • samples were mounted into syringes (as mentioned above, nucleic acid in the 3 mL syringe and lipids in the 1 mL syringe) on a NanoAssemblr Benchtop and preheated to 65 °C for the DSPC formulations or at room temperature (about 25 °C) for the DOPC formulations.
  • Samples were mixed using the NanoAssemblr Benchtop microfluidic chip system with a flow rate of 6 mL/min. pH was neutralized with 300 mM HEPES buffer at pH 7.5. Ethanol was removed using dialysis overnight. Samples were concentrated using Amicon Ultra -4 with a 100 kDa molecular weight cutoff.
  • Example 2 Transfection of exemplary delivery vehicles of this disclosure
  • transfection efficiency of exemplary delivery vehicles were assessed.
  • HEK cells cultured to confluency between 50-80% were used for transfections.
  • 1 pg of Gaussia luciferase expressing plasmid DNA encapsulated in a lipid nanoparticle was used per well in a 24 well plate.
  • Transfection efficiency was assessed by taking 30 m ⁇ of media after 24h and performing a flash luciferase assay (Pierce Gaussia Luciferase Assay Kit). Increased value of relative light units (RLU) corresponded to greater transfection efficiency.
  • RLU relative light units
  • Example 3 Stability of exemplary delivery vehicles of this disclosure
  • exemplary delivery vehicles were assessed.
  • the delivery vehicles used in this assay incorporated 0.5 mol% each of Dil and DiO.
  • Dil and DiO are fluorescent dyes that are FRET pairs.
  • Bile salts were simulated by using an equal mixture of cholic acid and deoxycholate at indicated concentrations (in FIGS. 2-4). It was expected that if the delivery vehicle was susceptible to being disrupted by the bile salts, it would result in decreased FRET intensity.
  • Relative fluorescence units (RFU) was determined by taking exciting at 465 nm and reading emission at 501 nm and 570 nm. The RFU reading at 570 nm was divided by the reading at 501 nm. The readings were normalized to the FRET intensity of the system without any treatment. Data is shown in FIG. 2, FIG. 3, and FIG. 4.
  • Example 4 Encapsulation of nucleic acid in exemplary delivery vehicles of this disclosure
  • a delivery vehicle containing lpg of DNA encapsulated by the lipid nanoparticle (Formulation No. 5 in Table 2) was loaded to lanes of an agarose gel, either untreated (lane 2 in FIG. 5), (ii) treated with 7% Triton-X 100 (lane 3 in FIG. 5), (iii) treated with 7% Triton-X 100 plus 70° C for 30 mins (lane 4 in FIG. 5), followed by electrophoresis.
  • SYBR Safe was used to detect the DNA by UV light.
  • Encapsulation of a nucleic acid cargo was performed as follows: lipids were dissolved in ethanol and heated above their phase transition temperature. The nucleic acid dissolved in an aqueous buffer heated above the phase transition temperature of the lipids.
  • the aqueous buffer pH was set at below the pKa of the bile salt and the cationic lipids. In this way, the lipids were strongly cationic when formulated with the nucleic acids.
  • the lipids and nucleic acids were mixed using microfluidic channels. The pH was raised to neutral and the sample was concentrated, and ethanol removed using dialysis.
  • DODMA (Sigma Aldrich), Deoxycholate (Sigma Aldrich), MVL5 (Avanti Polar Lipids), DSPC (Avanti Polar Lipids), DMG-PEG 2000 (Avanti Polar Lipids), DSG-PEG 2000 (Avanti Polar Lipids), DOPC (Avanti Polar Lipids), Dil (ThermoFisher Scientific), DiO (ThermoFisher Scientific), and glycerol monooleate (GMO) (MP Biomedicals).
  • DODMA Sigma Aldrich
  • Deoxycholate Sigma Aldrich
  • MVL5 Alvanti Polar Lipids
  • DSPC Advanti Polar Lipids
  • DMG-PEG 2000 Avanti Polar Lipids
  • DSG-PEG 2000 Advanti Polar Lipids
  • DOPC Advanti Polar Lipids
  • Dil ThermoFisher Scientific
  • DiO ThermoFisher Scientific
  • GMO glycerol monooleate
  • plasmid DNA encoding for gaussia luciferase under a CMV promoter was dissolved in a final volume of 3 mL 50 mM Sodium Acetate buffer (pH 4.8).
  • Appropriate moles of MVL5, DODMA, Deoxycholate, MVL5, DSPC, GMO, DMG-PEG 2000, DSG- PEG 2000, and/or DOPC were mixed in ethanol according to their mole and cationic lipid: Nucleic acid ratio.
  • the cationic lipid: nucleotide molar ratio remained constant at 16.
  • DSG has a stearic acid lipid tail that is present in the gel phase at 37°C.
  • DMG has a myristolic acid lipid tail that is in the liquid phase at 37°C.
  • DMG-PEG was present in the liquid phase portion(s) of the vehicle and thus stabilized the cationic lipids preventing aggregation whereas DSG-PEG was in the gel phase portion(s) and could not provide the same stabilization effect.
  • Example 6 In vivo administration of delivery vehicles [0256] Mice were dosed intrarectally with approximately 30 micrograms of DNA encapsulated in nanoparticles that were Dil and DiO labelled. 4 hours after dosing, mice were sacrificed, and the intestines were embedded in OCT and frozen in dry ice and stored at - 80°C. The tissues were cryosectioned into 30 micrometer slices and imaged using a BioTek Cytation 1. Dil fluorescence was measured in the RFP channel.
  • PEGylated particles fail to reach the intestinal epithelial cells
  • MVL5/DODMA/DSPC/Deoxycholate/DMG-PEG particles were formed with increasing amounts of DMG-PEG and the behavior of the particles was investigated in vivo.
  • An increasing amount of DMG-PEG resulted in decreased distribution at the intestinal tissue. This is in contradiction with the current dogma of increasing PEGylation to increase intestinal epithelial reach.
  • increased PEGylation reduces the exposure of the positive charge at the surface through its shielding properties. This reduces the dual nature of the particle, as shown in FIG. 6 (Particle 5), FIG. 7 (Particle 6), FIG. 8 (Particle 7), FIG. 9 (Particle 8), and FIG. 10 (Particle 9)
  • Example 7 Delivery Vehicle in vivo testing
  • the ratios of MVL5/DODMA were altered in DSPC/Deoxycholate/DMG-PEG) with Dil and DiO to investigate the effect of increasing positive charge.
  • the following ratios of MVL5/DODMA in the particles were formed (0%/25%), (6.25%/l 8.75%), (12.5%/12.5%), (18.75%, 6.25%), (25%/0%).
  • DODMA is mostly neutral at neutral pH and is monovalent
  • the negative charge of deoxycholate and the multivalent charges of MVL5 dominated the behavior of the particle.
  • Example 8 Zwitterionic delivery vehicles vs. dual phase delivery vehicles
  • FIG. 16A, FIG. 16B, FIG. 16C, and FIG. 16D for DOPC particles (particle 11, Table 3);
  • FIG. 17A, FIG. 17B, FIG. 17C, and FIG. 17D for GMO particles (particle 12, Table 3);
  • FIG. 18A, FIG. 18B, FIG. 18C, and FIG. 18D for DSPC particles (particle 5, Table 3).
  • Results with PBS control particles are shown in FIG. 19A, FIG. 19B, FIG. 19C, and FIG. 19D
  • Example 9 Stability of delivery vehicles with bile salts
  • bile salt component was either ursodiol, deoxycholate, lithocholate, isolithocholate, alloisolithocholate, dehydrolithocholate or 5beta-cholanic acid. No nucleic acid was incorporated into the lipid nanoparticles. Alternate formulations can also be generated such as those provided in Table 4.
  • Stability of the lipid nanoparticles in bile salt was measured as discussed previously up to 10 g/L. FRET signals from Dil and DiO were normalized to no treatment. Levels of stability of the vehicles, in salt form, is shown in FIG. 20.
  • Nanoparticles incorporating various bile salts according to formulations listed in Table 5 were made using methods previously described in Example 1. Mole % values shown are based on percent of total lipid. Bile salts indicated were included at 33.4% and indicated PEG-conjugated lipid at 1%. No nucleic acid was incorporated into lipid nanoparticles N001- N004. Lipid nanoparticles D001 and D002 were prepared with plasmid DNA at a cationic lipidmucleic acid ratio of 16.
  • Lipid nanoparticles prepared with plasmid DNA cargo were also assessed for stability in different levels of bile salt as described previously.
  • FRET signals from Dil and DiO were normalized to samples assayed in solutions without bile salts. Resulting values showing vehicle stability levels are shown in Table 7. Standard deviation values are shown in parenthesis.
  • Nanoparticles according to those described in Example 9 are prepared with therapeutic cargo and administered orally (or by other route that introduces the nanoparticles to the gastrointestinal tract, e.g., intrarectally) for local (gastrointestinal) or systemic delivery of the cargo or cargo expression products.
  • Cargo are selected from nucleic acids, polypeptides, protein biologies (e.g., mAh, enzymes, etc.), short half-life biologies (e.g., hormones), immunogens, and genetic or epigenetic editing system components.
  • Local gastrointestinal delivery includes nanoparticle targeting of intestinal epithelial cells, lamina limba cells, intestinal muscle cells, enteric neurons, and/or other cell types present in intestinal tissue.
  • Systemic delivery includes nanoparticle targeting of gastrointestinal epithelial cells and basal secretion of nanoparticle cargo or cargo expression products into circulation.
  • Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding cell signaling factors (e.g., cytokines). Nanoparticles are introduced to subject gastrointestinal tracts orally or intrarectally to transfect and induce factor expression in gastrointestinal cells. Expressed factors are secreted locally or into the circulation for treatment of systemic disorders.
  • cell signaling factors e.g., cytokines
  • Nanoparticles with nucleic acids encoding interleukin (IL)-2 or IL-2 mutein Fc-fusion are administered to provide low doses of either factor: (1) systemically to treat immune-related disorders, including graft versus host disease (GVHD), systemic lupus erythematosus (SLE), and type I diabetes; or (2) locally to treat gastrointestinal immune-related disorders, including irritable bowel disease (IBD), ulcerative colitis, and Crohn’s disease.
  • GVHD graft versus host disease
  • SLE systemic lupus erythematosus
  • IBD irritable bowel disease
  • Crohn’s disease irritable bowel disease
  • Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding anti-IL18 receptor 1 (IL-18R1) antibody. Nanoparticles are introduced to subject gastrointestinal tracts orally or intrarectally to transfect and induce antibody expression in gastrointestinal cells. Expressed antibodies are secreted locally or into the circulation for treatment of systemic disorders. Antibodies block IL-18 cell signaling activity and resulting inflammation associated with immune-related disorders. Local secretion of the antibody treats or prevents gastrointestinal immune-related disorders, including IBD, ulcerative colitis, and Crohn’s disease.
  • IL-18R1 anti-IL18 receptor 1
  • Example 13 Nanoparticle treatment of gastrointestinal disorders
  • Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding IL-10, IL-22, or muteins thereof. Nanoparticles are introduced to subject gastrointestinal tracts orally or intrarectally to transfect and induce factor expression in gastrointestinal cells. Expressed factors are secreted locally to treat gastrointestinal immune- related disorders, including IBD, ulcerative colitis, and Crohn’s disease.
  • Example 14 Nanoparticle-mediated delivery of granulocyte-macrophage colony- stimulating factor (GM-CSF)
  • Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding GM-CSF. Nanoparticles are introduced to subject gastrointestinal tracts orally or intrarectally to transfect and induce factor expression in gastrointestinal cells. Expressed factors are secreted locally or into the circulation to promote myeloid recovery.
  • Subjects receiving treatment include patients with Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, acute lymphoblastic leukemia, or acute myelogenous leukemia (AML). Included are subjects that have received or are undergoing other forms of therapy, such as chemotherapy or stem cell transplantation (e.g., autologous or allogeneic stem cell transplantation from HLA-matched donors).
  • leukapheresis is used to collect hematopoietic progenitor cells mobilized as a result of treatment.
  • nanoparticles are administered at a dose and regimen sufficient to provide GM-CSF at a level of about 250 pg/nr/day and are administered until neutrophil blood levels reach 1000/pL.
  • Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding G-CSF. Nanoparticles are introduced to subject gastrointestinal tracts orally or intrarectally to transfect and induce factor expression in gastrointestinal cells. Expressed factors are secreted locally or into the circulation to promote granulocyte production and neutrophil regulation.
  • Subjects receiving treatment include patients with neutropenia (e.g., chemotherapy-induced febrile neutropenia in non-myeloid malignancies or congenital or acquired severe chronic neutropenia).
  • neutropenia e.g., chemotherapy-induced febrile neutropenia in non-myeloid malignancies or congenital or acquired severe chronic neutropenia
  • nanoparticles are administered at a dose and regimen sufficient to provide G-CSF at a level of 5 pg/kg/day and are administered until neutrophil blood levels reach 1000/pL.
  • Additional subjects receiving treatment include subjects undergoing bone marrow transplant therapy, subjects undergoing peripheral blood progenitor cell collection and engraft
  • Example 16 Nanoparticle-mediated delivery of adrenomedullin
  • Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding adrenomedullin.
  • Adrenomedullin reduces endothelial cell barrier disfunction associated with inflammation or other conditions. Nanoparticles are introduced to subject gastrointestinal tracts orally or intrarectally to transfect and induce adrenomedullin expression in gastrointestinal cells and local secretion from gastrointestinal cells.
  • Example 17 Nanoparticle-mediated factor secretion into the gastrointestinal lumen
  • Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding antimicrobial agents, in some cases intestinal alkaline phosphatase (LAP) or defensins. Nanoparticles are introduced to subject gastrointestinal tracts orally or intrarectally to transfect and induce expression in gastrointestinal cells. Expression products are secreted apically into the gastrointestinal lumen to target infectious agents.
  • LAP intestinal alkaline phosphatase
  • Nanoparticle use for transient gastrointestinal protein expression is prepared with nucleic acid cargo encoding proteins implicated in gastrointestinal diseases or systemic diseases with gastrointestinal axis interactions. Nanoparticles are introduced to subject gastrointestinal tracts orally or intrarectally to transfect and induce transient protein expression in gastrointestinal epithelial cells. In some subjects, gastrointestinal epithelial cells are transfected with nucleic acids encoding MY05B gene product for treatment of microvillus inclusion disease (MVID). In some subjects, gastrointestinal epithelial cells are transfected with nucleic acids encoding cystic fibrosis transmembrane regulator protein (CFTR) for treatment of cystic fibrosis.
  • MVID microvillus inclusion disease
  • CFTR cystic fibrosis transmembrane regulator protein
  • Example 19 Nanoparticle-mediated delivery of non-coding RNA to gastrointestinal cells
  • Nanoparticles according to those described in Example 9 are prepared with non coding RNA cargo (e.g., siRNA, miRNA, long ncRNA, piRNA, snoRNA, scaRNA, tRNA, rRNA and/or snRNA). Nanoparticles are introduced to subject gastrointestinal tracts orally or intrarectally to alter gene expression in gastrointestinal cells. In some subjects, the nanoparticles are introduced to treat gastrointestinal disorders. In some subjects, the nanoparticles are introduced to treat systemic disorders through gastrointestinal interactions. In some subjects, the non-coding RNA cargo represses SMAD7 gene expression for treatment of IBD.
  • non coding RNA cargo e.g., siRNA, miRNA, long ncRNA, piRNA, snoRNA, scaRNA, tRNA, rRNA and/or snRNA.
  • Nanoparticles are introduced to subject gastrointestinal tracts orally or intrarectally to alter gene expression in gastrointestinal cells. In some subjects, the nanoparticles are
  • Example 20 Nanoparticle-mediated delivery of gene editing systems
  • Nanoparticles according to those described in Example 9 are prepared with genetic or epigenetic editing system components. Nanoparticles are used to contact stem cells in vitro or introduced to subject gastrointestinal tracts orally or intrarectally to edit (e.g., via CRISPR base editing) or modify expression of (e.g., via modified Cas system) cellular genes. In some subjects, the nanoparticles used to correct mutations in epithelial cell genes, including CFTR gene mutations, GPR35 gene mutations, RNF186 A64T germline mutations associated with increased ulcerative colitis risk (see Beaudoin, M. et al. PLoS Genetics. 2013.
  • nanoparticles are used to delete genes encoding IL-18 and/or IL-18R1 in gastrointestinal stem cells to treat or prevent IBD.
  • nanoparticles are used to generate RNF186 (179X) mutations in gastrointestinal stem cells to confer protection against IBD.
  • nanoparticles are used to insert transgenes into gastrointestinal cell DNA (e.g., via CRISPR or RNA- mediated retrotransposons) to provide permanent sources for factor expression, including anti-TNF, anti-P19, or anti-IL-23 to treat or prevent IBD; or GLP-1 or FGF21 to treat or prevent metabolic diseases.
  • Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding antigens as immunogens for generation of local or systemic immune responses. Nanoparticles are introduced to subject gastrointestinal tracts orally or intrarectally to transfect and induce antigen expression in gastrointestinal cells. Expressed antigens are secreted locally or into the circulation to promote an immune response. Some subjects receive nanoparticles with cargo encoding antigens from different infectious agents, including influenza virus, SARS-CoV-2, Ebola, polio, or others (e.g., any of those described in Pasetti, M.F., et al. Immunol Rev. 2011. 239(1): 125-48) to immunize subjects against those agents.
  • infectious agents including influenza virus, SARS-CoV-2, Ebola, polio, or others (e.g., any of those described in Pasetti, M.F., et al. Immunol Rev. 2011. 239(1): 125-48) to immunize subjects against those agents.
  • Example 22 Nanoparticle-mediated delivery of neoantigens
  • Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding neoantigens for generation of local or systemic immune responses for oncology applications. Nanoparticles are introduced to subject gastrointestinal tracts orally or intrarectally to transfect and induce neoantigen expression in gastrointestinal cells. Expressed antigens are secreted locally or into the circulation to promote an immune response. Some subjects receive nanoparticles with cargo encoding neoantigens capable of generating immune responses against colorectal cancer and/or non-gastrointestinal cancers.
  • Example 23 Nanoparticle-mediated delivery of tolerance-promoting antigens
  • Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding antigens associated with allergies and/or auto-immune diseases. Nanoparticles are introduced to subject gastrointestinal tracts orally or intrarectally to transfect and induce antigen expression in gastrointestinal cells. Expressed antigens are secreted locally or into the circulation to promote immune system tolerance to the antigens and prevention of associated immune-related indications (e.g., peanut allergies, celiac disease, rheumatoid arthritis, and IBD).
  • associated immune-related indications e.g., peanut allergies, celiac disease, rheumatoid arthritis, and IBD.
  • Nanoparticle-mediated delivery of nucleic acids to intestinal immune cells [0283] Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding factors for intestinal immune cell expression (e.g., lamina intestinal mononuclear cells or intraepithelial lymphocytes), either exclusively or non-exclusively. Nanoparticles are introduced to subject gastrointestinal tracts orally or intrarectally to transfect and induce factor expression in the intestinal immune cells. In some subjects, nanoparticles with nucleic acid cargo encoding IL-10 are administered for delivery to gastrointestinal monocytes/macrophages to promote suppressive T regulatory (Treg) cell induction.
  • Treg suppressive T regulatory
  • nanoparticles with nucleic acid cargo encoding IL-22 are administered for delivery to gastrointestinal monocytes/macrophages to promote wound healing.
  • nanoparticles with nucleic acid cargo encoding transcription factors are administered for delivery to gastrointestinal monocytes/macrophages to modulate cellular activities, including nucleic acid cargo encoding peroxisome proliferator-activated receptor gamma (PPARy) for M2 macrophage polarization.
  • PPARy peroxisome proliferator-activated receptor gamma
  • Example 25 Nanoparticle-mediated delivery of nucleic acids to enteric neurons
  • Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding factors for expression in enteric neurons, either exclusively or non- exclusively. Nanoparticles are introduced to subject gastrointestinal tracts orally or intrarectally to transfect and induce factor expression in enteric neurons. Expressed factors act intracellularly, to influence cellular activities, or are secreted to act locally or systemically.
  • Example 26 Nanoparticles for use with intestinal organoids
  • Nanoparticles according to those described in Example 9 are prepared for cargo delivery to intestinal organoids ex vivo.
  • Some nanoparticle cargos include nucleic acids encoding factors for expression in intestinal organoids. These nanoparticles are introduced to organoid cultures ex vivo to transfect and induce factor expression.
  • Some nanoparticle cargos include genetic or epigenetic editing system components. These nanoparticles are introduced to organoid cultures to edit (e.g., via CRISPRbase editing) or modify expression of (e.g., via modified Cas system) genes in organoid cells.
  • exosomes from nanoparticle treated cells are isolated. The exosomes are used in some instances to deliver therapeutic cargo to other cells or tissues (in vivo or ex vivo).
  • Nanoparticles for use in animal models are prepared for cargo delivery to research animal subjects (e.g., mice or other species).
  • Some nanoparticle cargos include nucleic acids encoding factors for expression in subject gastrointestinal after oral or intrarectal administration.
  • Some nanoparticle cargos include genetic or epigenetic editing system components. These nanoparticles are introduced to subject gastrointestinal tracts orally or intrarectally to edit (e.g., via CRISPR base editing) or modify expression of (e.g., via modified Cas system) genes in gastrointestinal cells.
  • the nanoparticle delivery is used to create animal models, e.g., for researching specific diseases or effects associated with nanoparticle treatments.
  • exosomes from nanoparticle treated research animal subjects are isolated. The exosomes are used, in some instances, to deliver therapeutic cargo to other cells, tissues, and/or subjects, including other animal or human subjects.
  • Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding Factor VIII clotting factor. Nanoparticles are administered to subjects with hemophilia A to provide or replace Factor VIII clotting factor that is absent or defective due to mutation or other mechanism. Nanoparticles are administered orally or intrarectally for transfection and induction of Factor VIII expression in gastrointestinal cells. Factor VIII is secreted locally or into the circulation to restore clotting capabilities. In some subjects, circulating levels of Factor VIII reach from about 10 ng/mL to about 300 ng/mL after nanoparticle administration.
  • Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding b-glucocerebrosidase (GBA). Nanoparticles are administered to subjects with Gaucher’s disease to provide or replace GBA that is absent or defective due to mutation or other mechanism. Nanoparticles are administered orally or intrarectally to reach subject gastrointestinal tracts for transfection and induction of GBA expression in gastrointestinal cells. GBA is secreted locally or into the circulation to restore GBA enzymatic activity. In some subjects, steady-state GBA levels of about 6 ng/mL are achieved in plasma with nanoparticle treatment.
  • GBA b-glucocerebrosidase
  • Nanoparticle-mediated treatment of short bowel syndrome [0289] Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding glucagon-like peptide 2 (GLP-2) or an analog thereof (e.g., teduglutide). Nanoparticles are administered to subjects with short bowel syndrome (SBS) to improve intestinal absorption. Nanoparticles are administered orally or intrarectally to reach subject gastrointestinal tracts for transfection and induction of local GLP-2 expression in gastrointestinal cells. In some subjects, nanoparticle administration provides a maximum circulating GLP-2 concentration of about 36 ng/mL.
  • GLP-2 glucagon-like peptide 2
  • SBS short bowel syndrome
  • Nanoparticles are administered orally or intrarectally to reach subject gastrointestinal tracts for transfection and induction of local GLP-2 expression in gastrointestinal cells. In some subjects, nanoparticle administration provides a maximum circulating GLP-2 concentration of about 36 ng/mL.
  • Example 31 Nanoparticle-mediated adalimumab treatment
  • Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding adalimumab, an antibody that binds to tumor necrosis factor (TNF)a and prevents TNFa receptor binding. Nanoparticles are administered to subjects with immune- related disorders, including rheumatoid arthritis, IBD, and ankylosing spondylitis. Nanoparticles are administered orally or intrarectally for transfection and induction of adalimumab expression in gastrointestinal cells. Expressed antibodies are secreted locally and/or into the circulation for systemic treatment. In some subjects, maximum circulating antibody concentrations reach about 4-5 pg/mL after nanoparticle administration.
  • TNF tumor necrosis factor
  • Example 32 Nanoparticle-mediated human growth hormone treatment
  • Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding human growth hormone (HGH). Nanoparticles are administered to subjects with HGH deficiency or wasting. Nanoparticles are administered orally or intrarectally for transfection and induction of HGH expression in gastrointestinal cells. Expressed HGH is secreted locally and/or into the circulation for systemic treatment. In some subjects, circulating HGH levels achieved after nanoparticle administration are from about 1 to about 10 ng/mL in normal adults or from about 10 to about 50 ng/mL in children.
  • HGH human growth hormone
  • Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding glucagon-like peptide 1 (GLP-1), a peptide hormone agonist of the GLP- 1 receptor. Nanoparticles are administered to subjects with diabetes, cardiovascular disease, and/or non-alcoholic steatohepatitis (NASH). Nanoparticles are administered orally or intrarectally for transfection and induction of GLP-1 expression in gastrointestinal cells. Expressed GLP-1 is secreted locally and/or into the circulation for systemic treatment.
  • GLP-1 glucagon-like peptide 1
  • NASH non-alcoholic steatohepatitis
  • Nanoparticle-mediated treatment of hypo-parathyroidism [0293] Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding parathyroid hormone (PTH). Nanoparticles are administered to subjects with hypo-parathyroidism to raise circulating PTH concentration to normal levels. Nanoparticles are administered orally or intrarectally for transfection and induction of PTH expression in gastrointestinal cells. Expressed PTH is secreted locally and/or into the circulation. In some subjects, a maximum circulating PTH concentration of about 150 pg/mL is achieved after nanoparticle administration.
  • PTH parathyroid hormone
  • Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding antibody inhibitors of proprotein convertase subtilisin kexin 9 (PCSK9). Nanoparticles are administered to subjects to block PCSK9-dependent degradation of low density lipoprotein (LDL) receptors, resulting in lower levels of circulating LDL cholesterol and reduced risk of cardiovascular disease. Nanoparticles are administered orally or intrarectally for transfection and induction of antibody expression in gastrointestinal cells. Expressed antibodies (e.g., evolocumab and alirocumab) are secreted locally and/or into the circulation. In some subjects, a maximum circulating antibody concentration of about 18-19 pg/mL is achieved after nanoparticle administration.
  • PCSK9 proprotein convertase subtilisin kexin 9
  • Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding anti-CD3 bispecific antibodies. Nanoparticles are administered to subjects to direct T cells to tumor cells. Nanoparticles are administered orally or intrarectally for transfection and induction of antibody expression in gastrointestinal cells. Expressed antibodies are secreted locally and/or into the circulation. In some subjects, antibodies are secreted locally to target gastrointestinal tumor cells (e.g., those associated with colon cancer). In some subjects, antibodies are secreted into the circulation to target non- gastrointestinal tumor cells and/or tumor cells that are not gastrointestinal-specific.
  • target gastrointestinal tumor cells e.g., those associated with colon cancer
  • Example 37 Nanoparticle-mediated delivery of fibroblast growth factor 21
  • Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding fibroblast growth factor (FGF) 21. Nanoparticles are administered to subjects to promote metabolic balance, including subjects with NASH. Nanoparticles are introduced to subject gastrointestinal tracts orally or intrarectally for transfection and factor expression in gastrointestinal cells. Expressed factors are secreted locally and/or into the circulation to promote metabolic balance.
  • FGF fibroblast growth factor
  • Nanoparticles according to those described in Example 9 are prepared with nucleic acid cargo encoding relaxin. Nanoparticles are administered to subjects to promote relaxin- dependent anti-fibrotic activity, including to subjects with cardiovascular disease and/or liver fibrosis. Nanoparticles are introduced to subject gastrointestinal tracts orally or intrarectally for transfection and relaxin expression in gastrointestinal cells. Expressed factors are secreted locally and/or into the circulation to promote systemic activity.
  • LNPs with mRNA cargo were prepared following the methods previously described in Examples 1, 4, and 5.
  • Formulation lipid components are shown in Table 8 with lipid component levels provided in mole percentage units relative to total nanoparticle lipid.
  • Molar ratio of total LNP cationic lipids to total LNP nucleotides is presented as CL:N.
  • MPPs mucus penetrating peptides
  • TVDNDAPTKRASKLFAV SEQ ID NO: 17
  • Nanoparticles were further fluorescently labelled with Dil and DiO.
  • LNPs were prepared with mRNA via microfluidic mixing using a Precision Nanosystems NANOASSEMBLR®. Lipids were dissolved in ethanol and the mRNA in sodium acetate. The assembled particles were then dialyzed in HEPES buffer to reduce the % ethanol and spin concentrated using a 100 kDa Amicon filter.
  • Nanoparticle characteristics including size (diameter in nanometers), polydispersity index (PDI), and stability in various concentrations of bile salts was determined (see Table 9). Size and PDI were determined by dynamic light scattering (DLS) via a Malvern Zetasizer Nano ZS-90. For PDI, a value of 1 indicates maximum heterogeneity. Stability in different concentrations of bile salts (or TritonX 100 buffered control solution) was assessed as described previously in Example 3 by measuring the FRET intensity signals from Dil and DiO. Mean values for size, PDI, and FRET signal (relative light units) are reported in the Table, with standard deviation values shown in parenthesis.
  • LNPs tested displayed relative uniformity within each population (PDI around 2 or less for most formulations) and mean diameters of from about 30 to about 100 nm (smaller than typical gastrointestinal mucus pore size of about 200 nm).
  • LNPs D107-D112 transfection efficiency was assessed with mRNA cargo encoding G-CSF.
  • Human embryonic kidney (HEK) and Caco 2 intestinal epithelial cell lines were seeded at 10,000 cells/well in a 96 well plate in G-CSF free media. 24 hours later, LNPs were used to transfect cells in triplicate with incubation at 37°C for 24 hours. G-CSF protein levels in the media were then determined via standard immunological assay. Mean concentrations of G-CSF protein (pg/mL) detected are presented in Table 10 with standard deviations listed in parenthesis.
  • transfection efficiency assays were carried out generally according to the procedure described in Example 2 and utilizing mRNA cargo encoding luciferase.
  • HEK and Caco 2 cell lines were transfected with LNPs and cultured for 24 hours before being lysed with RIPA buffer and assayed for luciferase activity.
  • Mean luciferase activity (relative light units) associated with transfected cells is shown in Table 11. Standard deviation values are shown in parenthesis.

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Abstract

L'invention concerne des excipients d'administration et leurs procédés de fabrication et d'utilisation pour atteindre des cellules épithéliales, telles que des cellules dans des environnements contenant du mucus, ainsi que des excipients d'administration présentant une stabilité améliorée dans des environnements hostiles, y compris dans le tractus gastro-intestinal.
PCT/US2021/037011 2020-12-14 2021-06-11 Compositions et procédés pour excipients d'administration biologiques Ceased WO2022260678A1 (fr)

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EP21840333.5A EP4259099A1 (fr) 2020-12-14 2021-12-14 Systèmes de distribution de produits biologiques
CA3205059A CA3205059A1 (fr) 2020-12-14 2021-12-14 Systemes de distribution de produits biologiques
US18/267,206 US20240058454A1 (en) 2020-12-14 2021-12-14 Biological delivery systems
TW110146743A TW202241388A (zh) 2020-12-14 2021-12-14 生物遞送系統
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CN117442732A (zh) * 2023-12-20 2024-01-26 中国医学科学院基础医学研究所 lncRNA SCARNA2在抗病毒中的新用途
CN118576568A (zh) * 2024-08-06 2024-09-03 北京悦康科创医药科技股份有限公司 一种药物组合物及其制备方法和应用
WO2024250095A1 (fr) * 2023-06-03 2024-12-12 Qurcan Therapeutics Inc. Système hybride polymère-lipide à commutation de charge intelligent pour l'administration in vivo de molécules comprenant des arn
CN119139327A (zh) * 2024-11-04 2024-12-17 常州市第一人民医院 脱氢石胆酸在制备治疗糖尿病或糖尿病肾病药物中的应用

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WO2019222400A2 (fr) * 2018-05-15 2019-11-21 Dnalite Therapeutics, Inc. Peptides pénétrant dans le mucus, véhicules d'administration et méthodes de thérapie
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WO2020252375A1 (fr) * 2019-06-14 2020-12-17 Dnalite Therapeutics, Inc. Compositions et procédés pour véhicules d'administration biologiques

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US20090041833A1 (en) * 2005-04-18 2009-02-12 Bracco Research S.A. Composition comprising gas-filled microcapsules for ultrasound mediated delivery
US20160122759A1 (en) * 2013-05-06 2016-05-05 Alnylam Pharmaceuticals, Inc. Dosages and methods for delivering lipid formulated nucleic acid molecules
US20200129431A1 (en) * 2017-06-15 2020-04-30 National University Corporation Hokkaido University LIPID MEMBRANE STRUCTURE FOR DELIVERY INTO siRNA CELL
WO2019222400A2 (fr) * 2018-05-15 2019-11-21 Dnalite Therapeutics, Inc. Peptides pénétrant dans le mucus, véhicules d'administration et méthodes de thérapie
WO2020252375A1 (fr) * 2019-06-14 2020-12-17 Dnalite Therapeutics, Inc. Compositions et procédés pour véhicules d'administration biologiques

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024250095A1 (fr) * 2023-06-03 2024-12-12 Qurcan Therapeutics Inc. Système hybride polymère-lipide à commutation de charge intelligent pour l'administration in vivo de molécules comprenant des arn
CN117442732A (zh) * 2023-12-20 2024-01-26 中国医学科学院基础医学研究所 lncRNA SCARNA2在抗病毒中的新用途
CN117442732B (zh) * 2023-12-20 2024-03-19 中国医学科学院基础医学研究所 lncRNA SCARNA2在抗病毒中的新用途
CN118576568A (zh) * 2024-08-06 2024-09-03 北京悦康科创医药科技股份有限公司 一种药物组合物及其制备方法和应用
CN119139327A (zh) * 2024-11-04 2024-12-17 常州市第一人民医院 脱氢石胆酸在制备治疗糖尿病或糖尿病肾病药物中的应用

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