WO2001021789A1 - Ribozymes pour la prevention de la restenose - Google Patents

Ribozymes pour la prevention de la restenose Download PDF

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Publication number
WO2001021789A1
WO2001021789A1 PCT/EP1999/007049 EP9907049W WO0121789A1 WO 2001021789 A1 WO2001021789 A1 WO 2001021789A1 EP 9907049 W EP9907049 W EP 9907049W WO 0121789 A1 WO0121789 A1 WO 0121789A1
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Prior art keywords
sequence
sequence seq
molecule according
seq
rna molecule
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PCT/EP1999/007049
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German (de)
English (en)
Inventor
Gabriele Grassi
Anne Christine Kuhn
Reinhard Kandolf
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Eberhard-Karls-Universität Tübingen
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Application filed by Eberhard-Karls-Universität Tübingen filed Critical Eberhard-Karls-Universität Tübingen
Priority to PCT/EP1999/007049 priority Critical patent/WO2001021789A1/fr
Priority to JP2001525347A priority patent/JP2003535573A/ja
Priority to CA002385228A priority patent/CA2385228A1/fr
Priority to EP99948829A priority patent/EP1214408A1/fr
Publication of WO2001021789A1 publication Critical patent/WO2001021789A1/fr

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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/121—Hammerhead

Definitions

  • the present invention is concerned with the therapy of arteriosclerosis, in particular with the prevention of restenosis of blood vessels after stenoses treated with angioplasty techniques.
  • Coronary artery disease and other arteriosclerosis diseases are the leading cause of morbidity and mortality in western industrialized countries.
  • every second patient suffers from cardiovascular disease as a result of arteriosclerotic vascular changes.
  • angioplasty techniques have become increasingly important in recent years, in which, for example, balloon dilation (PTCA) mechanically removes the constriction or occlusion of the vessel, i.e. the stenosis.
  • PTCA balloon dilation
  • stents are often implanted in the vessel in connection with this treatment in order to keep their lumens open, ie to prevent restenosis.
  • restenosis the incidence of which is 30-50%.
  • the main cause of this restenosis is an abnormal, transient growth stimulation of the vascular smooth muscle cells.
  • the resulting uncontrolled growth of vascular smooth muscle cells and their migration into the intimal space as well as reduced death processes then lead to stenosis due to the resulting wealth of intimal cells.
  • This intimate abundance of smooth muscle cells has become the accepted key finding in human restenosis tissue.
  • percutaneous, non-operative catheter techniques offer the option of selective delivery of protective substances into the infarct vessel with the aim of reducing the size of the infarct area or preventing aneurysm formation.
  • the present invention proposes the prevention of restenosis by inhibiting the proliferation of vascular smooth muscle cells, using therapeutic genes which induce cell cycle arrest and can be applied directly after balloon dilatation.
  • This therapeutic approach is based on the knowledge that the massive local increase in various growth factors after balloon dilation with the resulting synthesis of Gl-phase cyclins results in the re-entry of the smooth muscle cells into the cell cycle and thus their uncontrolled proliferation.
  • Cyclin E and E2F1 which are part of many regulatory pathways, including of a regulatory feedback mechanism in which cyclin E releases the transcription factor E2F1 in an active form through interaction with other products.
  • E2F1 in turn activates the transcription of genes whose products are essential for the S phase and stimulates both the transcription of cyclin E and that of its own gene. Combined inactivation of Cyclin E and E2F1 therefore enables particularly efficient induction of cell cycle arrest.
  • cyclin E and E2F1 are described in various publications, for example by Koff et al., Cell (1991), volume 66, 1217-1228, Geng et al., Oncogene (1996), volume 12, 1173-1180, Heiin et al., Cell (1992), vol. 70, 337-350, Ohtani et al., PNAS (1995), vol. 92, 12146-50.
  • Koff et al. Cell (1991), volume 66, 1217-1228, Geng et al., Oncogene (1996), volume 12, 1173-1180, Heiin et al., Cell (1992), vol. 70, 337-350, Ohtani et al., PNAS (1995), vol. 92, 12146-50.
  • the catalytic RNA molecules used in the context of the invention are so-called hammerhead ribozymes, as are described, for example, in the review by Symons: Small Catalytic RNAs, Annu Rev Biochem (1992), volume 61, 641-671. Their property of binding complementary RNA molecules via base pairing and destroying them through site-specific cleavage makes the hammerhead ribozymes suitable candidates for the production of therapeutic agents.
  • hammerhead ribozymes are constructed in such a way that they have two sequence sections, the so-called binding arms which are responsible for the specific binding to the target RNA, and a more or less conserved, catalytically active sequence section which is arranged between the two binding arms.
  • this catalytic sequence section there are two conserved areas at the 3 'or 5' end, a further sequence section being present between these two areas, which is at least partially double-stranded and can at least theoretically have any length and sequence.
  • the specificity of the hammerhead ribozymes is determined by the sequences of the binding arms which bind to the RNA molecule to be cut in the region of the target triplet.
  • the determination of binding arms for ribozymes directed against target RNA thus requires on the one hand the identification of single-stranded regions in the target RNA molecule and the selection of suitable interface triplets with subsequent determination of the sequence of the binding arms.
  • RNA molecules such as, for example, the mRNA for cyclin E and E2F1
  • sequences of the binding arms cannot simply be determined on the basis of the possibly known sequence of the RNA molecule, since its secondary structure and those freely accessible to the ribozymes single-stranded areas cannot easily be derived from the sequence.
  • the inventors of the present application have recognized that this method for determining suitable interfaces for mRNA molecules for cyclin E and E2F1 cannot be used successfully.
  • the inventors of the present application have therefore used the RNase H technique described in more detail in the exemplary embodiments in order to determine favorable binding sites for hammerhead ribozymes. After the possible points of attack had been determined experimentally, possible binding arm sequences had to be derived therefrom and the corresponding ribozymes had to be produced. It has been found that the length of the Low binding is crucial for the effectiveness of the catalytic activity of the ribozymes, so that efficient ribozymes could only be determined experimentally.
  • sequences listed in the enclosed sequence listing SEQ ID No. 1 and SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10 as well as SEQ ID No. 11 and SEQ ID No. 12 represent binding arm pairs for enzymes against cyclin E mRNA
  • sequences SEQ ID No. 13 and SEQ ID No. 14, SEQ ID No. 15 and SEQ ID No. 16 as well as SEQ ID No. 17 and SEQ ID No. 18 represent binding arm pairs for ribozymes against E2F1 RNA. Ribozymes with these binding arms have a particularly good catalytic activity against the mRNA molecules mentioned, as the inventors of this application have been able to demonstrate experimentally.
  • the binding arms can also have sequences which bind to sequences which bind to the sequences mentioned in each case.
  • the catalytically active third sequence section has the sequence SEQ ID No. 19 at its 3 * end and the sequence SEQ ID No. 20 at its 5 'end, this third sequence section preferably having the sequence SEQ ID No. 21 includes.
  • sequences of ribozymes tested against Cyclin E are contained in the attached sequence listing as SEQ ID No. 22 to SEQ ID No. 27 and against E2F1 as SEQ ID No. 28 - SEQ ID No. 30.
  • the kinetic constants for these ribozymes are given in Table 1 given in the description of the figures, which are in the range of the best constants described for hammerhead ribozymes.
  • DNA molecules for this which comprise a sequence section which codes for the above-mentioned catalytic RNA molecules.
  • This DNA sequence section can be part of a vector plasmid or can be used to generate a vector, preferably an adenoviral vector for gene therapy use.
  • the introduction of the foreign DNA by means of recombinant adenoviruses has the advantage over other methods that adenoviruses infect target cells with high efficiency and there is no risk of insertion mutagenesis.
  • the therapeutic effect can thus be achieved either via selective transfer systems or via cell-specific gene expression, a selective and efficient expression being achieved by tissue-specific expression of the therapeutic genes by a promoter which is only expressed in smooth muscle cells and shows a high expression rate.
  • the present invention also relates to a therapeutic composition with the new catalytic RNA molecule or the new DNA molecule or the new vector plasmid.
  • the present invention further relates to a kit with the new DNA molecule which has at least one sequence section coding for the new RNA molecule.
  • the present invention relates to the use of the new, catalytically active RNA molecule, the new DNA molecule and / or the new vector plasmid for the prevention of restenosis by inhibiting the proliferation of vascular smooth muscle cells.
  • Figure 1 shows the schematic structure of hammerhead ribozymes; and 2 shows a table with the measured kinetic constants of the hammerhead ribozymes tested.
  • FIG. 1 shows a ribozyme hybridized with a substrate (target RNA), two double-stranded regions being formed between the substrate and the ribozyme, between which the cleavage site indicated by an arrow can be found on the substrate side.
  • a substrate target RNA
  • binding arm On the ribozyme side, between the two sequence sections I and II called “binding arm”, there is the catalytic area III, which comprises conserved areas and variable areas, the conserved areas being highlighted by boxes.
  • the target triplet NUH is also highlighted by a box on the substrate side. It should be noted that N denotes one of the four nucleotides, H one of the nucleotides A, U and C, Y a pyrimidine and R a purine.
  • the catalytic sequence section of the ribozyme contains the sequence SEQ ID No. 19 at its 3 'end and the sequence SEQ ID No. 20 from the attached sequence listing at its 5 * end.
  • An example of the catalytic sequence section III is shown in SEQ ID No. 21 from the attached sequence listing, where also the double-stranded region and the loop region from the third sequence section III are given by way of example with specific nucleotides.
  • the specificity for the target mRNA in the ribozyme from FIG. 1 results from the special sequence of the binding arms I and II, which must be selected such that they flank a target triplet NUH on the substrate, as indicated.
  • cleavage sites of cyclin E and E2F1 accessible to hammerhead ribozymes cannot be determined directly on the basis of the known sequences of the mRNAs for cyclin E (Koff et al., Loc. Cit., Geng et al., Loc. Cit.) And E2F1 (Heiin et al., Loc. Cit.) or using mathematical folding models of these mRNAs, so that experimental methods had to be used.
  • RNA fragments Based on the ability of RNase H to cleave such RNA / DNA hybrids, depending on the RNase H cleavage sites, a mixture of RNA fragments is obtained, the lengths of which reflect the positions of the open regions that are in principle accessible to ribozymes.
  • the length of the RNase H fragments was then determined electrophoretically, it being possible to confirm possible ribozyme cleavage sites by using sequence-specific oligodeoxynucleotides.
  • RNase H mapping is carried out using class III and class I transcripts, which differ from one another only in the absence of two exons in the class I transcript (exon I and II).
  • RNAs of different lengths were also used for E2F1 transcripts, although the shortened transcript has no physiological significance.
  • RNA regions experimentally determined for Cyclin E and E2F1 were compared with various computer-aided folding analyzes, whereby it was found that optimally matched predicted structures could not be found for experimentally mapped cleavage sites. Knowing the target triplet NUH mentioned in Example 1, different sequences for the binding arms I and II were then determined for the open RNA regions, with which hammerhead ribozymes were then produced and tested, as described in Example 3 below.
  • the experimentally confirmed binding arm pairs I and II are in the attached sequence listing as sequence SEQ ID No. 1 and 2, SEQ ID No. 3 and 4, SEQ ID No. 5 and 6, SEQ ID No. 7 and 8, SEQ ID No. 8 and 9, SEQ ID No. 9 and 10, SEQ ID No. 11 and 12, SEQ ID No. 13 and 14, SEQ ID No. 15 and 16 and SEQ ID No. 17 and 18, with the sequence the odd SEQ ID denotes the binding arm at the 5 'end and that with the even SEQ ID denotes the binding arm at the 3' end of the ribozyme.
  • DNA molecules coding for the catalytic RNA molecules from Examples 2 and 3 are cloned into an adenoviral vector, the recombinant adenoviruses being rendered replication-incompetent by mutation or deletion.

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Abstract

Molécule d'ARN à action catalytique, qui est dirigée contre des molécules d'ARNm codant pour les protéines cycline E ou E2F1 pertinentes pour le cycle cellulaire. Ladite molécule d'ARN à action catalytique est un ribozyme en tête de marteau qui induit l'inhibition du cycle cellulaire de cellules vasculaires de muscles lisses pour prévenir la resténose.
PCT/EP1999/007049 1999-09-22 1999-09-22 Ribozymes pour la prevention de la restenose WO2001021789A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
PCT/EP1999/007049 WO2001021789A1 (fr) 1999-09-22 1999-09-22 Ribozymes pour la prevention de la restenose
JP2001525347A JP2003535573A (ja) 1999-09-22 1999-09-22 再狭窄の予防用リボザイム
CA002385228A CA2385228A1 (fr) 1999-09-22 1999-09-22 Ribozymes pour la prevention de la restenose
EP99948829A EP1214408A1 (fr) 1999-09-22 1999-09-22 Ribozymes pour la prevention de la restenose

Applications Claiming Priority (1)

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PCT/EP1999/007049 WO2001021789A1 (fr) 1999-09-22 1999-09-22 Ribozymes pour la prevention de la restenose

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997010334A2 (fr) * 1995-09-12 1997-03-20 Immusol, Inc. Therapie ribozymiale pour le traitement et/ou la prevention de la restenose

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997010334A2 (fr) * 1995-09-12 1997-03-20 Immusol, Inc. Therapie ribozymiale pour le traitement et/ou la prevention de la restenose

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
GRASSI G ET AL: "E2F1 and cyclin E directed ribozymes as a tool to inhibit smooth muscle cell proliferation after percutaneous transluminal angioplasty.", EUROPEAN HEART JOURNAL, vol. 20, August 1999 (1999-08-01), pages 367, XP000907169, ISSN: 0195-668X *
GRASSI, G. ET AL.: "Growth inhibition of smooth muscle cells from human coronary plaque tissues by hammerhead ribozymes", PATHOLOGY RESEARCH AND PRACTICE., vol. 194, 1998, pages 267, XP000910797, ISSN: 0344-0338 *
GRASSI, M. ET AL: "Modeling of the in vitro hammerhead ribozymes reaction kinetics", PROC. 26TH INT. SYMP. CONTROLLED RELEASE BIOACT. MATER., July 1999 (1999-07-01), pages 62 - 63, XP002138280 *
OHTANI K. ET AL.: "Regulation of the cyclin E gene by transcription factor E2F1.", PROC NATL ACAD SCI U S A 92(26):12146-50, December 1995 (1995-12-01), XP002138279 *
WEI GL. ET AL.: "Temporally and spatially coordinated expression of cell cycle regulatory factors after angioplasty.", CIRC RES 1997 MAR;80(3):418-26, XP000907020 *
XXIST CONGRESS OF THE EUROPEAN SOCIETY OF CARDIOLOGY;BARCELONA, SPAIN; AUGUST 28-SEPTEMBER 1, 1999 *

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JP2003535573A (ja) 2003-12-02
CA2385228A1 (fr) 2001-03-29
EP1214408A1 (fr) 2002-06-19

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