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WO1999065529A1 - Complexes de cobalamine et d'acide nucleique et leur utilisation en therapie genique - Google Patents

Complexes de cobalamine et d'acide nucleique et leur utilisation en therapie genique Download PDF

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Publication number
WO1999065529A1
WO1999065529A1 PCT/US1999/012236 US9912236W WO9965529A1 WO 1999065529 A1 WO1999065529 A1 WO 1999065529A1 US 9912236 W US9912236 W US 9912236W WO 9965529 A1 WO9965529 A1 WO 9965529A1
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Prior art keywords
complex
nucleic acid
cobalamin
protein
cells
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PCT/US1999/012236
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English (en)
Inventor
Pamela K. Foreman
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Alza Corporation
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Priority to AU43283/99A priority Critical patent/AU4328399A/en
Publication of WO1999065529A1 publication Critical patent/WO1999065529A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT

Definitions

  • This invention relates to delivery of nucleic acid to cells for the production of proteins.
  • it relates to the use of receptor mediated endocytosis for introduction of nucleic acid into cells so that lower doses of nucleic acid are required to produce a given effect and undesirable immunogenic effects are reduced.
  • Viral vector-directed gene transfer shows high gene transfer efficiency but is deficient in several areas. For example, some viral vectors randomly integrate DNA into host genomes, while others, such as adenoviral vectors, induce host inflammatory and immune responses, rendering these vectors ineffective in repeated application. Further, retroviral vectors require dividing cells for stable integration, making these vectors unsuitable for gene therapy of terminally differentiated cells. Similarly, for efficient transduction, adeno-associated virus prefers cells in the S-phase to cells in stationary culture. The high multiplicity of infection of adeno-associated virus for efficient transduction coupled with difficulty in obtaining virus preparations with high titer has limited the use of this virus as a routine gene therapy vector. 1
  • molecular conjugates prepared by chemically linking receptor ligands with polycations can be prepared by chemically linking antibodies or fragments thereof with polycations.
  • the polycations serve as carriers of DNA while the ligands target the receptors on cell surfaces.
  • the conjugates along with the DNA are internalized via receptor-mediated endocytosis.
  • Receptor-mediated endocytosis has been used to introduce DNA into a variety of cells.
  • Examples of these include an asialoosomucoid/polylysine conjugate complexed with a plasmid which utilizes the asialoglycoprotein receptor found on hepatocytes 4 and Fab fragments complexed with plasmid DNA which enter epithelial cells through the polymeric immunoglobulin receptor (plgR).
  • Other ligands include mannose, 17 lactose, 21 insulin, 19 epidermal growth factor, 23 anti-thrombomodulin antibodies, 25 transferrin, 26 gluconyl groups, 20 lectins, 16 folate 22 and fibroblast growth factor. 18, 24
  • the protein ligands can be difficult to prepare and can induce an unwanted immune response in the patient.
  • Cobalamin also known as Vitamin B ⁇ 2 (VB 12 )
  • Vitamin B ⁇ 2 is a large molecule that cannot cross the epithelial barrier through simple or facilitated diffusion.
  • Most fat soluble molecules, such as vitamins A, D, E and K, are generally absorbed in combination with fats, while the small water soluble vitamins, such as vitamin C, thiamine, riboflavin and folic acid are mainly absorbed by facilitated diffusion.
  • Cobalamin is absorbed through a mechanism that involves a number of transport proteins, which is markedly different than that of other vitamins. During the process of absorption, cobalamin is first bound by R-protein found in saliva.
  • antisense oligonucleotides have been functionalized with a VB-
  • Major problems associated with the use of nonviral (protein) and/or viral vectors are that a patient can develop a protein-related immune response with repeated use of these vectors or that vectors, e.g., polylysine, may trigger the complement cascade, when delivered via a non-oral route.
  • vectors e.g., polylysine
  • high doses are required which can be toxic to the patient.
  • a vector which 1) may be used repeatedly without raising an immune response; 2) may be administered in high doses if needed; and 3) may be administered by a non-parenteral route.
  • This invention is directed to complexes comprising a nucleic acid bound to cobalamin, methods of delivering nucleic acid into a cell and pharmaceutical compositions comprising a nucleic acid/cobalamin complex.
  • nucleic acid encoding a protein complexed with cobalamin may be introduced into a cell via cobalamin receptor- mediated endocytosis to deliver the nucleic acid into the cell so that it may encode a protein, which protein may be used locally, e.g., in the gut, or systemically.
  • This may be a therapeutic protein, a nutritional protein or a protein used for research purposes.
  • cobalamin is a required nutrient, the development of an immune response following repeated doses is unlikely.
  • this invention provides a complex and compositions comprising a nucleic acid, which nucleic acid encodes at least one protein, bound to cobalamin.
  • the nucleic acid may be bound to the cobalamin with a suitable linker arm, especially one selected from the group consisting of polylysine and protamine.
  • the complex may further comprise a cobalamin binding protein selected from the group consisting of intrinsic factor, R-proteins and transcobalamin II.
  • the nucleic acid encodes at least one protein, preferably a therapeutic protein, a nutritional protein or a marker protein.
  • Marker proteins include, but are not limited to, ⁇ - galactosidase, secreted human placental alkaline phosphatase, chloramphenicol acetyl transferase (CAT), luciferase, green fluorescent proteins and derivatives thereof, blue fluorescent proteins and derivatives thereof and peptides or proteins conferring antibiotic resistance.
  • Another aspect of this invention is directed to a method for introducing nucleic acid into cells comprising administering to a subject an effective amount of a complex described above.
  • this invention is directed to a composition
  • a composition comprising the nucleic acid/cobalamin complex as described above and a pharmaceutically acceptable carrier.
  • the composition is adapted for administration via an oral, parenteral, mucosal, rectal or enteral route.
  • the present invention relates to a cobalamin construct useful for delivering nucleic acid into cells.
  • the invention is directed to a nucleic acid encoding at least one protein, which nucleic acid is bound to a cobalamin molecule to form a cobalamin-nucleic acid construct.
  • the binding of the nucleic acid to the cobalamin molecule may be directly or via a linker, preferably a polycation linker. Without being limited to any theory, it is believed that this construct delivers nucleic acid to cells via cobalamin receptor-mediated endocytosis.
  • the nucleic acid encodes at least one protein and may encode at least two proteins.
  • the nucleic acid may be DNA or RNA.
  • the encoded protein may be of therapeutic interest (therapeutic protein), may be a nutritional protein and/or may be a marker protein which may be used to indicate whether transfection has occurred.
  • the encoded protein is preferably chloramphenicol acetyl transferase, ⁇ -galactosidase, secreted human placental alkaline phosphatase.
  • Other encoded proteins include luciferase, green fluorescent proteins and derivatives thereof, blue fluorescent proteins and derivatives thereof and peptides or proteins conferring antibiotic resistance.
  • the cobalamin may preferably be cyanocobalamin or may be an analogue or derivative of cobalamin such as methylcobalamin, adenosylcobalamin or hydroxycobalamin.
  • the derivative must bind nucleic acid and the bound form must be taken up by a cell which presents a cobalamin receptor.
  • the cobalamin-nucleic acid complex may comprise a linker arm, for example, polylysine, protamine, polyethyleneimine, DEAE dextran, polybrene, cationic lipids and ethidium salts. Preferred linkers are polycation linkers.
  • the cobalamin-nucleic acid complex or the cobalamin-linker-nucleic acid complex may further be bound by a cobalamin binding protein, for example, intrinsic factor, R-proteins and transcobalamin II, or analogues thereof.
  • compositions and methods of the present invention are useful clinically, nutritionally and/or as research tools for the study of various aspects of the activity of nucleic acids and proteins. They may be used in many ways in vitro or in vivo, for example, to study cellular or organ ontogeny, to study polarized secretion and protein secretory signals, to transfect cells which may be refractory to other methods, e.g., Caco-2 cells to determine transfection efficiency, to study the length of time the nucleic acid is expressed, and to determine whether a cell secretes the protein encoded by the nucleic acid introduced into the cell is secreted or is produced intracellularly.
  • the complex may be adapted for administration to a subject via a route selected from the group consisting of oral, parenteral, mucosal, rectal and enteral.
  • Targeted delivery to cells which contain cobalamin receptors for example, upper gastrointestinal tract cells, lower gastrointestinal tract cells and liver cells, may be accomplished either by direct administration to the cells, e.g., by instillation or deposition in the intestine, or by the use of various delivery systems which provide targeted delivery, e.g., enteric coated dosage forms, colonic dosage forms, delivery directed to the liver, etc.
  • the effective amount of the nucleic acid/cobalamin complex delivered to a subject is that amount which results in satisfactory protein production levels, and is dependent upon the protein, subject, goal of administration (e.g., testing, treating or prevention), route of administration, condition treated, and the like.
  • goal of administration e.g., testing, treating or prevention
  • route of administration e.g., condition treated, and the like.
  • Cobalamin refers to the compound commonly known as either Vitamin B 12 (VB 12 ) or cyanocobalamin.
  • Cosmetic derivatives refers to hydroxycobalamin, adenosylcobalamin, methylcobalamin, aquocobalamin, 5-0- methylbenzylcobalamin, as well as the desdimethyl, monoethylamide and the methylamide analogues of all of the above. Also included are the various analogues and homologues of cobamide such as coenzyme B ⁇ 2 and 5'- deoxyadenosylcobalamin.
  • analogues include chlorocobalamin, sulfitocobalamin, nitrocobalamin, thiocyanocobalamin, benzimidazole derivatives such as 5,6-dichlorobenzimidazole, 5- hydroxybenzimidazole, trimethylbenzimidazole, as well as adenosylcyanocobalamin, cobalamin lactone, cobalaminlactam and the anilide, ethylamide, monocarbozylic and dicarboxylic acid derivatives of VB 12 or its analogues.
  • analogues include analogues which replace the D-ribose moiety of the nucleotide loop with an oligomethylene group or a trimethylene analogue containing imidazole instead of 5,6- dimethylbenzimidazole, as described in Ishida et al., Arch. Microbiol. (1994) 161 :293- 299, which is incorporated by reference in its entirety. Additionally included are analogues where the cobalt is replaced by zinc or nickel, or analogues where the corrin ring, or any part of the cobalamin molecule, has one or more substituents.
  • analogues which are useful in the present invention are those which bind to the nucleic acid and/or a linker arm, and retain the capacity to bind to the cobalamin-binding proteins, such as Intrinsic Factor (IF).
  • IF Intrinsic Factor
  • Assays to determine such binding are known to those of skill in the art. (See, for example, Habberfield, et al., Vitamin B12-mediated uptake of erythropoietin and granulocyte colony stimulating factor in vitro and in vivo, International Journal of Pharmaceutics 145:1-8 (1996).)
  • cyanocobalamin, hydroxycobalamin, adenosylcobalamin or methylcobalamin is used in this invention.
  • Cobalamin-binding proteins or “cobalamin-binding transport proteins” include intrinsic factor (IF), R-proteins such as transcobalamin I and transcobalamin III, and transcobalamin II (TCII) and analogues thereof which bind to and transport cobalamin into a cell.
  • Intrinsic factor a glycoprotein
  • transcobalamin II a polypeptide
  • the R-proteins a family of glycoproteins, are found in plasma, granulocytes and several glandular secretions.
  • These proteins bind to cobalamin in a macromolecular complex that involves a binding site on the protein which allows the cobalamin-binding protein/cobalamin complex to enter a cell via cobalamin receptor mediated endocytosis.
  • Receptors for intrinsic factor are found on microvillous membranes of ileal absorptive cells, e.g., ileal mucosal cells, (ileal enterocytes).
  • ileal enterocytes e.g., ileal enterocytes.
  • Linker refers to a structure which is covalently or non-covalently bound to cobalamin, or a derivative thereof, and which binds nucleic acid, permits the transport of cobalamin into the cell and does not interfere with the coding ability of the nucleic acid once the complex has entered the cell.
  • linker arms suitable for use in this invention include polylysine, protamine, polyethyleneimine (see Boussif et al., Proc. Natl. Acad. Sci., (1995) 92:7297-7301), DEAE dextran, polybrene, cationic lipids, and ethidium salts.
  • Avidin or streptavidin-biotin may be used as the linker structure, e.g., a nucleic acid-avidin linked to biotinylated VB-
  • “Cobalamin-linker conjugate,” also referred to as “conjugate,” refers to cobalamin or a cobalamin derivative conjugated, covalently or non-covalently, to a linker, for example polylysine or protamine.
  • Cobalamin-nucleic acid complex also referred to as “complex” refers to nucleic acid bound to a cobalamin molecule.
  • This complex may include a linker, where the linker is bound to the cobalamin and binds the nucleic acid.
  • This complex is capable of being bound to a cobalamin binding protein such as intrinsic factor, R-protein or transcobalamin II.
  • the cobalamin-nucleic acid complex bound to a cobalamin-binding protein is capable of utilizing a cobalamin receptor to enter a cell via receptor-mediated endocytosis.
  • Nucleic acid refers to unbranched (linear or circular) chains of nucleotides which encode a protein or peptide. This includes deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the nucleic acid construct may be in the form of a plasmid.
  • the nucleic acid may include promoter, leader, signal, polyadenalyation or intron sequences, locus control regions, markers and the like.
  • the nucleic acid may utilize bacterial or mitochondrial codons. Nucleic acid containing modified, derivatized or non-naturally occurring nucleotides are also included within this definition.
  • a “protein” refers to polymers which comprise amino acid residues bound together by amide linkages (CONH). Both naturally-derived or recombinantly produced moieties are included in this definition. This includes post-translationally modified forms, e.g., glycosylated proteins and peptides.
  • a “nutritional protein” is a protein or peptide which provides nutrition to a cell or organism.
  • a “therapeutic protein” is a protein or peptide which may provide a pharmacologic effect, generally beneficial, to an organism. This effect may be localized in the gastrointestinal tract or may be systemic.
  • therapeutic proteins that provide localized therapy to the gastrointestinal tract include, but are not limited to, pancreatic enzymes, lactase, cytokines, interleukin-1 receptor antagonist, tumor necrosis factor receptor, recombinant antibodies and antibody fragments, tumor suppressor proteins, cytotoxic proteins and the like.
  • the nucleic acid of this invention encodes at least one protein, which may or may not be of therapeutic interest.
  • Useful non-therapeutic proteins include marker proteins.
  • a "marker protein” is any protein which may be used, often as a research tool, to determine whether transfection has occurred or to monitor the length of time the nucleic acid remains active in the cell. Any protein for which expression can be determined may be used as a marker protein, even though the protein may also have a therapeutic use.
  • Suitable marker proteins can include, but are not limited to, luciferase, ⁇ - galactosidase, secreted human placental alkaline phosphatase (SEAP), CAT, blue and green fluorescent proteins and proteins conferring antibiotic resistance.
  • Encoded therapeutic peptides and proteins may include, but are not limited to insulin (for the treatment of diabetes), ⁇ 1-antitrypsin (for 1-antitrypsin deficiency); Factor VIII (for hemophilia), Factor IX and other coagulation factors (for bleeding disorders); growth hormones (such as human growth hormone for growth disorders); other peptide hormones; growth factors (such as fibroblast growth factor (FGF), platelet derived growth factor (PDGF)); pituitary hormones (such as adrenal cortical stimulating hormone (ACTH), thyroid stimulating hormone (TSH)); lymphokines and cytokines for systemic therapy; ⁇ -interferon (for granulomatous disease of childhood); ⁇ -interferon (for leukemia and chronic active hepatitis); erythropoietin; other hematologic growth factors (for chronic renal failure and other marrow suppressive disorders); tissue plasminogen activator (TPA) (for prevention of thrombosis in the pulmonary coronary arteries
  • agents that may be delivered include peptide hormones, adrenal cortical stimulating hormone, thyroid stimulating hormone, and other pituitary hormones, interferon ⁇ , ⁇ , and ⁇ , consensus interferon, erythropoietin, growth factors such as GCSF, GM-CSF, insulin-like growth factor 1 , CF4, dDAVP, pancreatic enzymes, lactase, interleukin-2, tumor suppresser proteins, cytotoxic proteins, retroviruses and other viruses, viral proteins, antibodies, recombinant antibodies, antibody fragments and the like.
  • Tumor antigens, allergens and bacterial or viral proteins, which induce an immune response or which results in tolerization are also examples of therapeutic proteins.
  • “Pharmaceutically acceptable carrier” refers to biologically acceptable carriers.
  • “Transfection enhancer” refers to compounds which enhance the efficiency of transduction of non-viral vectors. They may be given therapeutically. An example of a transfection enhancer useful for oral delivery of nucleic acid constructs is chloroquine.
  • compositions comprising a cobalamin-nucleic acid complex which binds in concert with a transport protein to a cobalamin receptor and is endocytosed into a cell, thereby allowing expression of the nucleic acid in the cell.
  • the cell is an epithelial cell and more preferably, an enterocyte and even more preferably, an ileal absorptive cell. It is understood that the compounds of this invention may be made into pharmaceutical formulations for delivery to a patient.
  • the cobalamin-nucleic acid complex may be administered alone, with a cobalamin-binding protein bound to the cobalamin-nucleic acid complex, or the complex may be co-administered with biologically active binding proteins, such as intrinsic factor.
  • biologically active binding proteins such as intrinsic factor.
  • the cobalamin-binding proteins normally found in the patient's stomach, plasma, granulocytes or glandular secretions are utilized to bind to the cobalamin-nucleic acid complex so that cobalamin receptor-mediated endocytosis can occur which transports the nucleic acid into the cell.
  • the cobalamin-nucleic acid complex compositions may be formulated in a variety of ways.
  • the cobalamin-nucleic acid complex or cobalamin-nucleic acid/cobalamin binding protein complex may be administered as a solid formulation.
  • Solid formulations may contain a pharmaceutically inert carrier, including conventional carriers such as lactose, starch, dextrin or magnesium stearate. These are conveniently presented in a tablet or capsule form.
  • the formulation is encapsulated in an enteric coated capsule, e.g., a coating which remains intact in the stomach, but dissolves and releases the contents of the capsule once it arrives in the small intestine.
  • the total delay time prior to the delivery of the cobalamin-nucleic acid construct is usually in the range of less than about 1 hour, usually within 45 minutes, and preferably in less than about 30 minutes for delivery of the complex to the small intestine.
  • the PULSINCAP TM which comprises an impermeable capsule and a hydrogel plug. The plug is designed to swell and dislodge from the devise after a particular time period, thereby delivering its contents in a bolus following a predetermined delay. See, for example, International Patent Application WO 96/40081, published 19 December 1996, which is incorporated by reference in its entirety. See also, Remington's Pharmaceutical Sciences, 18 th edition, 1990, especially Chapter 89, for other oral pharmaceutical preparations which can be used in this invention.
  • the formulation can be a liquid formulation or semisolid form for oral dosage.
  • Liquid formulations include a pharmaceutically inert carrier such as water, saline or other pharmaceutically acceptable liquids for oral administration.
  • the liquid is compatible with the cobalamin-nucleic acid complex or the cobalamin-nucleic acid/cobalamin-binding protein complex.
  • a nasogastric tube may also be used to deliver the liquid compositions directly to the stomach. The use of such liquids is well known to those skilled in the art.
  • Semi-solid formulations may also be used. These may include the cobalamin-nucleic acid complex or cobalamin-nucleic acid/cobalamin- binding protein complex incorporated into gelatin, such as flavored JELLO®, and other soft foods such as applesauce, pudding or ice cream.
  • the compounds of this invention may also be administered directly to the ileum through surgical access.
  • oral administration of the cobalamin-nucleic acid complex provides a means of administering proteins to the cells of the epithelium.
  • This method provides a means of delivery of nucleic acid encoding proteins which are secreted into the lumen, interstitially or systemically, such as pancreatic enzymes, lactase (for lactose intolerance), etc.
  • pancreatic enzymes such as pancreatic enzymes, lactase (for lactose intolerance), etc.
  • lactase for lactose intolerance
  • the cobalamin- nucleic acid complex or cobalamin-nucleic acid/cobalamin binding protein complex may be suspended in a pharmaceutically acceptable carrier, such as water, saline, phosphate buffered saline, perfluorodecalin, etc. See Remington's Pharmaceutical Sciences, 18 th edition, 1990 for suitable carriers and formulations for parenteral administration.
  • Doses are selected to provide an effective amount of cobalamin-nucleic acid to cells. Depending upon the purpose, this can be a detected amount, an amount that causes or attenuates an immune response to the coded protein, or an amount effective to ameliorate or prevent a disease condition or disorder.
  • These compositions may be formulated in a unit dose form or in multiple or subunit doses.
  • enterocytes When oral administration is desired, the clinician should keep in mind that enterocytes have about a three day turnover in the ileum.
  • the dose level and schedule of administration may vary depending upon the particular condition treated, the age, weight and overall health of the patient. Determination of this dosage is well within the skill of the clinician or researcher.
  • cobalamin-nucleic acid complex compositions of this invention may be administered to animals in the manner and formulations described above. These include mammals such as humans, cattle, pigs, dogs, cats, horses, sheep, goats and rodents; avians such as chicken, turkey, duck and geese; and any other animal which has cobalamin receptors.
  • mammals such as humans, cattle, pigs, dogs, cats, horses, sheep, goats and rodents; avians such as chicken, turkey, duck and geese; and any other animal which has cobalamin receptors.
  • NA nucleic acid
  • DNA deoxyribonucleic acid
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • Cobalamin can be bound to nucleic acid either directly (covalently) or via an attached linker arm.
  • Cyanocobalamin monocarboxylic acids were prepared and purified as described in Anton et al., J. Amer. Chem. Soc. (1980) 102:2215-2219. Forty-five mg of the cyanocobalamin carboxylic acid (CNCb/IICOOH) was reacted with 50 mg hydroxybenzotriazole, 114 mg 1-ethyl, 3(3'dimethylaminopropyl)carbodiimide and 300 mg polylysine (MW 15,000-30,000, Sigma Chemical Co., St. Louis, MO) in a final volume of 15 ml of water. The pH was adjusted to 6.35 with NaOH and the reaction was allowed to proceed for 6-48 hours at room temperature.
  • CNCb/IICOOH cyanocobalamin carboxylic acid
  • the reaction mixture was then applied to a 43 X 3 cm Sephadex G-25 column.
  • This fraction may be further purified by passing over an affinity column (e.g., a 2.1 mm by 5 cm or 1.0 mm by 3 cm Poros perfusion column (PerSeptive Biosystems)) prepared with immobilized R-protein (non-intrinsic factor, Sigma Chemical Co.)
  • the cobalamin-polylysine conjugate is retained bound to the R-protein on the column and the underivatized polylysine passes through.
  • the column is then further washed with the running buffer (buffered isotonic HEPES solution) to remove residual underivatized polylysine and other reaction by-products.
  • the purified cobalamin-polylysine conjugate is eluted from the column by applying a mild organic modifier (such as MeOH or ACN) or by changing the pH of the running buffer.
  • a mild organic modifier such as MeOH or ACN
  • the eluted cobalamin-polylysine conjugate was then extensively dialyzed.
  • concentration of polylysine was determined by standard protein assay procedures (e.g., BCA kit using manufactures instructions (Pierce)).
  • concentration of cobalamin was determined by absorbance at 550 nm.
  • a 1 % solution of cobalamin has an absorbance of 64 at this wavelength. From the concentration of these two species, the average number of cobalamin molecules per polylysine was determined and found to be 3.7 cobalamin molecules per polylysine.
  • Polylysine is preferred as a linker, however, it is understood that other linkers may be conjugated to cobalamin including protamine, nucleic acid binding proteins, transcription factors, histones and the like.
  • Useful linkers are those able to bind to or associate with cobalamin and nucleic acid without preventing either binding to cobalamin receptors or interfering with the ability of the nucleic acid to encode a protein.
  • the above stoichiometry of reagents may be varied to alter the number of cobalamin residues bound to each polylysine molecule as needed, and that the length of the polylysine chain may be varied to optimize nucleic acid binding and cellular uptake of the nucleic acid of interest.
  • nucleic acid was visualized by staining the gel with ethidium bromide (Sigma). The unbound nucleic acid remaining in the mixture sample was observed to run at a similar rate as the control nucleic acid. The nucleic acid that was bound to the cobalamin-polylysine conjugate was unable to enter the gel and was retained in the well. The nucleic acid was found to bind quantitatively at charge ratios of
  • cobalamin-binding proteins to the cobalamin-nucleic acid complex may be evaluated as follows. Cobalamin (alone) and cobalamin-nucleic acid complexes are diluted in six-tenfold dilutions in intrinsic factor buffer (1 mg/ml BSA (VB 12 and IF deficient, Sigma) in 0.1 M phosphate buffer pH7.5). To 225 ⁇ l of the IF buffer is added
  • Co 57 VB 12 (0.25 ml, 0.25 ng in IF buffer) is then added to each sample.
  • Porcine IF (0.25 ml; 1 U/ml in IF buffer) is then added and the material allowed to incubate at room temperature for 20 minutes.
  • BSA-coated charcoal (0.25 ml; 0.5% BSA (B ⁇ 2 and IF free) plus 2.5% charcoal) is added to each sample, vortexed and centrifuged. The supernatant from each sample is then counted on a gamma counter
  • the binding capacity of the cobalamin-nucleic acid complex to the cobalamin-binding proteins is assessed by the ability of the complex to compete with the cobalamin-binding proteins
  • the binding capacity can be determined by using a number of commercially available kits, such as MAGIC® LITE VITAMIN B ⁇ 2 ASSAY, available from Ciba Corning Diagnostics Corporation. This assay is a competitive chemiluminescence receptor assay. Other commercially available assays which may be used to assess the binding capacity include MAGIC® Vitamin B-
  • the binding affinity of the cobalamin-nucleic acid complex is compared with that of the control, uncomplexed cobalamin.
  • the binding affinity for complexes preferred for use in this invention is at least 1% of control binding affinity.
  • Caco- 2 cells Fegh, J. Natl. Cancer Inst. (1977) 59:224-226) or other cell lines bearing cobalamin receptors, e.g., HepG2 cells (Hall et al., J. Cell Physiol. (1985) 124(3):507-
  • the nucleic acid is a plasmid nucleic acid encoding a marker protein such as chloramphenicol acetyl transferase (CAT). Alternatively, other linkers may also be used.
  • the cobalamin-polylysine-nucleic acid complex is mixed at various charge ratios as described above. Various concentrations of the complex, either in appropriate culture medium or in PBS containing 1.7mM CaCI 2 , is applied to subconfluent or confluent differentiated Caco-2 cells in culture. One to 24 hours later, the complex is removed and culture medium is applied. Twenty-four to 96 hours after the initial application of complex, the cells are evaluated for the expression of the marker protein, for example, CAT.
  • CAT chloramphenicol acetyl transferase
  • a variety of standard techniques can be used detect the expression of the marker protein.
  • histochemical staining can be used to determine the efficiency of transduction.
  • the protein product can be quantified in cell- free extracts by ELISA (CAT ELISA kit, Boehringer-Manneheim). Histochemical staining for CAT is accomplished by fixing cells using 50% methanol, 50% acetone for 2 minutes. Washed cells are incubated with rabbit anti-CAT antibody (5'-3') diluted 1 :150 in 10% fetal bovine serum, PBS for 1 hour at room temperature.
  • HRP horseradish peroxidase
  • Caltag goat anti-rabbit antibody
  • the concentration of the complex may be varied to optimize the efficiency of transfection.
  • Transfection of intestinal cells in vivo may be evaluated using the ileal loop model. Rats, and other species such as rabbits, may be used in this model. Twenty 300 to 350 g Sprague Dawley rats (Charles River) are anesthetized.
  • a midline abdominal incision is made and the ileum is located.
  • the intestinal contents are gently removed using warmed saline and the cobalamin- polylysine-nucleic acid complex is injected.
  • Intrinsic Factor (Sigma) bound to the cobalamin-polylysine-nucleic acid complex may also be used.
  • the treated segment is marked at each end using sutures to allow subsequent relocation.
  • Soft ligatures may be placed at each end of the flushed segment prior to injection of the complex. In these experiments, the ligated, treated segment is returned to the abdominal cavity for two hours where it is kept moist. The ligatures are then removed and the animal is closed and allowed to recover.
  • Tissues are rinsed in the lysis buffer provided (4mM Pefablock, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1mM benzamidine, 1 ⁇ g/ml pepstatin, 5 ⁇ g/ml aprotinin (Sigma), 0.5 mM EDTA and 0.5 mM dithiothreitol).
  • Tissues are homogenized in 0.5-1 ml lysis buffer using a Brinkman polytron. Following homogenization, extracts are centrifuged at 14,000 rpm for ten minutes. The supernatants are quick frozen and stored at -70° C.
  • the total protein concentration is determined using a BCA kit (Pierce) and adjusted to 25 mg/ml by diluting in lysis buffer containing protease inhibitors, ⁇ -galactosidase concentrations are determined by ELISA following manufacturer's instructions.
  • ⁇ -galactosidase concentrations are determined by ELISA following manufacturer's instructions.
  • Sections eight to ten ⁇ m long, are prepared and immediately fixed for 10 minutes in 0.5% glutaraldehyde, washed in PBS and immersed overnight in PBS containing 5mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCI 2 and 1 mg/ml 5-bromo-4-chloro- 3-indoyl ⁇ -D-galactopyranoside (X-gal, Sigma).
  • the sections are viewed under a microscope. The appearance of a blue stain indicate transfection has occurred, and no blue staining indicate that transfection has not occurred. Endogenous ⁇ -galactosidase activity does not interfere with this assay when performed under these conditions. See Foreman, P. et al, Adenoviral Gene Transfer to the Intestine, Human Gene Therapy, submitted for publication 3/9/98.
  • SEAP human placental alkaline phosphatase
  • Serum samples are obtained from rat tail veins prior to administration of the complexed DNA and at various times (e.g., 24 to 72 hours) after treatment. Following manufacturer's instruction, 10-25 ⁇ l of serum are diluted in 75-200 dilution buffer provided in the kit. The presence of secreted human placental alkaline phosphatase is determined using the Phospha-Light kit (Tropix, Inc.), a chemiluminescent reporter gene assay.
  • Phospha-Light kit Tropix, Inc.

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Abstract

L'invention concerne un complexe utilisé pour introduire de l'acide nucléique dans des cellules. Elle concerne, en particulier, des compositions et des procédés mettant en application un acide nucléique codant au moins une protéine et dont on a formé un complexe avec cobalamine. Ces compositions et ces procédés mettent en oeuvre une endocytose par l'intermédiaire du récepteur de cobalamine afin d'introduire l'acide nucléique codant la protéine dans les cellules. Ces compositions et ces procédés permettent d'utiliser des doses inférieures d'acide nucléique et de limiter les effets immunogènes indésirables.
PCT/US1999/012236 1998-06-15 1999-06-02 Complexes de cobalamine et d'acide nucleique et leur utilisation en therapie genique WO1999065529A1 (fr)

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AU43283/99A AU4328399A (en) 1998-06-15 1999-06-02 Nucleic acid-cobalamin complexes and their use in gene therapy

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US8929198P 1998-06-15 1998-06-15
US60/089,291 1998-06-15

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000074721A1 (fr) * 1999-06-02 2000-12-14 Biotech Australia Pty Limited Vitamine pour therapie a double ciblage
WO2001048006A1 (fr) * 1999-12-27 2001-07-05 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine de liaison de la cobalamine 10, et polynucleotide codant pour ce polypeptide
WO2003025139A2 (fr) * 2001-09-17 2003-03-27 Mayo Foundation For Medical Education And Research Administration d'acides nucleiques, d'analogues et de derives de ceux-ci induite par cobalamine
EP1377220A1 (fr) * 2001-03-16 2004-01-07 University of Utah Research Foundation Cobalamines fluorescentes et utilisations
US6919076B1 (en) 1998-01-20 2005-07-19 Pericor Science, Inc. Conjugates of agents and transglutaminase substrate linking molecules
US7462345B2 (en) 1995-11-13 2008-12-09 Mayo Foundation For Medical Education And Research Radionuclide labeling of vitamin B12 and coenzymes thereof
US7468432B2 (en) 1999-04-16 2008-12-23 Mayo Foundation For Medical Education And Research Cobalamin conjugates useful as antitumor agents
US7531162B2 (en) 2000-10-25 2009-05-12 Mayo Foundation For Medical Education And Research Transcobalamin receptor binding conjugates useful for treating abnormal cellular proliferation
US7591995B2 (en) 1999-10-15 2009-09-22 Mayo Foundation For Medical Education And Research Cobalamin conjugates useful as imaging and therapeutic agents

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0220030A2 (fr) * 1985-10-10 1987-04-29 Biotechnology Australia Pty. Ltd. Système oral de distribution
WO1992017167A1 (fr) * 1991-04-02 1992-10-15 Biotech Australia Pty. Ltd. Systemes de liberation orale de microparticules
WO1993007883A1 (fr) * 1991-10-24 1993-04-29 Isis Pharmaceuticals, Inc. Oligonucleotides derives presentant diverses qualites dont une meilleure facilite d'absorption

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0220030A2 (fr) * 1985-10-10 1987-04-29 Biotechnology Australia Pty. Ltd. Système oral de distribution
WO1992017167A1 (fr) * 1991-04-02 1992-10-15 Biotech Australia Pty. Ltd. Systemes de liberation orale de microparticules
WO1993007883A1 (fr) * 1991-10-24 1993-04-29 Isis Pharmaceuticals, Inc. Oligonucleotides derives presentant diverses qualites dont une meilleure facilite d'absorption

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GUY M ET AL: "Evaluation of coupling of cobalamin to antisense oligonucleotides by thin-layer and reversed-phase liquid chromatography", JOURNAL OF CHROMATOGRAPHY B: BIOMEDICAL SCIENCES & APPLICATIONS, vol. 706, no. 1, 27 February 1998 (1998-02-27), pages 149-156, XP004110841, ISSN: 0378-4347 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7462345B2 (en) 1995-11-13 2008-12-09 Mayo Foundation For Medical Education And Research Radionuclide labeling of vitamin B12 and coenzymes thereof
US6919076B1 (en) 1998-01-20 2005-07-19 Pericor Science, Inc. Conjugates of agents and transglutaminase substrate linking molecules
US7468432B2 (en) 1999-04-16 2008-12-23 Mayo Foundation For Medical Education And Research Cobalamin conjugates useful as antitumor agents
WO2000074721A1 (fr) * 1999-06-02 2000-12-14 Biotech Australia Pty Limited Vitamine pour therapie a double ciblage
US7591995B2 (en) 1999-10-15 2009-09-22 Mayo Foundation For Medical Education And Research Cobalamin conjugates useful as imaging and therapeutic agents
WO2001048006A1 (fr) * 1999-12-27 2001-07-05 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine de liaison de la cobalamine 10, et polynucleotide codant pour ce polypeptide
US7531162B2 (en) 2000-10-25 2009-05-12 Mayo Foundation For Medical Education And Research Transcobalamin receptor binding conjugates useful for treating abnormal cellular proliferation
EP1377220A1 (fr) * 2001-03-16 2004-01-07 University of Utah Research Foundation Cobalamines fluorescentes et utilisations
EP1377220A4 (fr) * 2001-03-16 2008-06-18 Univ Utah Res Found Cobalamines fluorescentes et utilisations
WO2003025139A2 (fr) * 2001-09-17 2003-03-27 Mayo Foundation For Medical Education And Research Administration d'acides nucleiques, d'analogues et de derives de ceux-ci induite par cobalamine
WO2003025139A3 (fr) * 2001-09-17 2003-08-21 Mayo Foundation Administration d'acides nucleiques, d'analogues et de derives de ceux-ci induite par cobalamine

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