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EP1196618A2 - Nouveaux vecteurs complexes et leur utilisation en therapie genique - Google Patents

Nouveaux vecteurs complexes et leur utilisation en therapie genique

Info

Publication number
EP1196618A2
EP1196618A2 EP00947840A EP00947840A EP1196618A2 EP 1196618 A2 EP1196618 A2 EP 1196618A2 EP 00947840 A EP00947840 A EP 00947840A EP 00947840 A EP00947840 A EP 00947840A EP 1196618 A2 EP1196618 A2 EP 1196618A2
Authority
EP
European Patent Office
Prior art keywords
component
vector
complex according
complex
cationic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00947840A
Other languages
German (de)
English (en)
Inventor
Hans-Harald Sedlacek
Sabine BRÜSSELBACH
Thomas Kissel
Rolf Müller
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sanofi Aventis Deutschland GmbH
Original Assignee
Aventis Pharma Deutschland GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Aventis Pharma Deutschland GmbH filed Critical Aventis Pharma Deutschland GmbH
Publication of EP1196618A2 publication Critical patent/EP1196618A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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

  • vectors for gene therapy have so far been nucleic acid sequences which are complexed with a non-viral carrier (e.g. cationic lipids or cationic polymers) or inserted into a virus.
  • a non-viral carrier e.g. cationic lipids or cationic polymers
  • RES reticuloendothelial system
  • the causes of rapid elimination are varied. They can be too large a negative or positive charge, a too large volume or an opsonization of the vector particles by blood proteins.
  • viral vectors they can furthermore be the binding of the virus envelope proteins to virus-specific receptors in the organs and / or also antibodies or immune cells specific for the viruses which bind to the vectors and thereby eliminate them.
  • Vector particle size inhibited the aggregation of the vectors with themselves or with blood cells, reduced the opsonization of vectors by binding immunoglobulins, complement factors, fibrinogen or fibronectin, protected (adeno-) viral vectors from being eliminated by antibodies (Chillon et al., Gene Ther 5: 995, 1998) and caused an increase in the blood retention time of
  • the invention relates to a new vector for gene therapy consisting of the following components:
  • Component a) can be an unmodified or modified DNA sequence or an unmodified or modified RNA sequence.
  • the nucleotide sequence can perform an anti-DNA (triplex) or anti-RNA (antisense; ribozyme) function or code for an RNA sequence acting in this way or for a protein.
  • the nucleotide sequences and their modification can be such that the
  • Nucleotide sequence is largely resistant to degradation by DNAsen or RNAsen. Examples of such nucleotide sequences and their modifications are in Breaker, Nature Biotechnol. 15: 427, 1997; Gerwik, Critical Reviews in Oncogenesis 8: 93, 1997; Mukhopadhyay et al., Crit. Rev. Oncogen. 7: 151, 1996; Mercola et al., Cancer Gene Ther. 2: 47, 1995; Frank-Kamenetski, Annu. Rev. Biochem. 64: 65, 1995 and Fraser et al., Exp. Opin. Invest. Drugs 4: 637, 1995.
  • the DNA sequence can be linear or circular, for example in the form of a plasmid.
  • Component a) can also be a virus, preferably a virus, into which a nucleic acid sequence foreign to the virus has been inserted using the methods known to those skilled in the art.
  • viruses are RTV, AV, AAV, HSV, vaccinia viruses, influenza viruses.
  • RTV Radio Transmission Tube
  • AV Gene Ther. 7
  • AAV AAV
  • HSV vaccinia viruses
  • influenza viruses Such and further examples are from Vile, Nature Biotechnol. 15: 840, 1997; McKeon et al., Human Gene Ther. 7: 1615, 1996; Flotte et al., Gene Ther. 2: 357, 1995; Jolly, Cancer Gene Ther. 1:51, 1994; Dubensky et al., J. Virol. 70: 508, 1996.
  • Component b) represents any cationic carrier.
  • cationic carriers are cationic lipids, for example described by Kao et al., Oncology Reports 5: 625, 1998, Liu et al., J. Biol. Chem. 270: 24864, 1995; Feigner, Human Gene Ther. 7: 1791, 1996; Ledley, Human Gene Ther. 6: 1129, 1995; Goyal et al., J. Liposom. Res. 5: 49, 1995; Thierry et al., Gene Ther. 4: 226, 1997; Schofield et al., Br. Med. Bull. 51: 56, 1995; Behr, Bioconj. Chem. 5: 382, 1994; Cotten et al.,
  • Cationic polymers also include, for example, cationized albumin. Production and use of cationized albumin has been used in the
  • Patent application EP-A 0 790 312 described.
  • component b) is a polyethyleneimine (PEI), in a further particular embodiment of this invention the polyethyleneimine has a molecular weight in a range from 500-20,000 Da and in a further embodiment has an average molecular weight of about 2000 Da and was prepared as described in patent application EP-A 0 905 254.
  • PEI polyethyleneimine
  • Component c) represents any polymer with more than 3 cationic and more than 3 anionic charges.
  • the ratio of cationic to anionic charges should only be in the range between 3:10 and Fluctuate 10: 3.
  • the ratio 1 ( ⁇ 20%): 1 ( ⁇ 20%) should preferably be used.
  • the charges can be non-uniform, random or evenly distributed over the polymer.
  • preference is given to polymers with a uniform distribution of the cationic and anionic charge.
  • preference is furthermore given to polymers with cationic and anionic charge, the isoelectric point of which is in the pH range from 3-9.
  • the polymer according to the invention is albumin.
  • This albumin can be isolated from the blood or produced recombinantly.
  • the albumin is purified human albumin from the serum. The purification and properties of albumin have been described, for example, by T. Peters in FW Putnam "The Plasma Proteins", Academic Press, New York 1975, pp. 133-181.
  • Human serum albumin is a single chain polypeptide chain (610 amino acids) with an isoelectric point of 4.7 and approximately 101 positive and 101 negative charges, which are evenly distributed over the entire polypeptide chain.
  • component a) is complexed with component b) and this complex is in turn complexed with component c).
  • Another embodiment of the invention is that the complexes of components a) and b) in liposomes, preferably anionic liposomes [prepared, for example, as in U.S. Patent No. 4,946,787, U.S. Patent No. 4,245,737, U.S. Patent No. 5,480,463, Heywood and Eanes, Calc , Tissue int. 40: 149, 1992; Lee and Huang, J. Biol. Chem. 271: 8481, 1996; Balicki and Beutler, Blood 88: 3884, 1996; Lucie et al., J. Lip. Res.
  • the invention furthermore relates to the addition of the preparation according to the invention by adding a component d).
  • This component d) represents a ligand which binds with component a) or component b) or component c) and at the same time has a binding site for the target cell.
  • ligands can be
  • component d) can also be inserted into a liposome, preferably an anionic liposome, for example as described in US Pat. Nos. 5,252,348 and 5,753,258.
  • the target cell-specific protein or peptide selected from one of the aforementioned groups, is to be conjugated to a lipid, for example as described in US Pat. No. 5,662,930.
  • a lipid for example as described in US Pat. No. 5,662,930.
  • Invention is the target cell-specific protein or peptide associated with a fusiogenic peptide and this in turn with a lipid.
  • fusogenic peptides are described in detail in patent applications EP-A 0 846 772 and DE19850987.1 (not yet published).
  • the conjugation of the target cell-specific protein or peptide with a fusiogenic peptide is preferably carried out by expression as a recombinant fusion protein using the methods known to the person skilled in the art.
  • the vector according to the invention consisting of components a), b) and c) or a), b), c) and d) is produced, for example, in such a way that in the first step component d) with component a), b) and / or c) in the molar ratio [d): a), b) and / or c)] of 1: 1 to 1000: 1, preferably between 10 : 1 and 100: 1 is mixed, hereinafter
  • component a) [either alone or in a complex with component d)] is mixed with component b) [in a complex with component d) or alone], preferably in a molar ratio [a) ( ⁇ d): b ) ( ⁇ d)] from 1: 1 to 1: 1000, the mixing ratio being adjusted so that the net charge of the resulting overall complex is preferably either cationic or anionic and below
  • the complex resulting from step 2) is mixed with component c) [in complex with d) or alone] in such a way that component c) is in excess, measurable by the fact that the net charge of the from step 2) resulting complex was completely neutralized by step 3), preferably that the mixture of free albumin and albumin-containing complexes resulting from step 3) has a slightly anionic to neutral net charge, further preferably the albumin-containing complex has its isoelectric point in pH Has a value range of 4-7.
  • Such vector complexes according to the invention are largely shielded by their component c), i.e. their binding and transfection and transduction of cells that do not carry a receptor for component d) within the vector complex according to the invention is largely reduced.
  • the residence time is significantly extended, after administration in the blood circulation, for example, up to several Hours to a few days.
  • the vector complexes according to the invention accumulate, for example, in the tumor vessel bed due to the so-called "passive targeting" (Unezaki et al., Int. J. Pharmaceutics 114: 11, 1996; Sadzuka et al., Cancer Lett. 127: 99, 1998 and Wunder et al., Int. J. Oncol. 11: 497, 1997).
  • the vector complexes according to the invention bind via their component d) to the target cell and transfect them to release the nucleic acid sequence in the vector complex according to the invention.
  • this nucleic acid sequence can develop its effect in the target cell, i.e. For example, inhibit the transcription or translation of a particular gene or a particular RNA, or transduce the cell to express the RNA or the protein encoded by this nucleic acid sequence.
  • the vector complexes according to the invention are therefore preferably suitable for in vivo administration with the aim of prophylaxis or therapy of diseases.
  • Example 1 Preparation of a vector complex with a plasmid and a target cell-specific multivalent protein.
  • the plasmid expression system "pGL3" from Promega was used as component a) and contains the following nucleotide sequences:
  • the plasmid was introduced into E. coli bacteria, the bacteria were propagated in culture medium and the plasmids were isolated. Production of component b)
  • Low molecular weight polyethyleneimine (PEI-2000) was produced as described in patent application EP-A 0 905 254.
  • a 10% strength ethyleneimine monomer solution in water (5 ml of ethyleneimine monomer + 45 ml of distilled water, dissolution with stirring) with the addition of 1% (0.5 ml) of concentrated hydrochloric acid (37 ° C.) as catalyst for 4 days at 50 ° C stirred, evaporated and dried under vacuum at room temperature.
  • the molecular weight determinations were carried out by means of laser scattered light measurement (Wyatt Dwan DSP light scattering photometer) at 633 nm after direct injection into a K5 measuring cell. The molar masses are determined on the basis of the calibration constants determined in toluene and the known sample weight.
  • Human albumin 20% from Centeon was used as the polymer.
  • a multi-functional ligand produced as described in patent application EP-A 0 846 772 is used as component d).
  • the hybridoma of the anti-NCAM monoclonal antibody 575/100/2 serves as the starting material for the ZS ligand. About 10 7 cells of this hyridome are removed and the mRNA is extracted from these cells with the aid of an mRNA extraction kit (eg from Pharmacia, Gibco, Qiagen). This mRNA is then transcribed into cDNA by reverse transcription using a cDNA synthesis kit and "random" hexaoligonucleotides (Pharmacia).
  • This cDNA serves as a starting material for using variable primers (Clackson et al., Nature 352: 624, 1991) to use the variable heavy chain or the variable light chain of the immunoglobulins by means of a polymerase chain reaction (Saiki et al., Science 230: 1350, 1985 ) to amplify. Restriction sites are simultaneously introduced through the primers in order to clone the fragments into a bacterial expression vector (eg pHENIS, which is derived from pHENI) (Hoogenboom et al., Nucl. Acids Res. 19: 4133, 1991).
  • pHENIS which is derived from pHENI
  • pelB signal sequence for periplasmic secretion
  • histidine tag for purification using immobolized metal affinity chromatography (IMAC)
  • IMAC immobolized metal affinity chromatography
  • cloning region for the heavy and light chain between a short sequence for a 14 amino acid glycine serine linker coded.
  • the heavy and light chains are digested with the appropriate restriction enzymes (VH with Sfil and Xhol; VL with ApaL1 and Notl) and successively cloned into the vector. This creates a recombinant single-chain Fv fragment consisting of the variable heavy chain and light chain, which are covalently linked by a short peptide sequence.
  • Recombinant antibodies with specificity for N6-methyladenine are selected from native or semi-synthetic antibody libraries (Nissim et al., EMBO J. 13: 692, 1994) by biopanning on N6-methyladenine-BSA or KLH conjugates.
  • the Positive antibody fragments are identified by ELISA in antigen-coated microtiter plates (Nissim et al., EMBO J. 13: 692, 1994).
  • Antibodies from these libraries are already in the desired single-chain Fv format and can be used directly for further cloning.
  • a fusogenic peptide with the amino acid sequence GLFEALLELLESLWELLLEA (SEQ ID NO .: 1) serves as the linker.
  • the coding DNA for this peptide is produced as a double-stranded synthetic oligonucleotide, with suitable restriction cleavage sites (Ascl and Xbal) being appended to the ends.
  • Aptl and Xbal suitable restriction cleavage sites
  • O1 (5'GGCCGCAGGCTTATTTGAGGCCCTTCTGGAATTGCTAGAGAGCCTCTGGG AATTGCTTCTGGAGGCAT; SEQ ID NO .: 2) and O2 (5'CTAGATGCCTCCAGAAGCAATCCCAGAGGCCTCTAGCAATTCCAGAAGGGo3Co with the manufacturer; Enter 5) for the manufacturer (eg warmed and slowly cooled to room temperature. This double-stranded DNA fragment is used directly for further cloning.
  • the complete ligand system is inserted into the expression vector pAB1 (which is similar to pHENIS but has no fusion with g3p) e.g. made in the form of a 3-fragment cloning.
  • the starting material is the anti-NCAM single-chain Fv fragment (ZS ligand), which was cut with the restriction enzymes Sfil and Notl, the synthetic linker fragment which contains the cloning sites Notl and Xbal, and the anti-N6-methyladenine single-chain Fv Fragment (GS ligand).
  • ZS ligand anti-NCAM single-chain Fv fragment
  • Sfil and Notl the restriction enzymes Sfil and Notl
  • the synthetic linker fragment which contains the cloning sites Notl and Xbal
  • the anti-N6-methyladenine single-chain Fv Fragment GS ligand
  • the GS fragment is reamplified with primers which have the restriction sites Xbal or Nbal at the N and C terminus.
  • Insert ascl are cloned into the pAB1 vector cut with the restriction enzymes Sfil and Ascl.
  • the construct is transformed into suitable bacterial strains, for example E. coli TG1.
  • the regulation of the expression of the Ligand system takes place via the bacterial lacZ promoter and is induced by adding IPTG (as described, for example, in McCafferty et al., Appl. Biochem. Biotech. 47: 157, 1994).
  • the expressed protein is purified from periplasmic preparations using IMAC (see Griffiths et al., EMBO J. 13: 3245, 1994).
  • the total protein has a molecular weight of approximately 55,000 and is present as a monomer.
  • the protein recognizes NCAM-expressing tumor cells such as that of small cell bronchial carcinoma and it binds to N6-methylated DNA, which is produced by propagation of the plasmids in bacteria.
  • the ligand system is made by incubating the fusion protein with the DNA. The binding to the tumor cell or DNA can be checked by immunofluorescence or ELISA.
  • a reporter gene e.g. Luciferase (see component a)
  • a ligand system is created that is able to bind to the tumor cells.
  • the linker-mediated release from the endosomes takes place, which leads to the transcription and expression of the effector gene.
  • the binding of the ligand system to the cell and uptake into the cell can be carried out
  • Detect the use of fluorescence-labeled fusion protein and the functionality of the system is based on the detection of the enzymatic activity of luciferase.
  • Plasmids [component a)] are suspended in a concentration of 10 9 plasmids / ml in physiological saline.
  • the multifunctional ligand [component d)] dissolved in physiological saline, is added in portions to the suspension while shaking, until a molar ratio of about 1:10 (plasmid: multifunctional ligand) is reached.
  • the component a) / component d) complex or component a) is then [component b)] PEI-2000 (1 mg / ml physical saline with 1 N HCl adjusted to pH 7.4) in portions until a complete Cationization of the resulting complexes [component a) + d) + b) or component a) + b)] is reached.
  • the cationization is determined in the agarose shift assay, in which 50 ⁇ l aliquots are applied to an approximately 0.5 cm thick gel of 1% (w / v) agarose and in Tris-EDTA buffer pH 7.4 at 80 mV Be developed for 2 hours. The location of the DNA was visualized at 254 nm after reaction with ethidium bromide.
  • the cationic complexes from components a) + d) + b) are then suspended in an excess of component c) and incubated at 4 ° C. for at least 48 hours. In this excess, complexes are formed from components a) + d) + b) + c), which have a neutral to slightly anionic charge.
  • Charge is checked in the agarose shift assay and should be in the range between pH 4 and 7.
  • the vector complex according to the invention [component a) + d) + b) + c)] can then be used.
  • the vector complex component a) + b) is used as a control.
  • Tumor cells expressing NCAM small cell bronchial carcinoma
  • melanoma cells MeWo
  • fibroblasts 3T3
  • Components a) + b) are added to the cells in excess (ratio approx. 20: 1) and the mixture is incubated at 37 ° C. for 1 hour. Below are the Washed cells and incubated for another 60 hours in fresh cell culture medium.
  • the successful uptake of the complexes into the cell, the transcription and the expression of the reporter gene in the plasmid are then determined by detecting the luciferase with the aid of the method known to the person skilled in the art and according to information from Promega.
  • NCAM-positive cells which are incubated with the vector complex according to the invention [component a) + d) + b) + c)] show a clear expression of luciferase. In contrast, no or only a slight expression is found in the control cells.
  • Component c) in the vector complex according to the invention thus prevents the non-ligand-specific transfection of cells.
  • mice are injected with the complexes according to the invention [component a) + d) + b) + c)] or, as a control, complexes with components a) + b) into the tail vein.
  • the dose is 50 ⁇ g of component a) in the respective complexes per mouse, the suspension medium is physiological saline, the application volume is 250 ⁇ l.
  • the animals are anesthetized and bled 30 minutes and 2 hours after the injection.
  • the collected blood is mixed with sodium citrate (final concentration 25 mM) and the blood plasma is separated from the blood cells by centrifugation (10 min, 1000 g).
  • the DNA is isolated from the whole blood or from the blood plasma using the QIAamp Tissue Kit (Qiagen, Hilden).
  • 10 ⁇ l heparin 1000 IU7 ml Novo Nordisk
  • Samples of the isolated DNA were applied to 0.8% agarose gel and analyzed by Southern blot as described in detail by Obris et al. 1999 described.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)
  • Peptides Or Proteins (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne un vecteur complexe qui contient les composants suivants: a) une séquence d'acide nucléique d'une longueur quelconque; b) un support cationique; c) un polymère possédant plus de trois charges cationiques et plus de trois charges anioniques; et d) éventuellement un ligand qui se fixe au composant a) ou au composant b), ou bien au composant c) et comporte également un site de liaison pour une cellule cible. L'invention concerne en outre la production de ce vecteur et son utilisation dans la prophylaxie et la thérapie de maladies.
EP00947840A 1999-06-24 2000-06-10 Nouveaux vecteurs complexes et leur utilisation en therapie genique Withdrawn EP1196618A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE1999129104 DE19929104A1 (de) 1999-06-24 1999-06-24 Neue Vektorkomplexe und deren Verwendung für die Gentherapie
DE19929104 1999-06-24
PCT/EP2000/005371 WO2001000241A2 (fr) 1999-06-24 2000-06-10 Nouveaux vecteurs complexes et leur utilisation en therapie genique

Publications (1)

Publication Number Publication Date
EP1196618A2 true EP1196618A2 (fr) 2002-04-17

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Family Applications (1)

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EP00947840A Withdrawn EP1196618A2 (fr) 1999-06-24 2000-06-10 Nouveaux vecteurs complexes et leur utilisation en therapie genique

Country Status (5)

Country Link
EP (1) EP1196618A2 (fr)
JP (1) JP2003503362A (fr)
AU (1) AU6149600A (fr)
DE (1) DE19929104A1 (fr)
WO (1) WO2001000241A2 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1711074B (zh) * 2002-11-15 2010-10-06 尼普洛株式会社 脂质体
JP2005298486A (ja) * 2004-03-15 2005-10-27 Nipro Corp リポソームを含む癌治療用医薬組成物

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4119590A (en) * 1975-05-22 1978-10-10 Toyo Soda Manufacturing Co., Ltd. Process for producing a polyion complex having a nucleic acid base
US4847240A (en) * 1978-01-16 1989-07-11 The Trustees Of Boston University Method of effecting cellular uptake of molecules
US5354844A (en) * 1989-03-16 1994-10-11 Boehringer Ingelheim International Gmbh Protein-polycation conjugates
US5574142A (en) * 1992-12-15 1996-11-12 Microprobe Corporation Peptide linkers for improved oligonucleotide delivery
US5908777A (en) * 1995-06-23 1999-06-01 University Of Pittsburgh Lipidic vector for nucleic acid delivery
FR2739292B1 (fr) * 1995-09-28 1997-10-31 Rhone Poulenc Rorer Sa Composition pharmaceutique utile pour la transfection d'acides nucleiques et ses utilisations
DE19605279A1 (de) * 1996-02-13 1997-08-14 Hoechst Ag Zielzellspezifische Vektoren für die Einschleusung von Genen in Zellen, Arzneimittel enthaltend derartige Vektoren und deren Verwendung
EP0920339A2 (fr) * 1996-07-09 1999-06-09 The Johns Hopkins University Systeme d'administration de genes
US5948681A (en) * 1996-08-14 1999-09-07 Children's Hospital Of Philadelphia Non-viral vehicles for use in gene transfer
GB9623051D0 (en) * 1996-11-06 1997-01-08 Schacht Etienne H Delivery of DNA to target cells in biological systems
DE19649645A1 (de) * 1996-11-29 1998-06-04 Hoechst Ag Mehrfach funktionelles Ligandensystem zur zielzellspezifischen Übertragung von Nukleotidsequenzen

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0100241A3 *

Also Published As

Publication number Publication date
JP2003503362A (ja) 2003-01-28
DE19929104A1 (de) 2000-12-28
AU6149600A (en) 2001-01-31
WO2001000241A2 (fr) 2001-01-04
WO2001000241A3 (fr) 2001-08-09

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