WO1997014440A1 - Conjugaison d'oligonucleotides anti-sens c-myc avec le cholesterol pour renforcer de maniere importante leur effet inhibiteur sur l'hyperplasie de la neo-intima - Google Patents

Conjugaison d'oligonucleotides anti-sens c-myc avec le cholesterol pour renforcer de maniere importante leur effet inhibiteur sur l'hyperplasie de la neo-intima Download PDF

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Publication number
WO1997014440A1
WO1997014440A1 PCT/US1996/016796 US9616796W WO9714440A1 WO 1997014440 A1 WO1997014440 A1 WO 1997014440A1 US 9616796 W US9616796 W US 9616796W WO 9714440 A1 WO9714440 A1 WO 9714440A1
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Prior art keywords
cholesterol
conjugated
antisense
odns
oligomers
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PCT/US1996/016796
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English (en)
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Angelica Fleser
Guy Leclerc
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Johnson & Johnson Interventional Systems
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Priority to AU75964/96A priority Critical patent/AU7596496A/en
Publication of WO1997014440A1 publication Critical patent/WO1997014440A1/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
    • C12N15/1135—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 against oncogenes or tumor suppressor genes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol

Definitions

  • Antisense oligodeoxynucleotides have recently been successfully used as "informational drugs" to prevent smooth muscle cell proliferation in vitro and neointimal hyperplasia after balloon angioplasty.
  • ODNs Antisense oligodeoxynucleotides
  • other investigators delivered oligonucleotides through the use of a pluronic gel surgically applied on the adventitial layer of the artery to permit sustained and prolonged DNA delivery to the angioplastied vessel. This approach is, however, inapplicable in human clinical angioplasty procedures.
  • SMC vascular smooth muscle cell
  • oligonucleotides have been covalently linked to a variety of hydrophobic groups, such as cholesterol. This lipidic conjugation of oligomers have proven efficient for in vitro inhibition of HIV multiplication. In vitro studies have also documented enhanced cellular uptake in neutrophils and HeLa cells. Furthermore, two to three fold improved oligomers nuclease resistance was associated with cholesterol conjugation in hepatoma cells.
  • the purpose of this invention evaluates, first, in vitro, the effect of conjugation of c-myc antisense phosphorothioate oligomers with cholesterol on ODNs pharmacokinetics properties and effect on smooth muscle cell proliferation. Then, in vivo, following direct arterial transfection, evaluate the long term outcome of transfected cholesterol-antisense oligomers and their effect on neointimal hyperplasia formation.
  • Figure 1 Sense, antisense and scrambled oligonucleotides conjugated with cholesterol moieties.
  • FIG. 3 Sucrose gradient distribution of fractions derived from in vitro transfected SMCs with 10 ⁇ M of either cholesterol-conjugated or unconjugated antisense oligonucleotides. Each experiment was repeated in triplicates producing similar results. Nucleotidase activity of each fraction is presented as a ratio of the highest value obtained within the sucrose gradient. For the comparison between 35 S-labeled cholesterol- conjugated and unconjugated ODNs association with gradient fractions, a S radioactivity quantified was standardized for a same amount of evaluated 5'-nucleotidase activity.
  • FIG. 5 Photomicrograph of histological section derived from rabbit carotid artery treated with 80 ⁇ M of fluoresceine-conjugated cholesterol antisense ODNs for 30 minutes in an isolated arterial segment ( 1 cm, 20 mg of tissue). "IEL” indicates internal elastic lamina and “M” indicates medial layer. Original magnification x 1000.
  • Figure 7 5 S-labeled oligonucleotides extraction from normal arterial segments exposed to oligomers (80 ⁇ M, 30 minutes). Each experiment was repeated in triplicata, producing similar results.
  • Phosphorothioate antisense ODNs 1 5 mer in length, complementary to c-myc (5'-CAC GTT GAG GGG CAT-3'), the corresponding sense sequence (5'-ATG CCC CAC AAC GTG-3') and scrambled sequence (5'- CAC TGT TAG GGG AAG-3') were synthesized on a 392 DNA/RNA synthesizer following standard procedure (Applied Biosystems) . This target was selected to inhibit smooth muscle cell proliferation in vitro.
  • oligomers were removed from the column with 30% NH ⁇ OH (1 hour at room temperature), and then deprotected for 8 hours at 60°C. Oligomers were purified and detritylated with oligonucleotide purification cartridges (Applied Biosystems), and then lyophilized with a centrifugal evaporator (Savant Speed Vac). Prior to transfection, oligomer concentration was assessed by spectrophotometry at 260 nm. Following ethidium bromide staining (0.3 ⁇ g/ ⁇ l), the comparison of oligomer migration with standard DNA, under UV light, showed only 15 mer ODNs in the final preparation to be transfected.
  • these oligomers were internally radiolabeled by replacing the standard sulfurizing step of base 3, 6, and 9 with a radiolabelling °°S solution (200 ⁇ l of TEDT/Acetonitrile + 5 ⁇ l of 35 S [0.373 mCi]).
  • ODN purification was performed as described above, with oligonucleotide purification cartridges.
  • 35 S radioactivity quantification was evaluated with a Beckman LS 8100 scintillation counter, and 35 S-ODN integrity was verified, as described above, by electrophoresis on a 20% oc urea-polyacrylamid gel.
  • radioactivity emission of S-ODN was trapped on a film (Kodak) for several days (in darkness at -80°C), and oligomer location was indicated by a dark band. Only 15 mer oligomers were detected on these radiographic films. Assessment of smooth muscle cell proliferation index in vitro
  • D-MEM medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, penicillin (100 U/ml) and streptomycine (100 U/ml). Cells from passage 3 to 5 were used in this study.
  • FBS fetal bovine serum
  • 2 mM glutamine penicillin (100 U/ml)
  • streptomycine 100 U/ml
  • Inhibitory effect of oligonucleotides on smooth muscle cell proliferation was assessed by thymidine incorporation index of untreated cells and also cells treated with 5 ⁇ M or 10 ⁇ M c-myc antisense (cholesterol-conjugated or not), and c-myc sense (cholesterol-conjugated or not).
  • Cells were plated at a density of 5000 cells/cm 2 , and allowed to recover 24 hours in D-MEM (Gibco-BRL) supplemented with 10% FBS.
  • Cells were synchronized to a same state of proliferation by supplementing the medium with 0.5 % FBS for 48 h. Increase of serum content to 10% FBS activated cell proliferation and oligonucleotides were added to the medium. After 1 2 hours of incubation, H thymidine was added (5 uCi/ml) and 12 hours later the medium was removed. Cells were washed with cold phosphate buffered saline, fixed for 10 minutes in ethanol:acetic acid (3: 1 ), washed with distilled water and incubated for 1 5 minutes in ice-cold 0.5N perchloric acid.
  • the reaction was stopped with successive addition of 39.5 ⁇ l of ZnSO 4 750 mM and 474 ⁇ l of Ba(OH) 2 100 mM (final concentration of 37.5 mM) at 4°C.
  • 5' nucleotidase activity was quantified on a spectrophotometer at 260 nm.
  • New Zealand rabbits male or female (2 Kg) were intramusculary sedated with xylazine (2 mg/Kg) and anesthetized with ketamine (100 mg/Kg) prior to surgical exposure of left carotid artery. Additional doses of anesthesia were given intramusculary throughout the experiment, as needed. Segments ( 10 mm, corresponding approximately to 20 mg of tissue) of carotids were transiently isolated by temporary ligatures and rinsed with 0.9% sodium chloride via a cannula until there was no more visible evidence of blood components.
  • the volume infused was 100 ⁇ l, and no visible loss of volume was noted throughout the incubation period .
  • the treated segments were rinsed with 0.9% sodium chloride (3 x 100 ⁇ l) and upon cannula removal, the arteriotomy site was repaired with microsutures (100 microns nylon monofilament, Pike Surgical Inc. Calgary, Canada), restoring normal blood flow.
  • Oligonucleotide physical integrity was assessed following oligomers extraction from the arterial segments at 72 hours and 168 hours post- transfection.
  • the transfected segments were removed surgically and oc crushed into fine powder following exposure to liquid nitrogen.
  • Oligos recovered in the aqueous phase were then reextracted with a 50:50 mixture of phenol and equilibrated chloroform:isoamyl (24:1 ), followed by an equal volume of chloroform:isoamyl.
  • the recovered 35 S-labeled ODN were desalted with oligonucleotide purification cartridges (Applied Biosystems), the cartridges being rinsed with 5 ml of acetonitrile (99%) of 5 ml of TEAA 2M prior to oligo application. Oligomers retained by the cartridges were rinsed with 10 ml of H2O and were eluted with 20% of acetonitrile, llyophilized and then solubilized in deionized water. Recovery of 3 ⁇ S-labeled oligos applied on the cartridge varied between 80% and 90%.
  • Oligomers physical integrity was verified by electrophoresis on a 20% urea-polyacrylamid gel, oligos migraton being compared to standard DNA following ethidium bromide coloration. Furthermore, visualization by autoradiography indicated that - li ⁇ the totality of 3 ⁇ S radioactivity recovered from the transfected arteries was coupled to the 15 mer oligos.
  • Oligos biodistribution in the vessel wall was evaluated by dot counting on histological sections derived from transfected arteries. Representative sections of carotid arteries were fixed in 4% paraformalin and embedded in paraffin, and 5- ⁇ m sections were cut. S radioactivity emission from the vascular cells was trapped by photographic emulsion (in darkness at 4°C, Kodak autoradiography emulsion). Following development (2.5 minutes in Kodak developer) and fixation (5 minutes in Kodak fixer), 35 S-labeled oligomers were visualized as black dots. Histological sections were stained with hematoxylin/eosin (for cells identification) and examined under light microscopy for oligomers transfection into the arterial wall.
  • Fluorescine-conjugated antisense ODNs (80 ⁇ M) were locally transfected into rabbit carotid arteries for 30 minutes and histological sections were derived from these carotid arteries. Representative sections were fixed in 4% paraformalin, embedded in paraffin.
  • the active molecule In order to efficiently prevent in vivo neointimal hyperplasia formation following balloon angioplasty, the active molecule must be efficaciously delivered to the vessel wall and preferably impose its presence to the injured area as long as possible during the proliferation window that occurs following the trauma. Accordingly, we examined in this series of experiments the level of ODNs successfully transfected in the target vessel wall, its tissue distribution, and vascular retention.
  • arterial neointimal hyperplasia was specifically reduced by 34.7% following transfection of unconjugated antisense c-myc ODNs, and by 84.7% following transfection of cholesterol-conjugated antisense ODNs (Fig. 12). Not only was the antiproliferative potential of antisense ODNs increased
  • Antisense oligonucleotide transfection has recently emerged as a potential therapeutic pathway to prevent neointimal proliferation component of restenosis.
  • Several studies have demonstrated that antisense directed against c-myc mRNA not only inhibits smooth muscle cell proliferation but also smooth muscle cell migration, which constitutes another critical component of smooth muscle cell response to vascular injury and, presumably, therefore of the restenosis process.
  • the development of antisense oligonucleotide therapy has not been as simple as first believed, and many critical issues have been highlighted. These include concerns about cellular uptake, sequence-specific and non- sequence-specific biological effects , pharmacokinetics and pharmacodynamics. Key elements of antisense strategy's success may rely on high transfection efficiency but also, importantly, on sufficient retention of this therapeutic molecule during the proliferation window of SMCs following angioplasty, which occurs mostly during the first week.
  • This approach permitted, potentially through advantageous pharmacokinetic properties of the conjugate, to significantly improve the efficiency to inhibit in vitro smooth muscle cells proliferation and in vivo neointimal hyperplasia formation.
  • 35 S-labeled phosphorothioates oligonucleotides were shown to accumulate in vesicular structures and in the nucleus but significantly less in the cytoplasm. Consistent with this invention, conjugated as well as unconjugated oligonucleotides entering in vivo SMCs migrate rapidly into the nucleus. It appears that cholesterol-conjugated ODNs are anchored to cellular membranes via cholesterol moieties, rendering oligomers available in the cytoplasmic and/or the nucleoplasm compartment.
  • oligonucleotides For many in vivo applications, oligonucleotides must be administered repeatedly in order to obtain the desired biological effects, partly because oligonucleotides are rapidly eliminated, and also because the target gene products have long half-lives. It appears reasonable to infer that if antisense therapy is to be effective in inhibiting the SMCs proliferation process, the antisense molecules should be actively available in the injured region for at least the duration of the proliferative window period. Thus, by improving oligomer phamacokinetics following 3'-end conjugation with cholesterol, the need for repeated oligomer delivery may be eliminated.

Abstract

Les oligonucléotides anti-sens ont été récemment proposés comme une voie pharmacologique pour prévenir l'hyperplasie de la néo-intima consécutive à l'angioplastie par ballonnet. La présente étude rapporte le renforcement important obtenu, pour la prévention de la resténose in vivo, par la conjugaison de cholesterol à un oligomère anti-sens c-myc par rapport à un oligonucleotide non conjugué. Le potentiel inhibiteur du conjugué a été multiplié par 2,4, ce qui a permis d'obtenir une réduction de 84,7 % de l'hyperplasie de l'intima par rapport aux artères témoins. Le renforcement de la prévention de l'hyperplasie peut être mis en relation avec la multiplication par 2,7 de la rétention vasculaire d'oligomères produite par les fragments de cholesterol, qui est suffisante pour couvrir une partie importante de la fenêtre de prolifération qui est caractéristique de ce qui suit l'angioplastie par ballonnet.
PCT/US1996/016796 1995-10-19 1996-10-18 Conjugaison d'oligonucleotides anti-sens c-myc avec le cholesterol pour renforcer de maniere importante leur effet inhibiteur sur l'hyperplasie de la neo-intima WO1997014440A1 (fr)

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US554495P 1995-10-19 1995-10-19
US60/005,544 1995-10-19
US1203796P 1996-02-21 1996-02-21
US60/012,037 1996-02-21

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998023299A2 (fr) * 1996-11-26 1998-06-04 Angiogene Inc. Oligonucleotide d'adn radiomarque, procede de preparation et utilisations therapeutiques associes
US7498315B2 (en) 2004-06-01 2009-03-03 Pronai Therapeutics, Inc. Methods and compositions for the inhibition of gene expression
US7635686B2 (en) 2001-10-03 2009-12-22 Bioniche Life Sciences, Inc. Therapeutically useful triethyleneglycol cholesteryl oligonucleotides
US8367628B2 (en) 2005-12-01 2013-02-05 Pronai Therapeutics, Inc. Amphoteric liposome formulation
US8785409B2 (en) 2007-01-30 2014-07-22 Geron Corporation Compounds having anti-adhesive effects on cancer cells
US8815599B2 (en) 2004-06-01 2014-08-26 Pronai Therapeutics, Inc. Methods and compositions for the inhibition of gene expression

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WO1994015646A1 (fr) * 1993-01-07 1994-07-21 Thomas Jefferson University Inhibition antisens du proto-oncogene c-myc permettant de moduler la proliferation des cellules des muscles lisses
WO1994023699A1 (fr) * 1993-04-19 1994-10-27 Medisorb Technologies International L.P. Traitement a action prolongee par apport a liberation lente d'oligodesoxyribonucleotides anti-sens a partir de microparticules biodegradables
WO1996024334A1 (fr) * 1995-02-10 1996-08-15 Nexstar Pharmaceuticals, Inc. Produits de synthese lipidique destines a la diffusion cytoplasmique d'agents

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Publication number Priority date Publication date Assignee Title
WO1994015646A1 (fr) * 1993-01-07 1994-07-21 Thomas Jefferson University Inhibition antisens du proto-oncogene c-myc permettant de moduler la proliferation des cellules des muscles lisses
WO1994023699A1 (fr) * 1993-04-19 1994-10-27 Medisorb Technologies International L.P. Traitement a action prolongee par apport a liberation lente d'oligodesoxyribonucleotides anti-sens a partir de microparticules biodegradables
WO1996024334A1 (fr) * 1995-02-10 1996-08-15 Nexstar Pharmaceuticals, Inc. Produits de synthese lipidique destines a la diffusion cytoplasmique d'agents

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BASIC RES. CARDIOL., 1992, Vol. 87, EBBECKE et al., "Antiproliferative Effects of a c-myc Antisense Oligonucleotide on Human Smooth Muscle Cells", pages 585-591. *
CIRCULATION REASEARCH, February 1995, Vol. 76, No. 2, EDELMAN et al., "c-myc in Vasculoproliferative Disease", pages 176-182. *
CIRCULATION, 15 October 1995, Vol. 92, No. 8, FLESER et al., "Conjugation of C-Myc Antisence DNA With Cholesterol Significantly Increases in Vivo Oligomer Vascular Retention", Abstract Number 1407, page I-296. *
CIRCULATION, September 1993, Vol. 88, No. 3, SHI et al., "Downregulation of c-myc Expression by Antisense Oligonucleotides Inhibits Proliferation of Human Smooth Muscle Cells", pages 1190-1195. *
FASEB JOURNAL, 1994, Vol. 8, No. 7, WICKSTROM et al., "Longterm Antisense DNA Administration in C-MYC Transgenic Mice Bearing C-HA-RAS Induced Tumors", Abstract Number 1102, page A1448. *
FEBS LETTERS, August 1989, Vol. 254, Number 1, 2, BOUTORIN et al., "Synthesis of Alkylating Oligonucleotide Derivatives Containing Cholesterol or Penazinium Residues at Their 3'-terminus and Their Interaction With DNA Within Mammalian Cells", pages 129-132. *
NUCLEIC ACIDS RESEARCH, 1992, Vol. 20, No. 3, OBERHAUSER et al., "Effective Incorperation of 2'-O-methyl-oligoribonucleotides Into Liposomes and Enhanced Cell Association Through Modification With Thiocholesterol", pages 533-538. *
PHARMACEUTICAL RESEARCH, 1995, Vol. 12, No. 4, STULL et al., "Antigene, Ribozyme and Aptamer Nucleic Acid Drugs: Progress and Prospects", pages 465-483. *
PROC. NATL. ACAD. SCI. USA, September 1989, Vol. 86, LETSINGER et al., "Cholesteryl-conjugated Oligonucleotides: Synthesis, Properties and Activity as Inhibitors of Replication of Human Immunodeficiency Virus in Cell Culture", pages 6553-6556. *
PROC. NATL. ACAD. SCI. USA., January 1993, Vol. 90, BIRO et al., "Inhibitory Effects of Antisense Oligonucleotides Targeting c-myc mRNA on Smooth Muscle Cell Proliferation and Migration", pages 654-658. *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998023299A2 (fr) * 1996-11-26 1998-06-04 Angiogene Inc. Oligonucleotide d'adn radiomarque, procede de preparation et utilisations therapeutiques associes
WO1998023299A3 (fr) * 1996-11-26 1998-12-17 Angiogene Canada Inc Oligonucleotide d'adn radiomarque, procede de preparation et utilisations therapeutiques associes
US7635686B2 (en) 2001-10-03 2009-12-22 Bioniche Life Sciences, Inc. Therapeutically useful triethyleneglycol cholesteryl oligonucleotides
US7498315B2 (en) 2004-06-01 2009-03-03 Pronai Therapeutics, Inc. Methods and compositions for the inhibition of gene expression
US8815599B2 (en) 2004-06-01 2014-08-26 Pronai Therapeutics, Inc. Methods and compositions for the inhibition of gene expression
US9393258B2 (en) 2004-06-01 2016-07-19 Pronai Therapeutics, Inc. Methods and compositions for the inhibition of gene expression
US8367628B2 (en) 2005-12-01 2013-02-05 Pronai Therapeutics, Inc. Amphoteric liposome formulation
US8785409B2 (en) 2007-01-30 2014-07-22 Geron Corporation Compounds having anti-adhesive effects on cancer cells
US9732114B2 (en) 2007-01-30 2017-08-15 Geron Corporation Compounds having anti-adhesive effects on cancer cells

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AU7596496A (en) 1997-05-07
CA2241835A1 (fr) 1997-04-24

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