CA2241040A1 - Improved pharmaceutical compositions - Google Patents
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- CA2241040A1 CA2241040A1 CA002241040A CA2241040A CA2241040A1 CA 2241040 A1 CA2241040 A1 CA 2241040A1 CA 002241040 A CA002241040 A CA 002241040A CA 2241040 A CA2241040 A CA 2241040A CA 2241040 A1 CA2241040 A1 CA 2241040A1
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
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- 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
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- 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
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- 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
- C07K16/2896—Immunoglobulins [IGs], 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
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- 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
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- 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
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Abstract
The invention is based on the discovery of a gene delivery vehicle for delivery of a therapeutic gene to cells or tissues bearing the insulin receptor.
Description
W097/22363 1 PCTtGB96103137 INPROVED P~ARNACEUTICAL COMPO~ITION8 FIELD OF THE INVENTION
The present invention relates to a pharmaceutical composition for delivery of DNA to cells or tissues bearing the insulin receptor.
BACKGROUND ~F THE INVENTION
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.
Early experiments on introducing DNA into mammalian cells ln vitro utilized DNA in precipitated form with low efficiency of transfection and required selectable mar~er genes (Wigler et al. (1977) Cell 16, 777-85; Graham and Van der Erb (lg79) Proc. Natl. Acad. Sci. USA 77, 1373-76 and (1973) Virology 52, 456)). Since this time molecular biologists have developed many other more efficient techniques for introducing DNA into cells, such as electroporation, complexation with asbestos, polybrene, DEAE-Dextran, liposomes, lipopolyamines, polyornithine, particle bombardment and direct microinjection (reviewed by Kucherlapati and Skoultchi (1984) Crit. Rev. Biochem. 16, 349-79; Keown et al. (1990) Methods Enzymol. 185, 527). Many of these methods are unsuitable for use clinically since they give highly variable and relatively poor levels of transfection. Another obstacle to the wider use of existing gene delivery vehicles resides in their instability in vivo.
It has been shown that particles of a similar size to the gene delivery vehicles of the prior art are rapidly and 3S efficiently removed from the blood by the reticuloendothelial system (Posse and Kirsch, Bio/Technology 1, 869 (1984)).
Loyter and Volsky ~Cell Sur. Rev. 8, 215-266 (1982)) SUBSTITUTE SHEET(RULE 26) CA 02241040 1998-06-lg and Kaneda et al. (Exp. Cell Res. 173, 56-69 (1987)) describe the reconstitution of viral envelopes as biological carriers including carriers of DNA. In this approach, naturally occurring viruses are isolated, dissolved in detergent containing solvents, the viral nucleic acid removed and the remaining viral components reconstituted in the presence of plasmid DNA. However, this technology has proven to be extremely expensive and difficult to scale up.
Moreover, serious safety concerns are connected with the pharmaceutical use of extracted viruses.
Other non-viral gene delivery systems described in the literature merely extend observations on transfection using DNA condensed by synthetic polymers, for example, soluble DNA/polylysine complexes can be generated (Li et al., Biochem. J. 12, 1763 (1973)). Polylysine complexes tagged with asialoglycoprotein have been used to target DNA
to hepatocytes in vitro (Wu and Wu, J. Biol. Chem. 262, 4429 (1987); U.S. Patent 5,166,320). Lactosylated polylysine (Midoux et al. (1993) Nuc. Acids Res. 21, 871-878) and galactosylated histones (Chen et al. (1994) Human Gene Therapy 5, 429-435) have been used to target plasmid DNA to cells bearing lectin receptors, and insulin conjugated to polylysine (Rosenkrantz et al. (1992) Exp. Cell Res. 199, 323-329) to cells bearing insulin receptors. However, Wagner et al. ( ibid) have shown that the latter approach is even less efficient than standard methods of transfection, and may therefore be considered unsuitable for pharmaceutical development. Monoclonal antibodies have been used to target DNA to particular cell types (Machy et al.- (1988) Proc.
Natl. Acad. Sci. USA 85, 8027-8031; Trubetskoy et al. (1992) Bioconjugate Chem. 3, 323-27 and WO 91/17773 and Wo 92/19287).
The insulin receptor has a}so 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 SUBSTITUTE SHEET (RULE 26) CA 02241040 1998-06-lg 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.
SUMMARY OF THE INVEN~ION
The invention is based on a gene delivery vehicle that is capable of targeting cells or tissue types which bear the insulin receptor on t~eir surface, and delivering a gene to that cell or tissue.
The invention encompasses a gene delivery vehicle which includes a nucleic acid binding peptide, HzN-Thr-Lys18-(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 invention also encompasses a pharmaceutical preparation for use in gene therapy, comprising a gene delivery vehicle comprising a therapeutic nucleic acid associated with Insulin-N~-Co-CH2-O-N=CH4-CO-Lysl8-Cys~S-Pyridyl)-OH in combination with a pharmaceutically acceptable carrier.
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 proqression of the cycle of cell division (e.g., SUv~ TE SHEET(RULE 26) CA 02241040 1998-06-lg W097~2363 4 PCTIGB96103137 CDK genes and the p53 gene), and the LDL receptor gene.
As used herein, "associated with" or 'l~ound 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. Alternatively, 'associated 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.
The protein hormone insulin or a derivative of insulin acts as the targeting ligand to direct the nucleic acid delivery vehicle to cells expressing the insulin receptor, where the insulin or insulin derivative retains receptor binding properties when conjugated to a nucleic acid binding component. As used herein, "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.
The invention also encompasses methods of ex vivo, in vitro, and in vivo cell-type specific targeting. As used herein, ex vivo targeting refers to targeting of a nucleic acid to an insulin receptor-bearing cell that has been removed from a patient; in vitro targeting refers to targeting of a nucleic acid to an insulin receptor-bearing cell from a cultured cell line; and in vivo cell targeting SUBSTITUTE SHEET (RULE 26) CA 02241040 1998-06-l9 refers to targeting of an ins~lin 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 a~ove-described delivery vehicle. -The invention thus also encompasses a method of treating a disease of a patient, comprising the steps of:
(a) providing a pharmaceutical preparation comprising a gene delivery vehicle comprising a therapeutic nucleic acid associated with Insulin-NH-CO-CH2-O-N=CH-CO-Lys1a-Cys(S-Pyridyl)-OH in combination with a pharmaceutically acceptable carrier; and (b) administering said pharmaceutical preparation to a patient suffering from a genetic disease Ex vivo and in vltro 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. As used herein, 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 SUBSTITUTE SHEET (RULE 26) ~VO 97122363 6 Thr-~ysl8-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.
Further features and advantages of the invention will become more fully apparent in the following description of the embodiments and drawings thereof, and from the claims.
DETAILED DESCRIPTION OF THE INVENTION
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.
Z0 The Structure of Insulin or an Insulin Derivative 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, In practice, the most useful forms of insulin are wild type insulin, or fraqments of insulin that are capable of binding to the insulin receptor and being internalized. There are over 500 fragments and derivatives of insulin having biological activity which are known in the art. 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 SlJb;~ JTE SHEET (RULE 26) WO 91n2363 by Martin et al., 1984, Diabetologia pages 118-120.
Described below is the insulin derivative amino-oxy-acetyl-Insulin.
5 Structure of the r)NA Bindinq Component The DNA binding component of the gene delivery vehicle is H2N-Thr-Lysl8-(S-Acetimidomethyl-cys)-COOH.
EXAMPLE I
8ynthe~is of Nucleic Acid ~inding Peptide 1. Preparation of H2N-Thr-Lysl8-(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. After 20 deprotection of the of the Fmoc group using 20% piperidine in DMF, the subsequent amino acids were coupled in four-fold excess using o- (lH-benzotriazol-1-yl) -tetramethyluronium tetraf luoroborate ( T B T U ) / 1 - h y d r o x y b e n z o t r i a z o l e a n d 25 N,N'-diispropylCarbOdiimide as activating agents. When necessary, a four-fold excess of the amino acid, 0-(lH-7-aza- benzotriazol-l-yl) -tetramethyluronium hexafluorophosphate and diisopropy1ethylamine was used to ensure complete coupling to the growing peptide. After the 30 synthesis of the peptide was complete, the N-terminal Fmoc group was removed as described above to give the free amino side chain protected peptide bound to the resin. This was c l e a v e d f r o m t h e r e s i n u s i n g a TFA/water/phenol/thioanisole/1, 2-ethanedithiol 35 (82.5:5:5:5:2.5) mixture. Following precipitation with ether and centrifugation, the peptide was purified using gel filtration to give the desired product. When necessary, the Acetimidomethyl (Acm) thiol protecting group may be removed SU~ ITE SHEET (RULE 26) CA 0224l040 l998-06-l9 Wo97~2363 8 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 O-(lH-benzotriazOl-l-yl)-tetramethyluronium tetrafluoroborate (TI3TU)/l-hydroxybenzotriazole and N,N'diisopropylcarbodiimide as activating agents. When necessary, a four-fold excess of the amino acid O-(lH-7-aza-benzotriazol-l-yl)tetramethyluronium hexafluorophosphate and diisopropylethylamine was used to ensure complete coupling to the growing peptide. After the synthesis of the peptide was complete, the N-terminal Fmoc group was removed as described above to give the free amino side chain protected peptide bound to the resin. This peptide was cleaved from the resin using a TFA/water/phenollthioanisole/l,2- ethanedithiol t82.5:5:5:5:2.5) mixture. Following precipitation with ether and centrifugaticn, the peptide was purified using gel 2S filtration to give the desired product. When necessary, 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 O.lN sodium acetate buffer containing 50% acetonitrile. A~ter 2h incubation at room temperature the product was purified by reverse phase hplc.
2. Synthesis of F_ller Component SUBSTITUTE SHEET (RULE 26) P~T/~B96/03137 W097t22363 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 ~inding component. The filler NBC-II (H2N-NBC-II-(Acetimidomethyl-Cys)-COOH) has the following sequence: NH2 PRKxRxvEKKspKKAE~RpARspAKA}tA~vKpKAAKpKK~tKKK
RKVEKKSPKKAKKPAAC-COOH.
EXAMPLE II
Functionalization of In~ulin Insulin may be chemically modified according to previously described methods (Offord, ~.E. "Semisynthetic Proteins" pp. 235, Wiley, Chichester and New York (1980)l, with slight modifications. Briefly, lOO mg Zn-free insulin is dissolved in l mL of lM NaHCO3, diluted with 4 mL
dimethylformamide (DMF) and reacted with an equimolar amount of MSC-ONSu (~-~ydroxy succinimide derivative of Methylsulfonyloxycarbonate, Tesser, 1975, in ~Peptides", John Wiley, NY, pp 53-56) relative to protein amino groups.
After lh incubation at room temperature, the mixture is acidified and subjected to prepara~ive HP~C on a Waters Prep Nova-Pak Hz C~ column ~flow rate 20ml min using a 25-50% B
gradient (B ~radient is a mixture of O.l tw/v) aqueous TFA
and acetonitrile:TFA water 900:l:l00 tv/w/v) over 50 min.
The peak corresponding to all-substituted insulin (as judged by subsequent ESI-MS) 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-~, A1-MSC, N- -B30MSC 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 SUBSTITUTE SHEET(RULE 26~
CA 02241040 1998-06-lg structure.
mg MSC2-insulin are dissolved in 1 mL
N-methyl-pyrrolidone and reacted with a lO-fold molar excess of Boc-AoA-OSu (Vilaseca et al., Bioconjugate Chem. 4 515-520 tl993)), in the presence of equimolar amounts of HOBt and of sufficient N-ethylmorpholine to bring the pH to approximately 8. After lh incubation at room temperature, the reaction medium is acidified and diluted with 0.1% TFA, and the derivatized insulin isolated by semipreparative HPLC
on a C8 reverse phase column equilibrated in 0.1% TFA in conjunction with a 35-45B% gradient (described above) over 20 min.
The ~SC 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 + O.l) and is deprotected by TFA treatment (30 minutes at room temperature~ just before conjugation to a polylysine peptide.
EXAMPLE III
Oxidation of the Thr-Lysl8-Cy~(S-Pyridyl) Peptide and Conjugation to Amino-oxy-acetyl ~AOA)-Insulin The cys-protected peptide (10 mg/ml) is dissolved in 50 mM imidazole, pH 6.9, and O.2 M methionine in water is added as a anti-oxidant scavenger to a lO-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) SUBSTITUTE SHEET (RULE 26) CA 02241040 1998-06-lg 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. mtz 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 pea~
fraction was dried in a speedvac (yield is approximately 4 mg of conjugate).
EXAMPLE IV
During gene transfer a fusogenic peptide may be included in the transfection mix in order to enhance efficiency of transfer of the therapeutic gene. A fusogenic peptide FP useful according to the invention includes the follow sequence: NH2- GLFEAIAGFIENGWEGMIDGGGC(Acm)-COOH, and is synthesized as follows.
1. Synthesis of H2N-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. After deprotection of the Fmoc group using 20% piperidine in DMF, the subsequent amino acids were coupled in four-fold excess using o-(lH-benzotriazol-l-yl)-tetramethyluronium tetrafluoroborate (TBTU)/1- hydroxybenzotriazole and N,N'-diisopropylcarbodiimide as activating agents. When necessary, a four fold excess of the amino acid,O(lH-7-aza-benzotriazol-1-yl) -tetramethlyroniumhexafluorophosphate and diisopropyleth~lamine was used to ensure coupling to the growing peptide. After the synthesis of the peptide was complete, the N-terminal Fmoc group was removed as described above to give the free amino side chain-protected peptide bound to the resin. This was cleaved from the resin using a TFA/water/phenol/thioanisole/1,2- ethanedithiol (82.5:5:5:5:2.5) mixture. Following precipitation with ether and centrifugation, the peptide can be purified using gel SUBSTITUTE SHEET (RULE 26) CA 02241040 1998-06-lg filtration to give the desired product.
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.
EXAMPLE V
8ynthe~is of Transfection ComplexeQ
DNA is made up to 20 mg/ml in a transfection buffer (0.15 M to 1.0 NaCl; 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 NH2-Thr-Lys~8-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.
Gel Retardation A~say for DNA Condensation 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 NaCl; 25 mM
HEPES, pH 7.4, or in 0.6 M NaCl; 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 O.l and 5.0 is calculated. This amount is made up to an equal volume to SUBSTITUTE SHEET (RULE 26) 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 O.l 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 W light. Condensed DNA remains in the well of the gel and does not migrate in the electric Field.
EXAMPLE VI
A~say for Gen~ Transfer The transfection complexes may be assayed for their ability to transfer genes into hepatocytes (HepG2 cells) expressing the insulin receptor. For studies aimed at determining transfection efficiency, the plasmid DNA
contains a marker gene for firefly luciferase. For pharmaceutical applications, 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 lO6 cells per ml. The cells are then aliquoted into tubes and .75 ml of RPMI medium added, followed by 0.04 -0.08ml of lOO ~m SU~S 111 ~JTE SHEET (RULE 26) CA 02241040 1998-06-lg W O 97~2363 14 PCT/GB96103137 Chloro~uine (CQ) or FP peptide and finally 0.25 ml of DNA-complex solution. 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 lml of RPMI and re-centrifuged. Finally, the cells are in 0.5 ~l 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 ~0 transferred to a well of a 12 well plastic culture plate containing l.~ml of RPMI 10% FBS. The original transfection tube is rinsed with a further lml 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% C02. 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 MgCl2, lmM E~TA, 1% Triton X-lO0 and 15% glycerol) and agitated with a pipette. 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. Light produced by the luciferase is integrated over 4 sec and is described as relative light units (RLU). The data are converted to RLU/ml of lysate, RLU/cell or RLU/mg protein (protein concentration of the lys~te having been determined in this case by the BioRAD Lowry assay).
The transfe~tion efficiency resulted in the delivery such that 200,000-500,000 relative luciferase units per mg of protein were expressed in the transfected cells.
EXAMP~E VII
Dosage and Pharmaceutical Formulation SUBSTITUTE SHEET (RULE 2~) CA 02241040 1998-06-l9 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. In the case of a pharmaceutical composition, the plasmid DNA
includes a gene whose expression would have some beneficial therapeutlc 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.
Use of this type of pharmaceutical composition in vivo or ex vivo with nucleic acid containing a gene of physiological importance, such as replacement of a defective gene or an additional potentially beneficial gene function, is expected to confer long term genetic modification of the cells and be effective in the treatment of disease. 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 dosaqes 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. Monitorinq levels of gene introduction, gene expression will assist in selecting and adjusting the dosages administered. Generally, a composition including a delivery vehicle will be administered in a single dose in the range of lo ng - lOo ug/kg body weight, preferably in the range of lon ng - 10 ug/kg body weight, SUBSTITUTE SHEET (RULE 26) l6 such that at least one copy of the therapeutic gene is delivered to each target cell.
Ex ~ivo treatment is also contemplated within the present invention. 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.
OTHER EMBODIMENTS
Other embodiments will be evident to those of skill in the art. It should be understood that the foregoing detailed description is provided for clarity only and is merely exemplary. The spirit and scope of the present invention are not limited to the above examples, but are encompassed by the following claims.
SUBSTITUTE SHEET (RULE 26)
The present invention relates to a pharmaceutical composition for delivery of DNA to cells or tissues bearing the insulin receptor.
BACKGROUND ~F THE INVENTION
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.
Early experiments on introducing DNA into mammalian cells ln vitro utilized DNA in precipitated form with low efficiency of transfection and required selectable mar~er genes (Wigler et al. (1977) Cell 16, 777-85; Graham and Van der Erb (lg79) Proc. Natl. Acad. Sci. USA 77, 1373-76 and (1973) Virology 52, 456)). Since this time molecular biologists have developed many other more efficient techniques for introducing DNA into cells, such as electroporation, complexation with asbestos, polybrene, DEAE-Dextran, liposomes, lipopolyamines, polyornithine, particle bombardment and direct microinjection (reviewed by Kucherlapati and Skoultchi (1984) Crit. Rev. Biochem. 16, 349-79; Keown et al. (1990) Methods Enzymol. 185, 527). Many of these methods are unsuitable for use clinically since they give highly variable and relatively poor levels of transfection. Another obstacle to the wider use of existing gene delivery vehicles resides in their instability in vivo.
It has been shown that particles of a similar size to the gene delivery vehicles of the prior art are rapidly and 3S efficiently removed from the blood by the reticuloendothelial system (Posse and Kirsch, Bio/Technology 1, 869 (1984)).
Loyter and Volsky ~Cell Sur. Rev. 8, 215-266 (1982)) SUBSTITUTE SHEET(RULE 26) CA 02241040 1998-06-lg and Kaneda et al. (Exp. Cell Res. 173, 56-69 (1987)) describe the reconstitution of viral envelopes as biological carriers including carriers of DNA. In this approach, naturally occurring viruses are isolated, dissolved in detergent containing solvents, the viral nucleic acid removed and the remaining viral components reconstituted in the presence of plasmid DNA. However, this technology has proven to be extremely expensive and difficult to scale up.
Moreover, serious safety concerns are connected with the pharmaceutical use of extracted viruses.
Other non-viral gene delivery systems described in the literature merely extend observations on transfection using DNA condensed by synthetic polymers, for example, soluble DNA/polylysine complexes can be generated (Li et al., Biochem. J. 12, 1763 (1973)). Polylysine complexes tagged with asialoglycoprotein have been used to target DNA
to hepatocytes in vitro (Wu and Wu, J. Biol. Chem. 262, 4429 (1987); U.S. Patent 5,166,320). Lactosylated polylysine (Midoux et al. (1993) Nuc. Acids Res. 21, 871-878) and galactosylated histones (Chen et al. (1994) Human Gene Therapy 5, 429-435) have been used to target plasmid DNA to cells bearing lectin receptors, and insulin conjugated to polylysine (Rosenkrantz et al. (1992) Exp. Cell Res. 199, 323-329) to cells bearing insulin receptors. However, Wagner et al. ( ibid) have shown that the latter approach is even less efficient than standard methods of transfection, and may therefore be considered unsuitable for pharmaceutical development. Monoclonal antibodies have been used to target DNA to particular cell types (Machy et al.- (1988) Proc.
Natl. Acad. Sci. USA 85, 8027-8031; Trubetskoy et al. (1992) Bioconjugate Chem. 3, 323-27 and WO 91/17773 and Wo 92/19287).
The insulin receptor has a}so 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 SUBSTITUTE SHEET (RULE 26) CA 02241040 1998-06-lg 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.
SUMMARY OF THE INVEN~ION
The invention is based on a gene delivery vehicle that is capable of targeting cells or tissue types which bear the insulin receptor on t~eir surface, and delivering a gene to that cell or tissue.
The invention encompasses a gene delivery vehicle which includes a nucleic acid binding peptide, HzN-Thr-Lys18-(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 invention also encompasses a pharmaceutical preparation for use in gene therapy, comprising a gene delivery vehicle comprising a therapeutic nucleic acid associated with Insulin-N~-Co-CH2-O-N=CH4-CO-Lysl8-Cys~S-Pyridyl)-OH in combination with a pharmaceutically acceptable carrier.
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 proqression of the cycle of cell division (e.g., SUv~ TE SHEET(RULE 26) CA 02241040 1998-06-lg W097~2363 4 PCTIGB96103137 CDK genes and the p53 gene), and the LDL receptor gene.
As used herein, "associated with" or 'l~ound 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. Alternatively, 'associated 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.
The protein hormone insulin or a derivative of insulin acts as the targeting ligand to direct the nucleic acid delivery vehicle to cells expressing the insulin receptor, where the insulin or insulin derivative retains receptor binding properties when conjugated to a nucleic acid binding component. As used herein, "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.
The invention also encompasses methods of ex vivo, in vitro, and in vivo cell-type specific targeting. As used herein, ex vivo targeting refers to targeting of a nucleic acid to an insulin receptor-bearing cell that has been removed from a patient; in vitro targeting refers to targeting of a nucleic acid to an insulin receptor-bearing cell from a cultured cell line; and in vivo cell targeting SUBSTITUTE SHEET (RULE 26) CA 02241040 1998-06-l9 refers to targeting of an ins~lin 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 a~ove-described delivery vehicle. -The invention thus also encompasses a method of treating a disease of a patient, comprising the steps of:
(a) providing a pharmaceutical preparation comprising a gene delivery vehicle comprising a therapeutic nucleic acid associated with Insulin-NH-CO-CH2-O-N=CH-CO-Lys1a-Cys(S-Pyridyl)-OH in combination with a pharmaceutically acceptable carrier; and (b) administering said pharmaceutical preparation to a patient suffering from a genetic disease Ex vivo and in vltro 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. As used herein, 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 SUBSTITUTE SHEET (RULE 26) ~VO 97122363 6 Thr-~ysl8-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.
Further features and advantages of the invention will become more fully apparent in the following description of the embodiments and drawings thereof, and from the claims.
DETAILED DESCRIPTION OF THE INVENTION
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.
Z0 The Structure of Insulin or an Insulin Derivative 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, In practice, the most useful forms of insulin are wild type insulin, or fraqments of insulin that are capable of binding to the insulin receptor and being internalized. There are over 500 fragments and derivatives of insulin having biological activity which are known in the art. 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 SlJb;~ JTE SHEET (RULE 26) WO 91n2363 by Martin et al., 1984, Diabetologia pages 118-120.
Described below is the insulin derivative amino-oxy-acetyl-Insulin.
5 Structure of the r)NA Bindinq Component The DNA binding component of the gene delivery vehicle is H2N-Thr-Lysl8-(S-Acetimidomethyl-cys)-COOH.
EXAMPLE I
8ynthe~is of Nucleic Acid ~inding Peptide 1. Preparation of H2N-Thr-Lysl8-(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. After 20 deprotection of the of the Fmoc group using 20% piperidine in DMF, the subsequent amino acids were coupled in four-fold excess using o- (lH-benzotriazol-1-yl) -tetramethyluronium tetraf luoroborate ( T B T U ) / 1 - h y d r o x y b e n z o t r i a z o l e a n d 25 N,N'-diispropylCarbOdiimide as activating agents. When necessary, a four-fold excess of the amino acid, 0-(lH-7-aza- benzotriazol-l-yl) -tetramethyluronium hexafluorophosphate and diisopropy1ethylamine was used to ensure complete coupling to the growing peptide. After the 30 synthesis of the peptide was complete, the N-terminal Fmoc group was removed as described above to give the free amino side chain protected peptide bound to the resin. This was c l e a v e d f r o m t h e r e s i n u s i n g a TFA/water/phenol/thioanisole/1, 2-ethanedithiol 35 (82.5:5:5:5:2.5) mixture. Following precipitation with ether and centrifugation, the peptide was purified using gel filtration to give the desired product. When necessary, the Acetimidomethyl (Acm) thiol protecting group may be removed SU~ ITE SHEET (RULE 26) CA 0224l040 l998-06-l9 Wo97~2363 8 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 O-(lH-benzotriazOl-l-yl)-tetramethyluronium tetrafluoroborate (TI3TU)/l-hydroxybenzotriazole and N,N'diisopropylcarbodiimide as activating agents. When necessary, a four-fold excess of the amino acid O-(lH-7-aza-benzotriazol-l-yl)tetramethyluronium hexafluorophosphate and diisopropylethylamine was used to ensure complete coupling to the growing peptide. After the synthesis of the peptide was complete, the N-terminal Fmoc group was removed as described above to give the free amino side chain protected peptide bound to the resin. This peptide was cleaved from the resin using a TFA/water/phenollthioanisole/l,2- ethanedithiol t82.5:5:5:5:2.5) mixture. Following precipitation with ether and centrifugaticn, the peptide was purified using gel 2S filtration to give the desired product. When necessary, 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 O.lN sodium acetate buffer containing 50% acetonitrile. A~ter 2h incubation at room temperature the product was purified by reverse phase hplc.
2. Synthesis of F_ller Component SUBSTITUTE SHEET (RULE 26) P~T/~B96/03137 W097t22363 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 ~inding component. The filler NBC-II (H2N-NBC-II-(Acetimidomethyl-Cys)-COOH) has the following sequence: NH2 PRKxRxvEKKspKKAE~RpARspAKA}tA~vKpKAAKpKK~tKKK
RKVEKKSPKKAKKPAAC-COOH.
EXAMPLE II
Functionalization of In~ulin Insulin may be chemically modified according to previously described methods (Offord, ~.E. "Semisynthetic Proteins" pp. 235, Wiley, Chichester and New York (1980)l, with slight modifications. Briefly, lOO mg Zn-free insulin is dissolved in l mL of lM NaHCO3, diluted with 4 mL
dimethylformamide (DMF) and reacted with an equimolar amount of MSC-ONSu (~-~ydroxy succinimide derivative of Methylsulfonyloxycarbonate, Tesser, 1975, in ~Peptides", John Wiley, NY, pp 53-56) relative to protein amino groups.
After lh incubation at room temperature, the mixture is acidified and subjected to prepara~ive HP~C on a Waters Prep Nova-Pak Hz C~ column ~flow rate 20ml min using a 25-50% B
gradient (B ~radient is a mixture of O.l tw/v) aqueous TFA
and acetonitrile:TFA water 900:l:l00 tv/w/v) over 50 min.
The peak corresponding to all-substituted insulin (as judged by subsequent ESI-MS) 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-~, A1-MSC, N- -B30MSC 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 SUBSTITUTE SHEET(RULE 26~
CA 02241040 1998-06-lg structure.
mg MSC2-insulin are dissolved in 1 mL
N-methyl-pyrrolidone and reacted with a lO-fold molar excess of Boc-AoA-OSu (Vilaseca et al., Bioconjugate Chem. 4 515-520 tl993)), in the presence of equimolar amounts of HOBt and of sufficient N-ethylmorpholine to bring the pH to approximately 8. After lh incubation at room temperature, the reaction medium is acidified and diluted with 0.1% TFA, and the derivatized insulin isolated by semipreparative HPLC
on a C8 reverse phase column equilibrated in 0.1% TFA in conjunction with a 35-45B% gradient (described above) over 20 min.
The ~SC 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 + O.l) and is deprotected by TFA treatment (30 minutes at room temperature~ just before conjugation to a polylysine peptide.
EXAMPLE III
Oxidation of the Thr-Lysl8-Cy~(S-Pyridyl) Peptide and Conjugation to Amino-oxy-acetyl ~AOA)-Insulin The cys-protected peptide (10 mg/ml) is dissolved in 50 mM imidazole, pH 6.9, and O.2 M methionine in water is added as a anti-oxidant scavenger to a lO-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) SUBSTITUTE SHEET (RULE 26) CA 02241040 1998-06-lg 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. mtz 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 pea~
fraction was dried in a speedvac (yield is approximately 4 mg of conjugate).
EXAMPLE IV
During gene transfer a fusogenic peptide may be included in the transfection mix in order to enhance efficiency of transfer of the therapeutic gene. A fusogenic peptide FP useful according to the invention includes the follow sequence: NH2- GLFEAIAGFIENGWEGMIDGGGC(Acm)-COOH, and is synthesized as follows.
1. Synthesis of H2N-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. After deprotection of the Fmoc group using 20% piperidine in DMF, the subsequent amino acids were coupled in four-fold excess using o-(lH-benzotriazol-l-yl)-tetramethyluronium tetrafluoroborate (TBTU)/1- hydroxybenzotriazole and N,N'-diisopropylcarbodiimide as activating agents. When necessary, a four fold excess of the amino acid,O(lH-7-aza-benzotriazol-1-yl) -tetramethlyroniumhexafluorophosphate and diisopropyleth~lamine was used to ensure coupling to the growing peptide. After the synthesis of the peptide was complete, the N-terminal Fmoc group was removed as described above to give the free amino side chain-protected peptide bound to the resin. This was cleaved from the resin using a TFA/water/phenol/thioanisole/1,2- ethanedithiol (82.5:5:5:5:2.5) mixture. Following precipitation with ether and centrifugation, the peptide can be purified using gel SUBSTITUTE SHEET (RULE 26) CA 02241040 1998-06-lg filtration to give the desired product.
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.
EXAMPLE V
8ynthe~is of Transfection ComplexeQ
DNA is made up to 20 mg/ml in a transfection buffer (0.15 M to 1.0 NaCl; 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 NH2-Thr-Lys~8-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.
Gel Retardation A~say for DNA Condensation 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 NaCl; 25 mM
HEPES, pH 7.4, or in 0.6 M NaCl; 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 O.l and 5.0 is calculated. This amount is made up to an equal volume to SUBSTITUTE SHEET (RULE 26) 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 O.l 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 W light. Condensed DNA remains in the well of the gel and does not migrate in the electric Field.
EXAMPLE VI
A~say for Gen~ Transfer The transfection complexes may be assayed for their ability to transfer genes into hepatocytes (HepG2 cells) expressing the insulin receptor. For studies aimed at determining transfection efficiency, the plasmid DNA
contains a marker gene for firefly luciferase. For pharmaceutical applications, 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 lO6 cells per ml. The cells are then aliquoted into tubes and .75 ml of RPMI medium added, followed by 0.04 -0.08ml of lOO ~m SU~S 111 ~JTE SHEET (RULE 26) CA 02241040 1998-06-lg W O 97~2363 14 PCT/GB96103137 Chloro~uine (CQ) or FP peptide and finally 0.25 ml of DNA-complex solution. 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 lml of RPMI and re-centrifuged. Finally, the cells are in 0.5 ~l 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 ~0 transferred to a well of a 12 well plastic culture plate containing l.~ml of RPMI 10% FBS. The original transfection tube is rinsed with a further lml 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% C02. 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 MgCl2, lmM E~TA, 1% Triton X-lO0 and 15% glycerol) and agitated with a pipette. 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. Light produced by the luciferase is integrated over 4 sec and is described as relative light units (RLU). The data are converted to RLU/ml of lysate, RLU/cell or RLU/mg protein (protein concentration of the lys~te having been determined in this case by the BioRAD Lowry assay).
The transfe~tion efficiency resulted in the delivery such that 200,000-500,000 relative luciferase units per mg of protein were expressed in the transfected cells.
EXAMP~E VII
Dosage and Pharmaceutical Formulation SUBSTITUTE SHEET (RULE 2~) CA 02241040 1998-06-l9 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. In the case of a pharmaceutical composition, the plasmid DNA
includes a gene whose expression would have some beneficial therapeutlc 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.
Use of this type of pharmaceutical composition in vivo or ex vivo with nucleic acid containing a gene of physiological importance, such as replacement of a defective gene or an additional potentially beneficial gene function, is expected to confer long term genetic modification of the cells and be effective in the treatment of disease. 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 dosaqes 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. Monitorinq levels of gene introduction, gene expression will assist in selecting and adjusting the dosages administered. Generally, a composition including a delivery vehicle will be administered in a single dose in the range of lo ng - lOo ug/kg body weight, preferably in the range of lon ng - 10 ug/kg body weight, SUBSTITUTE SHEET (RULE 26) l6 such that at least one copy of the therapeutic gene is delivered to each target cell.
Ex ~ivo treatment is also contemplated within the present invention. 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.
OTHER EMBODIMENTS
Other embodiments will be evident to those of skill in the art. It should be understood that the foregoing detailed description is provided for clarity only and is merely exemplary. The spirit and scope of the present invention are not limited to the above examples, but are encompassed by the following claims.
SUBSTITUTE SHEET (RULE 26)
Claims (5)
in combination with a pharmaceutically acceptable carrier.
2. A method of preparing a composition for transfecting insulin receptor-bearing cells, comprising
1. oxidizing Thr-Lys18-Cys(S-Pyridyl) peptide, and
2. conjugating said oxidized peptide to B1 amino-oxy-acetyl-insulin.
3. A method of treating a disease of a patient, comprising the steps of:
(a) providing a pharmaceutical preparation comprising a gene delivery vehicle comprising a therapeutic nucleic acid associated with Insulin-NHCO-CH2-O-N=CG-CO-Lys18-Cys(S-Pyridyl)-OH
in combination with a pharmaceutically acceptable carrier;
and (b) contacting insulin receptor-bearing cells with said pharmaceutical preparation.
(a) providing a pharmaceutical preparation comprising a gene delivery vehicle comprising a therapeutic nucleic acid associated with Insulin-NHCO-CH2-O-N=CG-CO-Lys18-Cys(S-Pyridyl)-OH
in combination with a pharmaceutically acceptable carrier;
and (b) contacting insulin receptor-bearing cells with said pharmaceutical preparation.
4. A method of targeting insulin receptor-bearing cells for delivery of a therapeutic nucleic acid, comprising the step of contacting insulin receptor-bearing cells with a pharmaceutical preparation comprising a gene delivery vehicle comprising a therapeutic nucleic acid associated with Insulin-NHCO-CH2-O-N=CH-CO-Lys18-Cys(S-Pyridyl)-OH under conditions sufficient to permit internalization and expression of said therapeutic nucleic acid in said insulin receptor-bearing cells.
5. The method of claim 4, said cells being liver cells.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9525955.2 | 1995-12-19 | ||
| GBGB9525955.2A GB9525955D0 (en) | 1995-12-19 | 1995-12-19 | Improved pharmaceutical compositions |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2241040A1 true CA2241040A1 (en) | 1997-06-26 |
Family
ID=10785695
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002241040A Abandoned CA2241040A1 (en) | 1995-12-19 | 1996-12-19 | Improved pharmaceutical compositions |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0873138A2 (en) |
| AU (1) | AU705060B2 (en) |
| CA (1) | CA2241040A1 (en) |
| GB (1) | GB9525955D0 (en) |
| WO (1) | WO1997022363A2 (en) |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB8820377D0 (en) * | 1988-08-26 | 1988-09-28 | Offord R E | Chemical compounds |
| GB8820378D0 (en) * | 1988-08-26 | 1988-09-28 | Offord R E | Chemical compounds |
| EP0788375A2 (en) * | 1994-11-09 | 1997-08-13 | Robin Ewart Offord | Functionalized polymers for site-specific attachment |
-
1995
- 1995-12-19 GB GBGB9525955.2A patent/GB9525955D0/en active Pending
-
1996
- 1996-12-19 CA CA002241040A patent/CA2241040A1/en not_active Abandoned
- 1996-12-19 WO PCT/GB1996/003137 patent/WO1997022363A2/en not_active Application Discontinuation
- 1996-12-19 EP EP96942490A patent/EP0873138A2/en not_active Withdrawn
- 1996-12-19 AU AU11850/97A patent/AU705060B2/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| AU705060B2 (en) | 1999-05-13 |
| WO1997022363A3 (en) | 1997-09-25 |
| EP0873138A2 (en) | 1998-10-28 |
| WO1997022363A2 (en) | 1997-06-26 |
| AU1185097A (en) | 1997-07-14 |
| GB9525955D0 (en) | 1996-02-21 |
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