Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2896—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
• • 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
• • 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
  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • 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

Definitions

• the present invention relates to a pharmaceutical composition for delivery of DNA to cells or tissues bearing the insulin receptor.
• Gene therapy relies on efficient delivery of DNA to target cells, and expression of the delivered DNA in the nucleus of such cells.
• Different modes of DNA delivery have been proposed, and these involve both viral and non-viral delivery of gene sequences.
• the insulin receptor has also been used to target gene delivery to cells derived from liver.
• Huchet et al. Biochem. Pharmacol. 40, 253 (1990) obtained low level transfection by using serum albumin derivatized with dimethylaminopropyl groups as a DNA carrier and crosslinked this complex to insulin.
• Higher transfection activity was obtained by Rosenkrantz et al. (Exp. Cell. Res. 199, 323 (1992)) in which the epsilon-amino group of the C-terminal lysine residue of the insulin beta chain was derivatized with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and coupled to derivatized polylysine.
• SPDP N-succinimidyl 3-(2-pyridyldithio)propionate
• the invention is based on a gene delivery vehicle that is capable of targeting cells or tissue types which bear the insulin receptor on their surface, and delivering a gene to that cell or tissue.
• the invention encompasses a gene delivery vehicle which includes a nucleic acid binding peptide, H 2 N-Thr- Lys 18 -(S-Acetimidomethyl-Cys)-COOH, linked to insulin or an insulin derivative, and associated with condensed nucleic acid (NA) coding for sequences of therapeutic benefit.
• the insulin or insulin derivative allows for targeting of the nucleic acid delivery vehicle to mammalian, preferably human, cells or tissue that bear the insulin receptor.
• the nucleic acid binding peptide thus allows the vehicle to form a complex with condensed nucleic acid and thus to deliver a selected nucleic acid to the insulin receptor- bearing target cell.
• the gene delivery vehicle is useful for treating diseases associated with the liver, such as cirrhosis of the liver, hypercholesterolemia, cancer, and infection by hepatitis A, B, C, D or E.
• Sequences of therapeutic benefit for treatment of such diseases include, for example, ribozymes directed against RNA of infectious organisms or sequences encoding such ribozymes, genes encoding growth factors and growth factor receptors, genes whose products influence progression of the cycle of cell division (e.g., CDK genes and the p53 gene) , and the LDL receptor gene.
• association with or “bound to” refer to noncovalent forms of molecular association, such as charge interactions, hydrogen bonding, and hydrophobic interactions; e.g., positively charged amino groups of the nucleic acid binding component are attracted to negatively charged phosphate groups on the nucleic acid phosphodiester backbone.
• association with or “bound to” may refer to base pairing; e.g., the hydrophobic and hydrogen bonding interactions found between two strands of DNA.
• the nucleic acid binding component of the invention includes an amino acid sequence that is capable of binding to nucleic acid.
• insulin derivative includes any form of insulin that is capable of specific binding to the insulin receptor and being internalized into the target cell when linked to the nucleic acid delivery vehicle; such as natural or synthetic fragments of the insulin molecule, chemically modified insulin molecules, or chemically modified synthetic or naturally occurring fragments of insulin.
• Examples of cells which bear the insulin receptor include but are not limited to hepatocytes, brain cells, adipocytes, lymphoid cells, muscle, epithelial cells, and cancerous tissue, all of which are known to bear a high density of the insulin receptor.
• ex vivo targeting refers to targeting of a nucleic acid to an insulin receptor-bearing cell that has been removed from a patient
• in vi tro targeting refers to targeting of a nucleic acid to an insulin receptor-bearing cell from a cultured cell line
• in vivo cell targeting refers to targeting of an insulin receptor-bearing cell in a mammal such as in a human being.
• the invention thus also includes methods of treating an infectious disease, such as is caused by infection by hepatitis virus, particularly hepatitis C, which method includes targeting and incorporating nucleic acids coding for anti-hepatitis C ribozyme genes, into insulin receptor-bearing cells using the above-described delivery vehicle.
• Ex vivo and in vitro methods will, include the step of contacting the vehicle with an insulin receptor-bearing target cell, whether that cell be in a substantially homogenous population of target cells or in a heterogenous cell population, for a time and under conditions sufficient to allow cell targeting and nucleic acid uptake to occur.
• One in vivo method includes administering a therapeutically effective amount of the gene delivery vehicle to a mammal, preferably a human.
• Another in vivo method includes administering a therapeutically effective amount of a homogenous or heterogenous insulin receptor-bearing cell population that has been prepared by contacting the gene delivery vehicle with a target insulin receptor-bearing cell for a time and under conditions sufficient to allow cell targeting and nucleic acid uptake to occur.
• a "therapeutically effective amount” is an amount which confers a therapeutic benefit on a patient.
• the invention also encompasses a method of making a delivery vehicle for delivery of a gene to an insulin receptor-bearing cell, comprising the steps of a) oxidizing Thr-Lysl ⁇ -Cys(S-Pyridyl) peptide, and b) conjugating the oxidized peptide to amino-oxy-acetyl-insulin.
• the invention also encompasses a method of making kits for carrying out therapeutic delivery of a gene to a target cell that expresses the insulin receptor, a kit comprising the gene delivery vehicle described herein and packaging materials therefore.
• the invention is based on the discovery of a vehicle for delivery of a gene to insulin receptor-bearing cells, which vehicle includes the ligand insulin or an insulin derivative to target those cells bearing the insulin receptor.
• the protein hormone insulin or an insulin derivative, is used to target the nucleic acid delivery vehicle to cells expressing the insulin receptor.
• Derivatives of insulin include any form of insulin that is capable of binding specifically to the insulin receptor and being internalized into the target cell when linked to the nucleic acid delivery vehicle.
• the insulin or insulin derivative must recognize and bind with high and specific affinity to the insulin receptor on the target cell type.
• the most useful forms of insulin are wild type insulin, or fragments of insulin that are capable of binding to the insulin receptor and being internalized.
• the invention encompasses those fragments and derivatives of insulin and proinsulin which have at least 10% of the receptor binding affinity of native insulin.
• the receptor binding affinity of native insulin is determined as taught by Martin et al., 1984, Diabetologia pages 118-120. Described below is the insulin derivative amino-oxy-acetyl-insulin.
• the DNA binding component of the gene delivery vehicle is H 2 N-Thr-Lys 18 -(S-Acetimidomethyl-Cys)-COOH.
• the peptide was synthesized using a Millipore 9050 plus peptide synthesizer in extended synthesis cycle mode (30 mins - 1.25 hour couplings increasing during the synthesis). Fmoc-Cys(Acm)-O-PEG-PS-Resin was used.
• the peptide was purified using gel filtration to give the desired product.
• the Acetimidomethyl (Acm) thiol protecting group may be removed using mercury (II) acetate in 30% acetic acid in water followed by precipitation of the mercury with 2-mercaptoethanol.
• the resulting free thiol peptide can be purified using gel filtration to give the desired product.
• the peptide was synthesized using Millipore 9050 plus peptide synthesizer in extended synthesis cycle mode (30 mins - 1.25 hour couplings increasing during the synthesis) .
• Fmoc-Cys(Acm) -O-PEGPS-Resin was used. After deprotection of the Fmoc group, using 20% piperidine in DMF, the subsequent amino acids were coupled in four fold excess using 0- (lH-benzotriazol-l-yl) -tetramethyluronium tetrafluoroborate (TI3TU) /l-hydroxybenzotriazole and N,N'diisopropylcarbodiimide as activating agents.
• the peptide was purified using gel filtration to give the desired product.
• the acetimidomethyl (Acm) thiol protecting group may be removed using mercury (II) acetate in 30t acetic acid in water followed by precipitation of the mercury with 2-mercaptoethanol.
• the resulting free thiol peptide can be purified using gel filtration to give the desired product.
• the S-pyridyl derivative was obtained by reaction with a 5 fold molar excess of dithiopyridine in 0.1N sodium acetate buffer containing 50% acetonitrile. After 2h incubation at room temperature the product was purified by reverse phase hplc.
• a filler component may be added to the assembly reaction during preparation of the gene delivery vehicle.
• the filler component may be synthesized according to the above-noted procedure for synthesis of the DNA binding component.
• the filler NBC-II H 2 N-NBC-II-
• Insulin may be chemically modified according to previously described methods (Offord, R.E. "Semisynthetic Proteins” pp. 235, Wiley, Chichester and New York (1980)), with slight modifications. Briefly, 100 mg Zn-free insulin is dissolved in 1 L of IM NaHC0 3 , diluted with 4 mL dimethylformamide (DMF) and reacted with an equimolar amount of MSC-ONSu (N-Hydroxy succinimide derivative of Methylsulfonyloxycarbonate, Tesser, 1975, in "Peptides", John Wiley, NY, pp 53-56) relative to protein amino groups.
• MSC-ONSu N-Hydroxy succinimide derivative of Methylsulfonyloxycarbonate, Tesser, 1975, in "Peptides", John Wiley, NY, pp 53-56
• the peak corresponding to all-substituted insulin is collected and desalted in a double Chromabond (C18 solid phase extraction cartridge (2xlg of resin in a polypropylene column) Macherey-Nagel, Dormstadt, Germany) equilibrated in 0.1% TFA.
• the derivative obtained in such reactions is known to be preponderantly the desired N- , A 1 -MSC, N- -B 30 MSC substituted molecule.
• Analysis of the modified protein after overnight incubation in 50 mM DTT allows identification of the B-chain with only a single MSC group (calcd. m/z 3547.8; found m/z, 3549.6 ⁇ 0.4) , which is in agreement with the desired structure.
• the MSC groups are then cleaved by treatment with sodium hydroxide as described by Offord (loc cit) and the material repurified on the C18 column using 35-45% gradient over 20 mins.
• the final compound, BOC-AoA-insulin may be characterized by ESI-MS (calcd. m/z 5950.6; found m/z 5948.1 ⁇ 0.1) and is deprotected by TFA treatment (30 minutes at room temperature) just before conjugation to a polylysine peptide.
• the Cys-protected peptide (10 mg/ml) is dissolved in 50 mM imidazole, pH 6.9, and 0.2 M methionine in water is added as a anti-oxidant scavenger to a 10-fold molar excess over peptide.
• 50 mM sodium periodate is added to a five-fold molar excess over peptide, and the solution allowed to stand in the dark for 5 minutes at 220°c
• the mixture is purified by semipreparative HPLC on a C8 reverse phase column using 0.1% aqueous TFA and a 10% to 60% gradient of 0.1% aqueous TFA in 90% acetonitrile over 25 min.
• the isolated oxidized peptide is dissolved into a solution of 5 mg of the AoA-insulin derivative (approximately 2-fold molar excess of peptide over insulin) made up in 0.5 mL 0.1 M sodium acetate buffer to which had been added 50 ⁇ L acetonitrile, followed by adjustment to pH 3.8 with glacial acetic acid. After 15h incubation at room temperature, the conjugate is isolated and characterized by ESI-MS (calcd. m/z 8426.1, found m/z 8429.3 ⁇ 0.5). 4 mg of material were isolated by semipreparative HPLC with a 30-45% gradient from the bulk of the reaction mixture. The peak fraction was dried in a speedvac (yield is approximately 4 mg of conjugate) .
• a fusogenic peptide FP useful according to the invention includes the follow sequence: NH 2 - GLFEAIAGFIENGWEGMIDGGGC(Acm) -COOH, and is synthesized as follows. 1. Synthesis of H 2 N-FP-(S-acetimidomethyl-Cys) -COOH: The FP peptide is synthesized using a Millipore 9050 plus peptide synthesizer in extended synthesis cycle mode (1 hour couplings) . Fmoc-Cys(Acm) -O-PEG-PS-was used.
• the acetamidomethyl (Acm) thiol protecting group on the peptide may be removed using mercury (II) acetate with water/acetonitrile tl:l, o.lt TFA) as solvent followed by precipitation of the mercury with 2-mercaptoethanol.
• the resulting free thiol peptide can be purified using gel filtration to give the desired product.
• DNA is made up to 20 mg/ml in a transfection buffer (0.15 M to 1.0 NaCI; 25 mM HEPES, pH 7.4)
• the conjugate and peptide is made to an equal volume to the DNA in safe buffer.
• the DNA is shaken or vortexed while the condensing agent is added at the rate of o.l volume per minute.
• the complex is left at room temperature for at least 30 minutes prior to adding to cells, and can be stored at 4° C if necessary.
• Transfection complexes consist of plasmid DNA containing the therapeutic gene or reporter gene, insulin conjugated to NH 2 -Thr-Lys 18 -Cys-COOH and unconjugated NBC-II.
• the transfection complex is synthesised by incubating the three components together, for 30 minutes to 24 hours at room temperature.
• the prepared complex is centrifuged to remove any aggregated material and then assayed for gene transfer.
• Conjugates or peptides are assayed for their ability to condense DNA using the following method:
• the DNA is made up to 20 mg/ml in 150 mM NaCI; 25 mM HEPES, pH 7.4, or in 0.6 M NaCI; mM HEPES, pH 7.4 and aliquoted between wells on a multiwell plate.
• the amount of conjugate or peptide required to give (positive charge:phosphate) ratios of between 0.1 and 5.0 is calculated. This amount is made up to an equal volume to the DNA aliquots (0.05-0.5 ml) in either 150 mM sodium chloride; 25 mM HEPES, pH 7.4 or 0.6 M sodium chloride; 25 mM HEPES, pH 7.4.
• the plate containing the DNA is placed on a plate shaker and shaken while the conjugate or peptide is added at a rate of 0.1 volume per minute. After addition of the condensing agent is complete, the solution is incubated at room temperature for at least 30 minutes. A sample for each (positive charge: phosphate) ratio is analyzed by electrophoresis on an agarose gel. The gel is stained with ethidium bromide and visualized under UV light. Condensed DNA remains in the well of the gel and does not migrate in the electric field.
• the transfection complexes may be assayed for their ability to transfer genes into hepatocytes (HepG2 cells) expressing the insulin receptor.
• the plasmid DNA contains a marker gene for firefly luciferase.
• the plasmid contains a gene whose expression will have a beneficial effect.
• the transfection complex is incubated with blood cells and the mixture is subjected to electroporation. After incubation, the cells are lysed and assayed for gene expression. In the case of the luciferase reporter gene, luciferin and ATP are added to lysed cells and the light emitted is measured with a luminometer.
• Cells are harvested on the day of assay by centrifugation at 1200 rpm for 5 min at room temperature.
• the cell pellet is resuspended in phosphate buffered saline (PBS) and re-centrifuged. This operation is performed twice.
• the cell pellet is then suspended in RPMI 1640 (Gibco Ltd.) to make up a suspension of approximately 2.7 x 10 6 cells per ml.
• the cells are then aliquoted into tubes and .75 ml of RPMI medium added, followed by 0.04 -0.08ml of 100 ⁇ m Chloroquine (CQ) or FP peptide and finally 0.25 ml of DNA-complex solution.
• CQ Chloroquine
• the transfection is then allowed to proceed by incubating the cells at 37°C for 4 h. After this time, the cells are harvested by centrifugation at 2000 rpm, suspended in 1ml of RPMI and re-centrifuged. Finally, the cells are in 0.5 ml RPMI containing 10% fetal bovine serum. At this stage, if necessary, the cells are electroporated at 300 V and 250 ⁇ F using conventional electroporation.
• Each 0.5 ml of transfected cell suspension is transferred to a well of a 12 well plastic culture plate containing 1.5ml of RPMI 10% FBS.
• the original transfection tube is rinsed with a further 1ml of medium and the wash transferred to the culture dish making a final volume of 3ml.
• the culture plate is then incubated at 37°C for 24-72h in an atmosphere of 5% C0 2 .
• the contents of each well in the culture dish are transferred to centrifuge tubes and the cells collected by centrifugation at 13,000 rpm.
• the pellet is resuspended in 0.12 ml of Lysis Buffer (100 mM sodium phosphate, pH 7.8, 8 mM MgCl 2 , ImM EDTA, 1% Triton X-100 and 15% glycerol) and agitated with a pipette.
• Lysis Buffer 100 mM sodium phosphate, pH 7.8, 8 mM MgCl 2 , ImM EDTA, 1% Triton X-100 and 15% glycerol
• the lysate is centrifuged at 13,000 rpm for 1 minute and the supernatant collected. 80 pl of the supernatant are transferred to a luminometer tube.
• the luciferase activity is then assayed using a Berthold Lumat L9501 luminometer.
• the assay buffer used is Lysis buffer containing lOmM Luciferin and 100 mM ATP.
• RLU relative light units
• the transfection efficiency resulted in the delivery such that 200,000-500,000 relative luciferase units per mg of protein were expressed in the transfected cells.
• the delivery vehicle and plasmid DNA may be formulated separately for parenteral administration or formulated together as the transfection complex. In the latter case the transfection complex may be assembled just prior to use.
• the plasmid DNA includes a gene whose expression would have some beneficial therapeutic effect on the cells of the recipient.
• the delivery vehicle and DNA are exchanged into isotonic phosphate free buffer and sterile filtered through a 0.45 or 0.22 ⁇ filter.
• the formulated solution or transfection complex (a mixture of the delivery vehicle, DNA and free DNA condensing component) may be sterile filled and aliquotted into suitable vials.
• the vials may be stored at 4°C, 20°C or 80°C or alternatively the DNA, delivery vehicle or transfection complex may be freeze dried from a buffer containing an appropriated carrier and bulking agent. In these cases, the dosage form is reconstituted with a sterile solution before administration.
• a delivery vehicle of the invention can be administered to the patient, preferably in a biologically compatible solution or a pharmaceutically acceptable delivery vehicle, by ingestion, injection, inhalation or any number of other methods.
• the dosages administered will vary from patient to patient; a "therapeutically effective dose" will be determined by the level of enhancement of function of the transferred genetic material balanced against any risk of deleterious side effects. Monitoring levels of gene introduction, gene expression will assist in selecting and adjusting the dosages administered.
• composition including a delivery vehicle will be administered in a single dose in the range of 10 ng - 100 ug/kg body weight, preferably in the range of 100 ng - 10 ug/kg body weight, such that at least one copy of the therapeutic gene is delivered to each target cell.
• a cell population comprising insulin receptor-bearing cells can be removed from the patient or otherwise provided, transduced with a therapeutic gene in accordance with the invention, then reintroduced into the patient.

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