WO2003047631A2 - Conjugue apn a pince pour le transport specifique d'acides nucleiques activables thermiquement - Google Patents

Conjugue apn a pince pour le transport specifique d'acides nucleiques activables thermiquement Download PDF

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Publication number
WO2003047631A2
WO2003047631A2 PCT/DE2002/004356 DE0204356W WO03047631A2 WO 2003047631 A2 WO2003047631 A2 WO 2003047631A2 DE 0204356 W DE0204356 W DE 0204356W WO 03047631 A2 WO03047631 A2 WO 03047631A2
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Prior art keywords
pna
nucleic acid
peptide
conjugate
lys
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PCT/DE2002/004356
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German (de)
English (en)
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WO2003047631A3 (fr
Inventor
Klaus Braun
Isabell Braun
Heike Corban-Wilhelm
Jürgen Debus
Jürgen JENNE
Ralf Rastert
Rüdiger Pipkorn
Ioannis Simiantonakis
Waldemar Waldeck
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Deutsches Krebsforschungszentrum DKFZ
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Deutsches Krebsforschungszentrum DKFZ
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Priority to AU2002351703A priority Critical patent/AU2002351703A1/en
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Publication of WO2003047631A3 publication Critical patent/WO2003047631A3/fr
Anticipated expiration legal-status Critical
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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6425—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 the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
    • 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
    • A61K47/66—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 the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • 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
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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

Definitions

  • the present invention relates to a PNA conjugate for the specific transport of a nucleic acid sequence into the cells and thermal activatability of the nucleic acid sequence at the desired target location, the conjugate having the following components:
  • clip PNA a clip peptide nucleic acid
  • ganciclovir e.g. ganciclovir; GCV
  • GCV ganciclovir
  • the invention is therefore essentially based on the technical problem of providing a gene transport system which is both efficient and independent of the desired cell type to be transfected, for example a tumor cell, and which has an action which is limited to the target cell / tissue.
  • Cell compartments preferably the cell nucleus
  • clip PNA a clip peptide nucleic acid
  • cleavable covalent disulfide coupling and a spacer (e.g. lysine-glycine between (b) and (c).
  • spacer e.g. lysine-glycine
  • the clamp PNA is hybridized with the corresponding nucleic acid and by means of components P and AP, an efficient and directed transport of the clamp PNA with the hybridized nucleic acid to the target location and thus gene therapy is made possible (see FIG. 1).
  • These components not only allow fast and effective transport of the staple PNA through cell membranes of living cells into the cytoplasm, but also, after cytoplasmic activation of address peptide sequences, an efficient transport into the cell nucleus.
  • clip PNAs protease and nuclease resistant Clip peptide nucleic acids
  • the nucleic acid sequence hybridized to the clamp PNA can dissociate by local heating and thus be converted from an inactive into a desired active form, which can be, for example, transcribed and translated, and thus a desired gene product is only in the desired target cell, ie the heated cell. This enables local gene therapy that requires low doses of administration and is largely free of undesirable side effects.
  • clamp conjugates according to the invention with regard to therapy can be seen in the fact that no viral vector is involved in the therapy, so there is no risk of infection and the other side effects are also lower.
  • the clip conjugates also show a high transfection effectiveness with various cell types.
  • the activation temperature can be set specifically (in one), and this can also be set according to the organ to be treated.
  • Activation temperature of 44 ° C is, for example, in a safe area far enough away from temperatures naturally occurring in the body, such as fever. In this way, incorrect activation can be avoided. Another advantage is the fact that only relatively short times (1 to 2 minutes) are required for activation, which is about 1/20 of the conventional heat inducible promoters required
  • the clip-PNA conjugates according to the invention represent a new type of pharmaceuticals which are effectively transported to the target site and, due to their high stability and excellent specificity, a stable and efficient activity control of the nucleic acid which is introduced (which is a “prodrug” represents), e.g. Transcription control, enable.
  • the clip-PNA conjugates according to the invention should be effective even at extremely low administration doses (below 100 pM final concentration), thus preventing or at least greatly reducing the occurrence of undesirable side effects.
  • clamp-PNA conjugates thus have the following advantages: (a) non-invasive transport system, (b) modular design, (c) cytoplasmic cleavage, (d) core addressing signal, (e) clamp-PNA for transporting the desired Nucleic acid sequence by local warming
  • the present invention thus relates to a PNA conjugate for the specific transport of a nucleic acid sequence into the cell nucleus and thermal activatability of the nucleic acid sequence at the desired target location, the conjugate having the following components:
  • the structure of the conjugate according to the invention is preferably: clip PNA (including the hybridized nucleic acid sequence to be transported) - transport module - address protein or address peptide.
  • the transport mediator for the cell membrane is preferably a peptide or protein that can overcome the plasma membrane.
  • the length of this peptide or protein is not limited as long as it has the above property.
  • Examples of transport mediators preferably come from the penetratin family (Derossi et al., Trends Cell Biol. 8 (1988), pp. 84-87), are transport agents or parts thereof (Pooga et al., The Faseb Journal 1_2 (1998 ), P. 68 ff.) Or the transmembrane peptide pAntp (43-58).
  • Transport peptide units TPU
  • Bacterial ⁇ ECo) 1A0P GeneBank
  • site specif. Recombinase- PDB
  • Viral (HIV-1 / TAT) 1TIV-HIV-1 GeneBank
  • transactivator protein fragment PDB
  • the selected transport mediator is produced biologically (purification of natural transport mediator proteins or cloning and expression of the sequence in a eukaryotic or prokaryotic expression system), but preferably synthetically, e.g. according to the Merrifield method (Merrifield, J. Am. Chem. Soc. 85 (1963), 2149).
  • the person skilled in the art can select the selection of the address protein or peptide on the basis of the known amino acid sequences for the import of peptides or polypeptides which control the cell nucleus.
  • the length of this address peptide or protein is not subject to any restriction, as long as it has the property of ensuring a cell nucleus-specific transport.
  • Address proteins or address peptides which contain a cell nucleus-specific recognition signal are generally selected for the introduction of the clamp PNAs with the hybridized nucleic acid thereby directing the staple PNAs into the cell nucleus.
  • the pure one is for the transport into the cell nucleus
  • the gap lies within the
  • Amino acids are added to ensure that the address sequence is split off after reaching the cell nucleus.
  • the selected "AP" sequence is produced biologically
  • Examples of suitable address proteins or peptides are: (a) -Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val and (b)
  • the conjugate can optionally contain a spacer (abbreviated SP above), which is preferably located between the address protein / peptide and the clamp peptide nuclear acid to be transported. However, it can additionally or alternatively also be present between the transport intermediary and the address protein.
  • the spacer serves to abolish or favorably influence any steric interactions that may exist between the components.
  • the spacer can be selected, for example, from Polylysm, polyethylene glycol (PEG), derivatives of poly-methacrylic acid or polyvinyl pyrrolidone (PVP).
  • a redox cleavage site for example -Cystem-SS-Cystem-ONH-, between the transport mediator and the address protein / peptide.
  • the bond formed between the transport agent and the address protein is a redox coupling (gentle cell-immanent connection using DMSO; Rietsch and Beckwith, Annu. Rev. Genet 32 (1998), 163-84):
  • the clip peptide nucleic acid (clip PNA) of the conjugates according to the invention allows the specific transport and the targeted release (and thus activation) of the transported nucleic acid sequence via local heating.
  • the nucleic acid sequence to be transported can be bound more efficiently than a monomer PNA using the PNA clamp.
  • the PNA clip consists of two PNAs complementary to target sequences, which preferably each contain two glycine residues via ⁇ -
  • Amino and ⁇ -amino lysine are linked.
  • the sequence specificity is achieved by the number of nucleobases, preferably 18-23 bases.
  • the higher stability of the H-bridge bonds between the PNA nucleobases and the DNA nucleobases is due to the formation of triple helix bonds PNA / DNA / PNA:
  • Areas suitable for binding the nucleic acid sequence to be transported can easily be determined by a person skilled in the art, care being taken to ensure that those sequences of the nucleic acid to be transported hybridize and are thus inactivated which are required, for example, for complete transcription / translation. These can be control sequences, for example, or sequence areas to be transcribed / translated.
  • the regions which hybridize with one another (clip PNA / nucleic acid sequence to be transported) become corresponding generally known methods chosen so that they are
  • the peptide nucleic acid monomers of the clamp PNA preferably have a length of at least 18 bases each and more preferably at least 21 bases, peptide nucleic acids with a length of at least 23 bases each are particularly preferred.
  • the peptide-nucleic acid clip may also be labeled, e.g. radioactive, with a dye, with biotin / avidin etc.
  • the conjugate constituents P and AP are preferably synthesized by the Merrifield method (Merrifield, J. Am. Che. Soc. 85 (1963), 2149).
  • the other constituents for example spacers and / or PNA
  • the redox gap between P and AP is chemically inserted through the redox coupling mentioned above.
  • a covalent bond preferably an acid amide bond, is also present between an optionally present spacer and the PNA or the address protein and the PNA.
  • Possible alternatives are ether or ester linkages, depending on the functional group (s) present in the substance to be conjugated.
  • the application of the mixture according to the invention of staple-PNA conjugate and nucleic acid sequence to be transported, which hybridizes with the staple PNA via triple helix formation, is preferably carried out in vitro directly into the culture medium (preferably with a final concentration of about 100 pM) and in vi vo intraperitoneal, intravenous or intratumoral.
  • bracket PNA comprises two identical PNA
  • the address protein or peptide is covalently bound to the C-terminus of a PNA molecule monomer.
  • the present invention also relates to a mixture of the staple-PNA conjugates described above and a nucleic acid sequence to be transported and hybridized to the staple PNA, which is preferably a double-stranded DNA (dsDNA), dsRNA or nucleic acid derivative (eg morpholinos), and a medicament containing this conjugate according to the invention, optionally together with a suitable carrier, and its use for local, non-invasive gene therapy, the nucleic acid being activated by dissociation of the staple PNA by local heating.
  • Administration is preferably carried out intraperitoneally, intravenously or intratumorally.
  • the mixture of clip-PNA conjugate according to the invention and nucleic acid sequence to be transported allows the activated nucleic acid sequence to be activated at the target site by local hyperthermia.
  • Local gene therapy is thus made possible, which is independent of the cell type, for example tumor type, ie the product encoded by the transported nucleic acid is only effective if it is activated locally.
  • suitable nucleic acid sequences or products encoded thereby are DNA which can be hybridized with the PNA with integrated VP22 HS v-sequence (Elliott and O'Hare, Gene
  • VP22 HSV facilitates the cell-cell transport of genetic material.
  • DNA sequences which encode HSV thymidine kinase (action in connection with GCV ganciclovir), cytosine deaminase (action in connection with FU fluorouracil) and the iodide symporter (action in connection with iodide).
  • the hybrid region is preferably selected so that the dissociation of the nucleic acid sequence to be activated from the staple PNA only occurs from temperatures above 43 ° C., ie the site of action and the desired time of action is achieved by local heating of the target area / target tissue.
  • the applied temperature should be such that cell death does not result solely from the temperature effect, which depends on both the temperature and the exposure time (Sapareto and Dewey, Int. J. Radiat. Oncol. Biol. Phys. 10 (1984), 787-800).
  • the target area is guided and the target area (e.g. tissue) is heated by absorption of the laser light.
  • the target area e.g. tissue
  • Microwaves There are various options. On the one hand, this can be accomplished by antennas outside the body, whereby a locally limited area can be heated by a certain focusing. However, complex target areas can only be heated homogeneously with difficulty become. On the other hand, microwaves can also be applied locally, for example by means of a microwave antenna pierced into the target area.
  • a non-invasive procedure is e.g. Magnetic resonance imaging (MRI), which allows a temperature resolution of approx. 2-5 ° C, alternatively methods with diagnostic ultrasound or computer tomography.
  • MRI Magnetic resonance imaging
  • the targeted non-invasive local tissue heating by focused ultrasound under MRI control is preferred.
  • the clip-PNA conjugates allow the transport of therapeutic genes (eg for defective gene substitution Genes).
  • the clip-PNA complexes according to the invention allow the transport of therapeutic genes (eg for defective gene substitution Genes).
  • the conjugate and the nucleic acid sequence to be transported are suitable, inter alia. for targeted tumor therapy, e.g. to destroy solid tumors, tumors resistant to chemotherapy, inoperable tumors, and e.g. also for
  • Hypercholesterolemia etc.
  • Other conceivable applications are in monitoring (e.g. MRI ("Magnetic Resonance Imaging")) and in diagnostics (e.g. for transferrin receptor genes (TfR), iodide
  • Figure 2 Representation of the introduction of the conjugate and the gene activation based on the GFP transcription via DIC / CLSM
  • Conjugate 100 pM; inactive control, CLSM.
  • the Fmoc strategy using a fully automated synthesizer (Syro II (Multisyntech, Witten, Germany) was used for the solid-phase synthesis.
  • the synthesis was carried out on a 0.05 mmol Fmoc-AS polystyrene resin (1% cross-linked).
  • the coupling reagent used was 2- (lH-benzotriazol-1-yl) -1, 1, 3, 3-
  • Tetramethyluronium hexafluorophosphate (HBTU) used.
  • the side chain protecting groups were Lys (Boc), Asp (Obut), Ser (But), Cys (Trt) and Asn (Trt).
  • the protected peptidyl resin was treated with 20% piperidine in dimethylformamide.
  • the cleavage and cleavage of the protective groups was achieved by treatment with 90% trifluoroacetic acid, 5% ethanedithiol, 2.5% thioanisole and 2.5% phenol (vol. / Vol. / Vol.) For 2.5 hours at room temperature. All products were precipitated in ether and by preparative HPLC (Shimazu LC-8A, Shimazu, Duisburg, Germany) on a YMC ODS-A 7A
  • Range of pDNA-EGFP-C3 (Clontech, Paolo Alto, CA, USA) can hybridize: Pos.542..ACG GTG GGA GGT CTA TAT
  • AAG..Pos.563 area of the expression of EGFP-controlling CMV promoter (total area: items 1-589;
  • Enhancer range pos. 59-465; TATA box: Item 554-560).
  • the linking was carried out according to the methods described above.
  • the clamp PNA module thus synthesized has the following structure:
  • the purification was carried out by reverse phase HPLC, followed by lyophilization. After determining the mass by means of MS-MS, the lyophilisate was dissolved in a defined volume of physiological saline to a stock solution of 10 ⁇ M.
  • DNA regions were hybridized in accordance with standard methods (Britten and Kohne, Science 1_61 (1968), 3841).
  • the conjugate was administered parenterally (i.v.) to various tumor cells (AT-1, rat prostate carcinoma cell line; DU-145, human prostate carcinoma cell line; HeLa cells).
  • the final drug concentration was in the range of 100 pM.
  • the activation was carried out by local heating of the tumor cells to 37-45 ° C. for 30, 60, 120 and 300 seconds using focused ultrasound waves 1, 2 or 3 hours after the entire conjugate had been applied. There was no local warming in a control experiment.
  • the location of the reporter gene and its release after local heating were determined by measuring the concentration of the fluorescent protein using a combination of DIC / CLSM (FIGS. 2 and 3) or a fluorescence measurement (FIG. 4).

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Abstract

L'invention concerne des conjugués APN pour le transport spécifique d'une séquence d'acide nucléique dans des compartiments cellulaires, de préférence des noyaux cellulaires, et l'activation thermique de la séquence d'acide nucléique en un emplacement cible recherché, ledit conjugué présentant les composants suivants : (a) un module peptidique de transport servant au transport de membranes cellulaires, (b) une protéine ou peptide d'adressage pour l'amenée dans des compartiments cellulaires, de préférence les noyaux cellulaires, et (c) un acide peptide-nucléique à pince (APN à pince) hybridisable spécifiquement avec la séquence d'acide nucléique à transporter.
PCT/DE2002/004356 2001-11-28 2002-11-27 Conjugue apn a pince pour le transport specifique d'acides nucleiques activables thermiquement Ceased WO2003047631A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002351703A AU2002351703A1 (en) 2001-11-28 2002-11-27 Clasp-pna-conjugate for the specific transport of nucleic acids which are thermally active

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10158331A DE10158331A1 (de) 2001-11-28 2001-11-28 Klammer-PNA-Konjugat zum spezifischen Transport thermisch aktivierbarer Nukleinsäuren
DE10158331.1 2001-11-28

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WO2003047631A2 true WO2003047631A2 (fr) 2003-06-12
WO2003047631A3 WO2003047631A3 (fr) 2004-01-29

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003006065A3 (fr) * 2001-07-12 2003-10-02 Deutsches Krebsforsch Conjugue pna pour traiter des affections associees au vih

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU9570898A (en) * 1997-09-18 1999-04-05 Gene Therapy Systems, Inc. Chemical modification of dna using peptide nucleic acid conjugates
DE19933492B4 (de) * 1999-07-16 2008-01-10 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Konjugat zur Vermittlung eines zell-, kompartiment- oder membranspezifischen Transports von Wirksubstanzen, Verfahren zu dessen Herstellung und dessen Verwendung
DE10133307A1 (de) * 2001-07-12 2003-02-06 Deutsches Krebsforsch PNA-Konjugat zur Therapie von mit HIV in Zusammenhang stehenden Erkrankungen

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003006065A3 (fr) * 2001-07-12 2003-10-02 Deutsches Krebsforsch Conjugue pna pour traiter des affections associees au vih
US7563761B2 (en) 2001-07-12 2009-07-21 Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts PNA conjugate for the treatment of diseases associated with HIV

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DE10158331A1 (de) 2003-06-12
WO2003047631A3 (fr) 2004-01-29
AU2002351703A8 (en) 2003-06-17
AU2002351703A1 (en) 2003-06-17

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