DE19541679A1 - Gene transfer systems containing nuclear localisation sequences and or magnetisable microparticles, - Google Patents

Gene transfer systems containing nuclear localisation sequences and or magnetisable microparticles,

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DE19541679A1
DE19541679A1 DE19541679A DE19541679A DE19541679A1 DE 19541679 A1 DE19541679 A1 DE 19541679A1 DE 19541679 A DE19541679 A DE 19541679A DE 19541679 A DE19541679 A DE 19541679A DE 19541679 A1 DE19541679 A1 DE 19541679A1
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receptor
gene
gts
gene transfer
cell
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Gerhard Dr Friedrich
Cai-Steffen Dr Kuhn
Klaus Dr Mittenbuehler
Kurt Appel
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KUHN CAI STEFFEN DR
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KUHN CAI STEFFEN DR
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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
    • 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
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • 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
    • A61K47/644—Transferrin, e.g. a lactoferrin or ovotransferrin
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5094—Microcapsules containing magnetic carrier material, e.g. ferrite for drug targeting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Abstract

Preparation, sale and application of gene transfer systems (GTS) that contain at least one of nuclear localisation sequences (NLS) and/or magnetisable microparticles (A) are new.

Description

Ziel ist es neuartige GTS mit optimierter Effektivität und Sicherheit zu entwickeln, die folgende Komponenten/Eigenschaften aufweisen:The aim is to develop new types of GTS with optimized effectiveness and security have the following components / properties:

  • 1. Antikörper/Ligand: Diese binden an ihre jeweiligen Rezeptoren, wodurch eine erhöhte Zellspezifität des Gentransfers erreicht wird. Die Internalisierung des Konstruktes erfolgt durch rezeptorvermittelte Endocytose.1. Antibody / Ligand: These bind to their respective receptors, causing an increased Cell specificity of gene transfer is achieved. The construct is internalized by receptor-mediated endocytosis.
  • 2. Membranaktive Peptide/replikationsdefiziente Adenoviren: Diese führen zur Freisetzung des GTS aus den Endosomen ins Cytosol und verhindern dadurch die lysosomale Degradation der DNA.2. Membrane-active peptides / replication-deficient adenoviruses: These lead to the release of the GTS from the endosomes into the cytosol and thereby prevent the lysosomal degradation of the DNA.
  • 3. "Nuclear Localization-Sequence" (NLS): Durch die Kopplung des GTS mit einer NLS soll ein gezielter Transport des Konstruktes in den Zellkern erreicht werden.3. "Nuclear Localization Sequence" (NLS): By coupling the GTS with an NLS targeted transport of the construct into the cell nucleus is to be achieved.
  • 4. Magnetisierbare Mikropartikel: Diese Komponente des GTS soll eine schnelle und einfache Separation der GTS enthaltenden Zellen ermöglichen was besonders bei der ex vivo Gentherapie ein bedeutender Vorteil ist. Außerdem kann so die zelluläre Internalisationsrate der Konstrukte näherungsweise bestimmt werden.4. Magnetizable microparticles: This component of the GTS is said to be fast and simple separation of the GTS-containing cells enables something especially in ex vivo Gene therapy is a significant benefit. It can also reduce the cellular internalization rate the constructs are approximately determined.
  • 5. Polykationischer Träger: Dieser ermöglicht die elektrostatische Bindung des Vektors. Die Komponenten 1)-4) sind kovalent an diesen gebunden.5. Polycationic carrier: This enables the vector to be bound electrostatically. The Components 1) -4) are covalently bound to it.
  • 6. Vektor: Mittels eines Reportergens wird der Einfluß der verschiedenen Komponenten des GTS auf die Expressionsrate ermittelt. Zusätzlich wird die Transfektionseffizienz über quantitative PCR bestimmt.6. Vector: Using a reporter gene, the influence of the various components of the GTS determined on the expression rate. In addition, the transfection efficiency is about quantitative PCR determined.

In weiteren Experimenten sollen verschiedene Enhancer Elemente zur spezifischen Steuerung der Transkription des integrierten Gens eingesetzt werden.In further experiments, various enhancer elements for specific control should be used the transcription of the integrated gene.

Stand der Wissenschaft und Technik; alternative Lösungen; LiteraturangabenState of science and technology; alternative solutions; References

Für den Transfer von DNA sind verschiedene Verfahren entwickelt worden. Die z.Zt. zur Verfügung stehenden Carriersysteme als auch Vektoren zur somatischen Gentherapie (z. B. Retro-, Adenoviren und kationische Liposomen etc.) lassen sowohl hinsichtlich ihrer Sicherheit als auch ihrer Effizienz für den Patienten zu wünschen übrig (1-4). Der retroviral vermittelte Gentransfer weist neben den Vorteilen der vergleichsweise hohen Transfektionseffizienz und der stabilen Integration der Fremd-DNA in das Genom der Zielzelle die Gefahr der Aktivierung von zellulären Onkogenen als auch der Inaktivierung von Suppressorgenen auf. Ein weiterer Nachteil liegt in der unzureichenden Spezifität. Außerdem besteht die Gefahr der Bildung replikationskompetenter Viren durch Rekombinationsvorgänge.Various methods have been developed for the transfer of DNA. The currently to Available carrier systems as well as vectors for somatic gene therapy (e.g. Retro viruses, adenoviruses and cationic liposomes etc.) leave both with regard to their safety as well as their efficiency for the patient (1-4). The retroviral mediated In addition to the advantages of the comparatively high transfection efficiency and gene transfer the stable integration of the foreign DNA into the genome of the target cell the risk of activation of cellular oncogenes and the inactivation of suppressor genes. Another The disadvantage is the insufficient specificity. There is also a risk of education replication-competent viruses through recombination processes.

Aufgrund der genannten Sicherheitserwägungen wurde an der Entwicklung anderer Vektorsysteme gearbeitet. Im Gegensatz zu Retroviren können Adenoviren sehr große Fragmente von Fremd-DNA aufnehmen und vor allem auch nicht proliferierende Zellen infizieren (Nervengewebe, Lunge). Nachteilig wirkt sich in diesem System die Notwendigkeit aus, relativ viele Adenovirusgene in den Vektor mit aufzunehmen, deren Expression eine Immunreaktion des Wirtsorganismus nach sich ziehen kann. Aus diesen Gründen gewinnen non-virale Systeme zunehmend an Bedeutung.Because of the security considerations mentioned, the development of others Vector systems worked. Unlike retroviruses, adenoviruses can be very large Take up fragments of foreign DNA and especially non-proliferating cells infect (nerve tissue, lungs). The necessity is disadvantageous in this system from including a relatively large number of adenovirus genes in the vector, the expression of which a Immune response of the host organism can result. Win for these reasons Non-viral systems are becoming increasingly important.

Bei ersten humanen Gentransferversuchen mit Hilfe non-viraler Carrier wurden kationische Liposomen (5-11) verwendet. Neben den Vorteilen der Sicherheit und der Möglichkeit große DNA Fragmente einzuschleusen, weisen diese non-viralen Carrier bisher gegenüber viralen GTS folgende Nachteile auf:The first human gene transfer attempts using non-viral carriers became cationic Liposomes (5-11) used. In addition to the advantages of security and the possibility of great Introducing DNA fragments has previously shown these non-viral carriers to be viral GTS has the following disadvantages:

  • a) geringe Transfektionseffizienz,a) low transfection efficiency,
  • b) geringe Spezifität hinsichtlich der Zellerkennung, Internalisation und Integration in das Wirtsgenom.b) low specificity with regard to cell recognition, internalization and Integration into the host genome.

Daher wird untersucht, ob diese derzeitigen Beschränkungen non-viraler GTS durch Kombination von rezeptorvermittelter Endocytose auf der Basis spezifischer Rezeptor- Liganden bzw. Antikörper (12-23) mit endosomolytisch wirkenden Peptiden (24-29) und "Nuclear Targeting" (30-40) überwunden werden können. Dieses stellt in der somatischen Gentherapie ein neuartiges Konzept dar, mit dem Ziel der gleichzeitigen Erhöhung von Spezifität, Effizienz und Sicherheit durch Targeting und Kontrolle des GTS auf folgenden Ebenen:Therefore, it is investigated whether these current restrictions result from non-viral GTS Combination of receptor-mediated endocytosis based on specific receptor Ligands or antibodies (12-23) with endosomolytic peptides (24-29) and "Nuclear Targeting" (30-40) can be overcome. This represents in the somatic Gene therapy represents a new concept, with the aim of increasing at the same time Specificity, efficiency and security by targeting and controlling the GTS on the following Levels:

  • a) höhere Spezifität des Carriersystems für die Zielzelle durch rezeptorvermittelte Endocytose (12-23)a) higher specificity of the carrier system for the target cell through receptor-mediated endocytosis (12-23)
  • b) Disruption der gebildeten Endosomen und Freisetzung des GTS ins Cytosol (24-29) mit Hilfe von membranaktiven Substanzenb) Disruption of the formed endosomes and release of the GTS into the cytosol (24-29) with Help from membrane-active substances
  • c) "Nuclear Targeting": "Nuclear Localization Sequence" vermittelter, aktiver Transport des GTS vom Cytosol in den Zellkern (30-40)c) "Nuclear Targeting": "Nuclear Localization Sequence" mediated, active transport of the GTS from the cytosol into the cell nucleus (30-40)
  • d) "Transcriptional Targeting": Spezifische Steuerung der Transkription des integrierten Gens (u. a. gewebsspezifische Promotoren/Enhancer Elemente) (7, 41-45)d) "Transcriptional Targeting": Specific control of the transcription of the integrated gene (including tissue-specific promoters / enhancer elements) (7, 41-45)
Ausführliche Beschreibung des VerfahrensDetailed description of the procedure

Das Verfahren gliedert sich in folgende Phasen.The process is divided into the following phases.

Phase 1Phase 1

Konstruktion von verschiedenen GTS bestehend ausConstruction of various GTS consisting of

  • 1) rezeptorspezifischen Liganden bzw. Antikörpern1) receptor-specific ligands or antibodies
  • 2) membranaktiven Peptiden/replikationsdefizienten Adenoviren2) membrane-active peptides / replication-deficient adenoviruses
  • 3) "Nuclear Localization Sequences" (NLS)3) "Nuclear Localization Sequences" (NLS)
  • 4) magnetisierbaren Partikeln4) magnetizable particles
  • 5) Polykationischem Träger5) Polycationic carrier
  • 6) Vektor.6) vector.

In dieser Phase werden die verschiedenen Systeme hinsichtlich ihrer Internalisationsrate und Spezifität evaluiert.In this phase, the different systems with regard to their internalization rate and Specificity evaluated.

Phase 2Phase 2

Die verschiedenen Konstrukte werden in vitro unter Verwendung von verschiedenen Zellkultursystemen verglichen.The different constructs are made in vitro using different Comparing cell culture systems.

Zur Optimierung der GTS hinsichtlich des gerichteten Transports in den Kern und der Expression des Reportergens werden verschiedene NLS-Konjugate synthetisiert. Anschließend erfolgt die Auswahl der geeignetsten GTS.To optimize the GTS with regard to the directed transport into the core and the Expression of the reporter gene, various NLS conjugates are synthesized. The most suitable GTS is then selected.

Untersuchungen zur Internalisation (rezeptorvermittelte Endocytose) und Integration in potentielle Zielzellen für eine somatische Gentherapie sollen an den Zellkultürmodellen Caco-2 und HepG2, die beide sowohl den Asialoglycoprotein- (46,47) als auch den Transferrinrezeptor (21, 48) aufweisen, vorgenommen werden. Letzterer ist weit verbreitet, wohingegen der Asialoglycoproteinrezeptor - soweit bekannt - nur auf Hepatocyten (46, 47) und intestinalen Epithelzellen (46) lokalisiert ist.Studies on internalization (receptor-mediated endocytosis) and integration in Potential target cells for somatic gene therapy are to be modeled on the Caco-2 cell culture models and HepG2, both of the asialoglycoprotein (46.47) and the Have transferrin receptor (21, 48) are made. The latter is common whereas the asialoglycoprotein receptor - as far as known - only on hepatocytes (46, 47) and intestinal epithelial cells (46).

Phase 3Phase 3

Orale, pulmonale oder intravenöse Applikation der ausgewählten GTS im Tierversuch in vivo. "Transcriptional Targeting": Entwicklung von Vektoren mit entsprechenden gewebsspezifischen Enhancer/Promotor-Regionen (41-45). Oral, pulmonary or intravenous application of the selected GTS in animal experiments in vivo. "Transcriptional Targeting": Development of vectors with corresponding tissue-specific enhancer / promoter regions (41-45).  

Zu Phase 1To phase 1

Vorgehensweise zur Konstruktion der Asialoglycoprotein- und Transferrinrezeptor spezifischen GTS.How to construct the asialoglycoprotein and transferrin receptor specific GTS.

1) Transferrinrezeptor-spezifische GTS (pentamer) mit kernspezifischer Targeting Sequenz1) Transferrin receptor-specific GTS (pentamer) with a core-specific targeting sequence

Im ersten Schritt werden sowohl die benötigten Transferrin-Polykation Konjugate in Anlehnung an Wagner et al. (17) als auch membranaktive Peptide-/replikationsdefiziente Adenoviren-Polykation Konjugate (20, 22, 23) synthetisiert. Parallel erfolgt die Synthese der NLS-Polykation-Konjugate und von polykationischen magnetisierbaren Mikropartikeln. Obige Komponenten werden mit dem Vektor (pCMVL/pCMVβ) vereinigt, der ein Luciferase/β-Galactosidase-Reportergen unter Kontrolle des Cytomegalovirus (CMV) Promotor/Enhancer enthalt (22, 23). Durch die polykationischen, magnetisierbaren Partikel wird eine Evaluierung der Internalisierung des pentameren Konjugates ermöglicht. Über das Reportergen wird die Transfektionsrate bestimmt. Die Kombination beider Daten ermöglicht eine Bewertung der verschiedenen GTS-Systeme. Sowohl bei der Synthese der einzelnen Polykation-Konjugate als auch bei der Zusammensetzung der GTS bestehen mehrere Möglichkeiten, die systematisch experimentell ausgetestet und optimiert werden.In the first step both the transferrin-polycation conjugates are needed Based on Wagner et al. (17) as well as membrane active peptide / replication deficient Adenovirus polycation conjugates (20, 22, 23) synthesized. The synthesis of NLS polycation conjugates and of polycationic magnetizable microparticles. The above components are combined with the vector (pCMVL / pCMVβ), which is a Luciferase / β-galactosidase reporter gene under the control of cytomegalovirus (CMV) Promoter / enhancer included (22, 23). Thanks to the polycationic, magnetizable particles an evaluation of the internalization of the pentameric conjugate is made possible. About the The transfection rate is determined by reporter gene. The combination of both data enables an assessment of the various GTS systems. Both in the synthesis of the individual There are several polycation conjugates as well as in the composition of the GTS Possibilities that are systematically experimentally tested and optimized.

Parallel werden pentamere Konjugate synthetisiert, bei denen das Transferrin durch monoklonale Transferrinrezeptor-Antikörper ersetzt wird.In parallel, pentameric conjugates are synthesized in which the transferrin passes through monoclonal transferrin receptor antibody is replaced.

2) Asialoglycoproteinrezeptor-spezifische GTS mit kernspezifischer Targeting Sequenz2) Asialoglycoprotein receptor-specific GTS with core-specific targeting sequence

Die Konstruktion der Asialoglycoproteinrezeptor-spezifischen GTS erfolgt analog wie unter 1) beschrieben mit folgenden Änderungen:The construction of the asialoglycoprotein receptor-specific GTS is carried out analogously as under 1) described with the following changes:

  • a) Transferrin wird durch ein Asialoglycoproteinrezeptor-spezifisches, synthetisches Glycoprotein ersetzt (43).a) Transferrin is activated by an asialoglycoprotein receptor-specific synthetic glycoprotein replaced (43).
  • b) Anti-Transferrinrezeptor Antikörper wird durch einen entsprechenden Asialoglycoproteinrezeptor Antikörper ersetzt.b) Anti-transferrin receptor is replaced by an appropriate antibody Asialoglycoprotein receptor antibody replaced.
Zu Phase 2To phase 2

Eine Optimierung der "Nuclear Targeting" Eigenschaften der in Phase 1 ausgewählten, gut internalisierten GTS soll durch Variation der Beschaffenheit des entsprechenden NLS- Konjugats im pentameren GTS Komplex erreicht werden.An optimization of the "Nuclear Targeting" properties of the selected in phase 1, good internalized GTS should by varying the nature of the corresponding NLS Conjugate in the pentameric GTS complex can be achieved.

Dazu werden folgende Konjugate synthetisiert und anschließend über die Expressionsrate des Reportergens, in situ Hybridisierung und Konfokale Laser Scanning Mikroskopie auf ihre Targeting Eigenschaften untersucht.For this purpose, the following conjugates are synthesized and then via the expression rate of the Reporter gene, in situ hybridization and confocal laser scanning microscopy on their Targeting properties examined.

Aufbau der NLS-KonjugateStructure of the NLS conjugates

Als NLS werden verwendet:The following are used as NLS:

  • 1) Pro-Lys-Lys-Lys-Arg-Lys-Val (NLS des SV40 large T-antigen) 1) Pro-Lys-Lys-Lys-Arg-Lys-Val (NLS of SV40 large T antigen)  
  • 2) Val-Ser-Lys-Arg-Pro-Arg-Pro (NLS des Polyoma Virus large T-antigen)2) Val-Ser-Lys-Arg-Pro-Arg-Pro (NLS of the Polyoma Virus large T antigen)
  • 3) Lys-Arg-Pro-Ala-Ala-Thr-Lys-Lys-Ala-Gly-Gln- Ala-Lys-Lys-Lys-Lys (NLS des Nukleoplasmin)3) Lys-Arg-Pro-Ala-Ala-Thr-Lys-Lys-Ala-Gly-Gln- Ala-Lys-Lys-Lys-Lys (NLS of nucleoplasmin)
1) Allgemeiner Aufbau der Konjugate: Polylysin-Protein/Spacer-(NLS)n1) General structure of the conjugates: polylysine protein / spacer (NLS) n

  • a) Polylysin-Spacer-NLS-Konjugate verschiedener Größea) Polylysine-spacer-NLS conjugates of various sizes
  • b) T- Antigen-Polylysin Konjugat
    Das SV40 T-Antigen wird über 5-5- bzw. Carbodiimid-Crosslinking oder mit dem Biotin-Streptavidin-System an Polylysin (variable Länge) gekoppelt.    b) T-antigen-polylysine conjugate
    The SV40 T antigen is coupled to polylysine (variable length) via 5-5 or carbodiimide crosslinking or with the biotin-streptavidin system.
  • c) Pyruvat-Kinase-NLS-Polylysin-Konjugat
    Kopplung wie unter b)    c) Pyruvate-kinase-NLS-polylysine conjugate
    Coupling as under b)
  • d) BSA-(NLS)x-Polylysin-Konjugat
    In Anlehnung an Dworetzky et al. (33) wird BSA mit NLS-Sequenzen zu den entsprechenden Konjugaten umgesetzt und über Carbodiimid-Chemie mit Polylysin gekoppelt.    d) BSA (NLS) x polylysine conjugate
    Based on Dworetzky et al. (33) BSA is implemented with NLS sequences to the corresponding conjugates and coupled with polylysine via carbodiimide chemistry.
  • e) Humanalbumin-(NLS)x-Polylysin
    Wie unter d) wird mit Humanalbumin ein NLS-Protein-Polylysin-Konjugat hergestellt.    e) Human albumin (NLS) x polylysine
    As in d), an NLS-protein-polylysine conjugate is produced with human albumin.

Die Konjugate a)-e) werden mit und ohne Fluoreszenzmarker synthetisiert und die Expressionsraten der resultierenden GTS bestimmt. Die Targeting Eigenschaften in den Kern werden über Konfokale Laser Scanning Mikroskopie/in situ Hybridisierung quantifiziert und mit den jeweiligen Expressionsraten verglichen. Dieses ermöglicht einen direkten Vergleich und damit die gezielte Optimierung von Internalisierung, Disruption der Endosomen, Targeting in den Kern und der Expression.The conjugates a) -e) are synthesized with and without fluorescent markers and the Expression rates of the resulting GTS were determined. The targeting properties in the core are quantified using confocal laser scanning microscopy / in situ hybridization and compared with the respective expression rates. This enables a direct comparison and thus the targeted optimization of internalization, endosome disruption, targeting in the core and expression.

Als Sonderfall eines NLS-Konjugats zur nachfolgenden Konstruktion eines quarternären GTS werden folgende Polylysin-Konjugate synthetisiert:As a special case of an NLS conjugate for the subsequent construction of a quaternary GTS the following polylysine conjugates are synthesized:

2) Magnetisierbare Partikel - (NLS) x-Polylysin2) Magnetizable particles - (NLS) x-polylysine

Magnetisierbare Mikropartikel (Durchmesser: 30-60 nm) auf Dextran-Basis werden mit NLS- Einheiten und Polylysin modifiziert.Magnetizable microparticles (diameter: 30-60 nm) based on dextran are Modified units and polylysine.

Nach Optimierung der "Nuclear Targeting" Eigenschaften erfolgt die Auswahl der effizientesten GTS.After optimization of the "Nuclear Targeting" properties, the selection is made most efficient GTS.

Zu Phase 3To phase 3

Um ein "Transcriptional Targeting" zu ermöglichen erfolgt parallel die Entwicklung von Vektoren mit gewebsspezifischen Promotor/Enhancer Regionen.To enable "transcriptional targeting", the development of Vectors with tissue-specific promoter / enhancer regions.

Zelluläre Promotoren weisen folgende Vorteile auf:Cellular promoters have the following advantages:

  • a) Reduktion des Verlusts von cDNA durch Inaktivierung aufgrund von Methylierung oder anderen Mechanismen die eine "long term expression" ermöglicht.a) Reduction of loss of cDNA due to inactivation due to Methylation or other mechanisms that have a "long term expression" enables.
  • b) "Tissue Restricted Expression": Transkription erfolgt nur in der Zielzelle. Gen wird - im Gegensatz zu viralen Promotoren - zum richtigen Zeitpunkt an und abgeschalten.b) "Tissue Restricted Expression": Transcription takes place only in the target cell. gene is - in contrast to viral promoters - at the right time and switched off.

Dazu können u. a. folgende Promotoren/Enhancer-Regionen in Betracht kommen:To do this, u. a. the following promoters / enhancer regions can be considered:

  • - PEPCK Promotor: Transcriptional aktiv in Leber, Niere und Fettgewebe (42, 43) und in HepG2-Zellen; induzierbar durch Glucagon, Glucocorticoide (Dexamethason)/kohlenhydratarme Diät, cAMP und Theophyllin- PEPCK promoter: Transcriptionally active in liver, kidney and adipose tissue (42, 43) and in HepG2 cells; inducible by glucagon, glucocorticoids (Dexamethasone) / low carbohydrate diet, cAMP and theophylline
  • - Kreatin Kinase Promotor: Limitiert Dystrophin cDNA Expression auf Skelett- und Herzmuskel (44)Creatine Kinase Promoter: Limits Dystrophin cDNA Expression on Skeleton and heart muscle (44)
  • - Albumin Promotor: Hepatomzellspezifität in vitro (41)- Albumin promoter: hepatoma cell specificity in vitro (41)
  • - Alpha-fetoprotein Promotor: Fötale Leber/Hepatomzellspezifität (42)- Alpha-fetoprotein promoter: fetal liver / hepatoma cell specificity (42)
  • - Von Willebrand Faktor Promotor: Endothelspezifisch (45)- Von Willebrand factor promoter: endothelium-specific (45)

Weiterhin wird in Phase 3 der Einfluß der Transfektion auf die Proliferation, Vitalität und den Metabolismus der verwendeten Zellinien getestet, z. B. durchIn phase 3, the influence of transfection on proliferation, vitality and Metabolism of the cell lines used tested, e.g. B. by

  • 1) BrdU-Zell-ELISA1) BrdU cell ELISA
  • 2) Messung der Aktivität der mitochondrialen Dehydrogenasen z. B. mittels des MTT-/XTT-Assays2) Measuring the activity of mitochondrial dehydrogenases e.g. B. using the MTT / XTT assay
  • 3) Messung der Aktivität von Markerenzymen für Caco-2, z. B. Sucrase/Isomaltase.3) Measurement of the activity of marker enzymes for Caco-2, e.g. B. Sucrase / Isomaltase.
  • 4) Messung der Albuminexkretion für HepG2.4) Measurement of albumin excretion for HepG2.
LITERATURLITERATURE

(1) Mulligan, R.C. (1993): The basic science of gene therapy. Science 260, 926-932.
(2) Morgan, R.A. and Anderson, W.F. (1993): Human Gene Therapy. Annu. Rev. Biochem. 62, 191-217.
(3) Brach M.A., Kiehntopf, M., and Herrmann F. (1995): Gentherapie in der Onkologie. Dtsch. med. Wschr. 120, 113-120.
(4) Finkel, T. and Epstein, S.E. (1995): Gene therapy for vascular disease. FASEB J. 9, 843-851.
(5) Ono, T., Fujino, Y., Tsuchiya, T., and Tsuda, M. (1990): Plasmid DNAs directly injected into mouse brain with lipofectin can be incorporated and expressed by brain cells. Neurosci. Lett. 117, 259-263.
(6) Gao, X. and Huang, L. (1991): A novel cationic liposome reagent for efficient transfection of mammalian cells. Biochem. Biophys. Res. Comm. 179, 280-285.
(7) Vile, R.G. and Hart, I.R. (1993): In vitro and in vivo targeting of gene expression to melanoma cells. Cancer Res. 53, 962-969.
(8) Alton, E.W.F.W., Middleton, P.G., Caplen, N.J., Smith, S.N., Steel, D.M., Munkonge, F.M., Jeffery, P.K., Geddes, D.M., Hart, S.L., Williamson, R., Fasold, K.I., Miller, A.D., Dickinson, P., Stevenson, B.J., McLachlan, G., Dorin, J.R., and Porteus, D.J. (1993): Non-invasive liposome-mediated gene delivery can correct the ion transport defect in cystic fibrosis mutant mice. Nature genetics 5, 135-142.
(9) San, H., Yang, Z., Pompili, V., Jaffe, M., P1autz, G., Xu, L., Felgner, J.H., Wheeler, C.J., Felgner, P.L., Gao, X., Huang, L., Gordon, D., Nabel, G.J., and Nabel, E.G. (1993): Safety and short-term toxicity of a novel cationic lipid formulation for human gene therapy. Human Gene Therapy 4, 781-788.
(10) Crystal, R.G. (1995): The gene as the drug. Nature medicine 1, 15-17.
(11) Caplen, N.J., Alton, E.W.F.W., Middleton, P.G., Dorin, J.R., Stevenson, B.J., Gao, X., Durham, S.R., Jeffery, P.K., Hodson, M.E., Coutelle, C., Huang, L., Porteous, D.J., Williamson, R., and Geddes, D.M. (1995): Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Nature medicine 1, 39-46.
(12) Goldstein, J.L., Brown, M.S., Anderson, R.G.W., Russel, D.W., and Schneider, W. J. (1985): RECEPTOR-MEDIATED ENDOCYTOSIS: Concepts Emerging from the LDL Receptor System. Ann. Rev. Cell Biol. 1, 1-39.
(13) Wu, G.Y. and Wu, C.H. (1987): Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. J. Biol. Chem. 262, 4429-4432.
(14) Wu, G.Y. and Wu, C.H. (1988): Receptor-mediated gene delivery and expression in vivo. J. Biol. Chem. 263, 14621-14624 (1988).
(15) Zenke M., Steiniein P., Wagner E., Cotten M., Beug H., and Birnstiel M.L. (1990):
Receptor-mediated endocytosis of transferrin-polycation conjugates: An efficient way to introduce DNA into hematopoietic cells. Proc. Natl. Acad. Sci. U.S.A. 87, 3655-3659.
(16) Cotten M., Längle-Rouault F., Kirlappos K., Wagner E . . Mechtler, M. Zenke M., Beug H., and Birnstiel M. L. (1990). Transferrin-polycation-mediated introduction of DNA into human leukämic cells: Stimulation by agents that affect the survival of transfected DNA or modulate transfemn receptor levels. Proc. Natl. Acad. Sci. U.S.A. 87, 4033-4037.
(17) Wagner E. , Zenke M., Cotten M., Beug H., and Birnstiel M.L. (1990). Transferrin­ polycation conjugates as carrier s for DNA uptake into cells. Proc. Natl. Acad. Sci. U.S.A. 87, 3410-3414.
(18) Wagner E., Cotten M., Foisner R., and Birnstiel M. L. (1991): Transferrin-polycations on the structure of the complex and DNA delivery to cells. Proc. Natl. Acad. Sci. U.S.A. 88, 4255-4259.
(19) Wu, G.Y., Wilson, J.M., Shalaby, F., Grossman, M., Shatritz, D.A., and Wu, C.H. (1991): Receptor-mediated gene delivery in vivo. Partial correction of genetic analbuminemia in nagase rats. J. Biol. Chem. 266, 14338-14342.
(20) Wagner E., Plank C., Zatloukal K., Cotten M., and Birnstiel, M. L. (1992): Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: Toward a synthetic virus-like gene-transfer vehicle. Proc. Natl. Acad. Sci. U.S.A. 89, 7934-7938.
(21) Rosenkranz, A. A., Yachmenev, S. V., Jans, D. A., Serebryakova, N. V., Muravev, V. I., Peters, R., and Sobolev, A. S. (1992): Receptor-mediated endocytosis and nuclear transport of a transfecting DNA construct. Exp. Cell Res. 199, 323-329.
(22) Cotten, M., Wagner, E., and Birnstiel, M.L. (1993): Receptor-Mediated Transport of DNA into Eukaryotic Cells. Methods in Enzymology 217, 618-644.
(23) Buschle, M., Cotten, M., Kirlappos, H., Mechtler, K., Schaffner, G. Zauner, W., Birnstiel, M. L., and Wagner, E. (1995): Receptor-mediated gene transfer into human T- lymphocytes via Binding of DNA/CD3 antibody particles to the CD3 T cell receptor complex. Hum. Gene Ther. 6, 753-761.
(24) Curiel, D.T., Agarwal, S., Wagner, E., and Cotten, M. (1991): Adenovirus enhancement of transferrin-polylysine-mediated gene delivery. Proc. Natl. Acad. Sci. U.S.A. 88, 8850-8854.
(25) Curiel, D.T., Wagner, E., Cotten, M., Birnstiel, M. L., Agarwal, S., Li, C.-M., Loechel, S., and Hu, P.-C.(1992): High-efficiency gene transfer by adenovirus coupled to DNA- polylysine complexes. Hum. Gene Ther. 3, 147-154.
(26) Cotten M., Wagner E., Zatloukal K., Phillips S., Curiel D. T., and Birnstiel M. L. (1992):
High-efficiency receptor-mediated delivery of small and large (48 kb) gene constructs using the endosome disruption activity of detective or chemically inactivated adenovirus particles. Proc. Natl. Acad. Sci. U.S.A. 89, 6094-6098.
(27) Plank, C., Zatloukal, K., Cotten, M., Mechtler, K., and Wagner, E. (1992). Gene transfer into hepatocytes using asialoglycoprotein receptor-mediated endocytosis of DNA complexed with an artificial tetra antennary galactose hgand. Bioconj. Chem. 3, 533-539.
(28) Wagner E., Zatloukal K., Cotten M., Kirlappos H., Mechtler K., Curiel, D.T., and Birnstiel, M.L. (1992). Coupling of adenovirus to transfernn-polylysin/DNA complexes greatly enhances receptormediated gene delivery and expression of transfected genes. Proc. Natl. Acad. Sci. U.S.A. 89, 6099-6103.
(29) Michael, S.I.m Huang, C H., Romer, M.U., Wagner. E., Hu, P C., and Cunel, D.T. (1993). Bmding-incompetent Adenovirus facilitates molecular-mediated gene transfer by the receptor mediated endocytosis pathway. J. Biol. Chem. 268, 6866-6869.
(30) Richardson, W.D., Roberts, B.L., and Smith, A.E. (1986). Nuclear location signals in polyoma virus large-T. Cell 44, 77-85.
(31) Goldfarb, D.S., Gariepy, J., Schoolnik, G., and Kornberg, R.G. (1986). Synthetic peptides as nuclear localization signals. Nature 322, 641-644.
(32) Lyons, R.H., FergUson, B.Q., and Rosenberg, M. (1987). Pentapeptide Nuclear Localization signal in adenovirus E1a. Mol. Cell. Biol. 7, 2451-2456.
(33) Dworetzky, S.I., Lanford, RE., and Feldherr, C.M. (1988). The effects of variations in thenumber and sequence of targeting signals on nuclear uptake. J. Cell. Biol. 107, 1279-1287.
(34) Silver, P.A. (1990): How proteins enter the nucleus. Cell 64, 489-497.
(35) Breeuwer, M., and Goldfarb. D.S. (1990): Facilitated nuclear transport of histone H1 and other small nucleophilic proteins. Cell 60, 999-1008.
(36) Robbins, J., Dilworth, S.M., Laskey, R.A., and Dingwall, C. (1991): Two Interdependent Basis Domains in Nucleoplasmin Nuclear Targeting Sequence: Identification of a Class of Bipartite Nuclear Targeting Sequence. Cell 64, 615-623.
(37) Dingwali, C. (1991): Transport Across the Nuclear Envelope: Enigmas and Explanations. BioEssays 13, 213-218.
(38) Osborne, M.A. and Silver, P.A. (1993): Nucleocytoplasmic Transport In Yeast Saccharomyces Cerevisiae. Annu. Rev. Biochem. 62, 219-54.
(39) Zhou, Z-X., Sar, M., Simental, J. A., Lane, M.V., and Wilson, E.M. (1994): A Ligand­ dependent bipartite nuclear targeting signal in the human androgen receptor. J. Biol. Chem. 269, 13115-13123.
(40) Simos, G. and Hurt, E.C. (1995): Nucleocytoplasmic transport: factors and mechanisms. FEBS Lett. 369, 107-112.
(41) Friedman, J.M., Babiss, L.E., Clayton, D.F., and Darnell, JR. (1986): Cellular promoters incorporated into the adenovirus genome: cell specificity of albumin and immunglobulin expression. Mol. Cell Biol. 6, 3791-3797.
(42) Hatzoglou, M., Lamers, W., Bosch, F., Wynshaw-Boris, A., Clapp, D.W., and Hanson R.W. (1990): Hepatic gene transfer in animals using retroviruses containing the promoter from the gene for phosphoenolpyruvate carboxykinase. J. Biol. Chem. 265, 17285-17293.
(43) Ferkol, T., Lindberg, G.L., Chen, J., Perales, J.C., Crawford, D.R., Ratnoff, O.D., and Hanson, R.W. (1993): Regnlation of the phosphoenolpyruvate carboxykinase/ human factor IX gene introduced into the livers of adult rats by receptor-mediated gene transfer. FASEB J. 7, 1081-1091.
(44) Cox, G.A., Cole, N.M., Matsumura, K., Phelps, S.F., Hauschka, S.D.,Campell, K.P., Faulkner, J.A., and Chamberlain, J.S. (1993): Overexpression of dystrophin in transgenic mdx mice eliminates dystrophic symptoms without toxicity. Nature 364, 725-729.
(45) Jahroudi, N. and Lynch, D.C. (1994): Endothelial-cell-specific regulation of von Willebrand factor gene expression. Mol. Cell Biol. 14, 999-1008.
(46) Mu, J.Z., Fallon, R.J., Swanson, P.E., Carroll, S.B., Danaher, M., and Alpers, D.H. (1994): Expression of an endogenous asialoglycoprotein receptorin a human epithelial cell line, Caco-2. Biochim. Biophys. Acta. 1222, 483-91.
(47) Spiess, M (1990: TheAsialoglycoprotein Receptor. A Model for Endocytic Transport Receptors. Biochemistry 29, 10009-10018.
(48) Shah, D. and Shen, W.C. (1994). The establishment of polanty and enhanced transcytosis of transferrin receptors in enterocyte-like Caco-2 cells. J. Drug Target. 2, 93-99. (1) Mulligan, RC (1993): The basic science of gene therapy. Science 260, 926-932.
(2) Morgan, RA and Anderson, WF (1993): Human Gene Therapy. Annu. Rev. Biochem. 62, 191-217.
(3) Brach MA, Kiehntopf, M., and Herrmann F. (1995): Gene therapy in oncology. German med. Wschr. 120, 113-120.
(4) Finkel, T. and Epstein, SE (1995): Gene therapy for vascular disease. FASEB J. 9, 843-851.
(5) Ono, T., Fujino, Y., Tsuchiya, T., and Tsuda, M. (1990): Plasmid DNAs directly injected into mouse brain with lipofectin can be incorporated and expressed by brain cells. Neurosci. Lett. 117, 259-263.
(6) Gao, X. and Huang, L. (1991): A novel cationic liposome reagent for efficient transfection of mammalian cells. Biochem. Biophys. Res. Comm. 179, 280-285.
(7) Vile, RG and Hart, IR (1993): In vitro and in vivo targeting of gene expression to melanoma cells. Cancer Res. 53, 962-969.
(8) Alton, EWFW, Middleton, PG, Caplen, NJ, Smith, SN, Steel, DM, Munkonge, FM, Jeffery, PK, Geddes, DM, Hart, SL, Williamson, R., Fasold, KI, Miller, AD, Dickinson, P., Stevenson, BJ, McLachlan, G., Dorin, JR, and Porteus, DJ (1993): Non-invasive liposome-mediated gene delivery can correct the ion transport defect in cystic fibrosis mutant mice. Nature genetics 5, 135-142.
(9) San, H., Yang, Z., Pompili, V., Jaffe, M., P1autz, G., Xu, L., Felgner, JH, Wheeler, CJ, Felgner, PL, Gao, X., Huang, L., Gordon, D., Nabel, GJ, and Nabel, EG (1993): Safety and short-term toxicity of a novel cationic lipid formulation for human gene therapy. Human Gene Therapy 4, 781-788.
(10) Crystal, RG (1995): The gene as the drug. Nature medicine 1, 15-17.
(11) Caplen, NJ, Alton, EWFW, Middleton, PG, Dorin, JR, Stevenson, BJ, Gao, X., Durham, SR, Jeffery, PK, Hodson, ME, Coutelle, C., Huang, L., Porteous, DJ, Williamson, R., and Geddes, DM (1995): Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Nature medicine 1, 39-46.
(12) Goldstein, JL, Brown, MS, Anderson, RGW, Russel, DW, and Schneider, WJ (1985): RECEPTOR-MEDIATED ENDOCYTOSIS: Concepts Emerging from the LDL Receptor System. Ann. Rev. Cell Biol. 1, 1-39.
(13) Wu, GY and Wu, CH (1987): Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. J. Biol. Chem. 262, 4429-4432.
(14) Wu, GY and Wu, CH (1988): Receptor-mediated gene delivery and expression in vivo. J. Biol. Chem. 263, 14621-14624 (1988).
(15) Zenke M., Steiniein P., Wagner E., Cotten M., Beug H., and Birnstiel ML (1990):
Receptor-mediated endocytosis of transferrin-polycation conjugates: An efficient way to introduce DNA into hematopoietic cells. Proc. Natl. Acad. Sci. USA 87, 3655-3659.
(16) Cotten M., Längle-Rouault F., Kirlappos K., Wagner E. . Mechtler, M. Zenke M., Beug H., and Birnstiel ML (1990). Transferrin-polycation-mediated introduction of DNA into human leukemic cells: Stimulation by agents that affect the survival of transfected DNA or modulate transfemn receptor levels. Proc. Natl. Acad. Sci. USA 87, 4033-4037.
(17) Wagner E., Zenke M., Cotten M., Beug H., and Birnstiel ML (1990). Transferrin polycation conjugates as carrier s for DNA uptake into cells. Proc. Natl. Acad. Sci. USA 87, 3410-3414.
(18) Wagner E., Cotten M., Foisner R., and Birnstiel ML (1991): Transferrin-polycations on the structure of the complex and DNA delivery to cells. Proc. Natl. Acad. Sci. USA 88, 4255-4259.
(19) Wu, GY, Wilson, JM, Shalaby, F., Grossman, M., Shatritz, DA, and Wu, CH (1991): Receptor-mediated gene delivery in vivo. Partial correction of genetic analbuminemia in nagase rats. J. Biol. Chem. 266, 14338-14342.
(20) Wagner E., Plank C., Zatloukal K., Cotten M., and Birnstiel, ML (1992): Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: Toward a synthetic virus-like gene-transfer vehicle. Proc. Natl. Acad. Sci. USA 89, 7934-7938.
(21) Rosenkranz, AA, Yachmenev, SV, Jans, DA, Serebryakova, NV, Muravev, VI, Peters, R., and Sobolev, AS (1992): Receptor-mediated endocytosis and nuclear transport of a transfecting DNA construct. Exp. Cell Res. 199, 323-329.
(22) Cotten, M., Wagner, E., and Birnstiel, ML (1993): Receptor-Mediated Transport of DNA into Eukaryotic Cells. Methods in Enzymology 217, 618-644.
(23) Buschle, M., Cotten, M., Kirlappos, H., Mechtler, K., Schaffner, G. Zauner, W., Birnstiel, ML, and Wagner, E. (1995): Receptor-mediated gene transfer into human T- lymphocytes via Binding of DNA / CD3 antibody particles to the CD3 T cell receptor complex. Hum. Gene Ther. 6, 753-761.
(24) Curiel, DT, Agarwal, S., Wagner, E., and Cotten, M. (1991): Adenovirus enhancement of transferrin-polylysine-mediated gene delivery. Proc. Natl. Acad. Sci. USA 88, 8850-8854.
(25) Curiel, DT, Wagner, E., Cotten, M., Birnstiel, ML, Agarwal, S., Li, C.-M., Loechel, S., and Hu, P.-C. (1992) : High-efficiency gene transfer by adenovirus coupled to DNA- polylysine complexes. Hum. Gene Ther. 3, 147-154.
(26) Cotten M., Wagner E., Zatloukal K., Phillips S., Curiel DT, and Birnstiel ML (1992):
High-efficiency receptor-mediated delivery of small and large (48 kb) gene constructs using the endosome disruption activity of detective or chemically inactivated adenovirus particles. Proc. Natl. Acad. Sci. USA 89, 6094-6098.
(27) Plank, C., Zatloukal, K., Cotten, M., Mechtler, K., and Wagner, E. (1992). Gene transfer into hepatocytes using asialoglycoprotein receptor-mediated endocytosis of DNA complexed with an artificial tetra antennary galactose hgand. Bioconj. Chem. 3, 533-539.
(28) Wagner E., Zatloukal K., Cotten M., Kirlappos H., Mechtler K., Curiel, DT, and Birnstiel, ML (1992). Coupling of adenovirus to transfernn-polylysin / DNA complexes greatly enhances receptormediated gene delivery and expression of transfected genes. Proc. Natl. Acad. Sci. USA 89, 6099-6103.
(29) Michael, SIm Huang, C H., Romer, MU, Wagner. E., Hu, PC, and Cunel, DT (1993). Bmding-incompetent adenovirus facilitates molecular-mediated gene transfer by the receptor mediated endocytosis pathway. J. Biol. Chem. 268, 6866-6869.
(30) Richardson, WD, Roberts, BL, and Smith, AE (1986). Nuclear location signals in polyoma virus large-T. Cell 44, 77-85.
(31) Goldfarb, DS, Gariepy, J., Schoolnik, G., and Kornberg, RG (1986). Synthetic peptides as nuclear localization signals. Nature 322, 641-644.
(32) Lyons, RH, FergUson, BQ, and Rosenberg, M. (1987). Pentapeptide Nuclear Localization signal in adenovirus E1a. Mol. Cell. Biol. 7, 2451-2456.
(33) Dworetzky, SI, Lanford, RE., And Feldherr, CM (1988). The effects of variations in thenumber and sequence of targeting signals on nuclear uptake. J. Cell. Biol. 107, 1279-1287.
(34) Silver, PA (1990): How proteins enter the nucleus. Cell 64, 489-497.
(35) Breeuwer, M., and Goldfarb. DS (1990): Facilitated nuclear transport of histone H1 and other small nucleophilic proteins. Cell 60, 999-1008.
(36) Robbins, J., Dilworth, SM, Laskey, RA, and Dingwall, C. (1991): Two Interdependent Basis Domains in Nucleoplasmin Nuclear Targeting Sequence: Identification of a Class of Bipartite Nuclear Targeting Sequence. Cell 64, 615-623.
(37) Dingwali, C. (1991): Transport Across the Nuclear Envelope: Enigmas and Explanations. BioEssays 13, 213-218.
(38) Osborne, MA and Silver, PA (1993): Nucleocytoplasmic Transport In Yeast Saccharomyces Cerevisiae. Annu. Rev. Biochem. 62, 219-54.
(39) Zhou, ZX., Sar, M., Simental, JA, Lane, MV, and Wilson, EM (1994): A Ligand dependent bipartite nuclear targeting signal in the human androgen receptor. J. Biol. Chem. 269, 13115-13123.
(40) Simos, G. and Hurt, EC (1995): Nucleocytoplasmic transport: factors and mechanisms. FEBS Lett. 369, 107-112.
(41) Friedman, JM, Babiss, LE, Clayton, DF, and Darnell, JR. (1986): Cellular promoters incorporated into the adenovirus genome: cell specificity of albumin and immunoglobulin expression. Mol. Cell Biol. 6, 3791-3797.
(42) Hatzoglou, M., Lamers, W., Bosch, F., Wynshaw-Boris, A., Clapp, DW, and Hanson RW (1990): Hepatic gene transfer in animals using retroviruses containing the promoter from the gene for phosphoenolpyruvate carboxykinase. J. Biol. Chem. 265, 17285-17293.
(43) Ferkol, T., Lindberg, GL, Chen, J., Perales, JC, Crawford, DR, Ratnoff, OD, and Hanson, RW (1993): Regnlation of the phosphoenolpyruvate carboxykinase / human factor IX gene introduced into the livers of adult rats by receptor-mediated gene transfer. FASEB J. 7, 1081-1091.
(44) Cox, GA, Cole, NM, Matsumura, K., Phelps, SF, Hauschka, SD, Campell, KP, Faulkner, JA, and Chamberlain, JS (1993): Overexpression of dystrophin in transgenic mdx mice eliminates dystrophic symptoms without toxicity. Nature 364, 725-729.
(45) Jahroudi, N. and Lynch, DC (1994): Endothelial-cell-specific regulation of von Willebrand factor gene expression. Mol. Cell Biol. 14, 999-1008.
(46) Mu, JZ, Fallon, RJ, Swanson, PE, Carroll, SB, Danaher, M., and Alpers, DH (1994): Expression of an endogenous asialoglycoprotein receptorin a human epithelial cell line, Caco-2. Biochim. Biophys. Acta. 1222, 483-91.
(47) Spiess, M (1990: TheAsialoglycoprotein Receptor. A Model for Endocytic Transport Receptors. Biochemistry 29, 10009-10018.
(48) Shah, D. and Shen, WC (1994). The establishment of polanty and enhanced transcytosis of transferrin receptors in enterocyte-like Caco-2 cells. J. Drug Target. 2, 93-99.

Anwendung für die TherapieApplication for therapy

Die beschriebenen Prinzipien zur Optimierung und Quantifizierung des Gentransfers können nach Klärung der Sicherheits- und Effizienzfragen unter Einsatz therapierelevanter Gene in der Gentherapie angewendet werden.The principles described for optimizing and quantifying gene transfer can after clarification of the safety and efficiency questions using therapy-relevant genes in the Gene therapy can be applied.

Claims (1)

Herstellung, Verkauf und Einsatz von Gentransfersystemen, die beide oder eine der folgenden Komponenten aufweisen:
  • a) "Nuclear Localization Sequences" (NLS)
  • b) magnetisierbare Mikropartikel
Manufacture, sale and use of gene transfer systems that have both or one of the following components:
  • a) "Nuclear Localization Sequences" (NLS)
  • b) magnetizable microparticles
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* Cited by examiner, † Cited by third party
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EP1046394A2 (en) * 1999-04-19 2000-10-25 ImaRx Pharmaceutical Corp. Novel compositions useful for delivering compounds into a cell
US6521456B1 (en) 1999-01-08 2003-02-18 Gregor Siebenkotten Cellular transport system for the transfer of a nucleic acid through the nuclear envelope and methods thereof
US6638767B2 (en) 1996-05-01 2003-10-28 Imarx Pharmaceutical Corporation Methods for delivering compounds into a cell
WO2003092735A2 (en) * 2002-05-01 2003-11-13 Pantheco A/S Peptide nucleic acid conjugates with beta-amino acid polymers or reptoids
US6743779B1 (en) 1994-11-29 2004-06-01 Imarx Pharmaceutical Corp. Methods for delivering compounds into a cell

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6743779B1 (en) 1994-11-29 2004-06-01 Imarx Pharmaceutical Corp. Methods for delivering compounds into a cell
US6638767B2 (en) 1996-05-01 2003-10-28 Imarx Pharmaceutical Corporation Methods for delivering compounds into a cell
US6521456B1 (en) 1999-01-08 2003-02-18 Gregor Siebenkotten Cellular transport system for the transfer of a nucleic acid through the nuclear envelope and methods thereof
EP1046394A2 (en) * 1999-04-19 2000-10-25 ImaRx Pharmaceutical Corp. Novel compositions useful for delivering compounds into a cell
EP1046394A3 (en) * 1999-04-19 2001-10-10 ImaRx Pharmaceutical Corp. Novel compositions useful for delivering compounds into a cell
WO2003092735A2 (en) * 2002-05-01 2003-11-13 Pantheco A/S Peptide nucleic acid conjugates with beta-amino acid polymers or reptoids
WO2003092735A3 (en) * 2002-05-01 2004-03-25 Pantheco As Peptide nucleic acid conjugates with beta-amino acid polymers or reptoids

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