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WO1996011266A2 - Procede d'inhibition de la proliferation des cellules musculaires lisses et oligonucleotides utilises dans celui-ci - Google Patents

Procede d'inhibition de la proliferation des cellules musculaires lisses et oligonucleotides utilises dans celui-ci Download PDF

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Publication number
WO1996011266A2
WO1996011266A2 PCT/US1995/012770 US9512770W WO9611266A2 WO 1996011266 A2 WO1996011266 A2 WO 1996011266A2 US 9512770 W US9512770 W US 9512770W WO 9611266 A2 WO9611266 A2 WO 9611266A2
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Prior art keywords
oligonucleotide
oligonucleotides
modified
smooth muscle
guanine residues
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PCT/US1995/012770
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English (en)
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WO1996011266A3 (fr
Inventor
Teresa L. Burgess
Catherine L. Farrell
Eric F. Fisher
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Amgen Inc.
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Priority to AU38895/95A priority Critical patent/AU3889595A/en
Publication of WO1996011266A2 publication Critical patent/WO1996011266A2/fr
Publication of WO1996011266A3 publication Critical patent/WO1996011266A3/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
    • C12N2310/33—Chemical structure of the base

Definitions

  • This invention relates to the field of therapeutics, and in particular the field of nucleic acid therapeutics.
  • restenosis involves the re-occlusion of blood vessels that have been at least partially cleared of occlusion and is, ironically, the result of the process employed to clear the occluded vessel(s) in the first place.
  • restenosis frequently occurs following balloon angioplasty or other catheter-based medical intervention (e. g. , close to 50% occurrence), but can also occur as the result of, e . g. , coronary by-pass surgery or any similar type of insult or injury to a blood vessel.
  • the pathological process of restenosis is a hyperproliferative response that includes both smooth muscle cell proliferation and extracellular matrix accumulation. It is a widely held belief that the reclosure of an artery subjected to angioplasty is due primarily to a process akin to wound healing.
  • activated smooth muscle cells play a major role in the reclosure/injury response involved in restenosis, and that smooth muscle cell proliferation is a key element in the process. Activated smooth muscle cells appear to proliferate and then migrate into the arterial lumen. causing restenosis. Thus, inhibition of smooth muscle cell proliferation is believed to be a key element in the prevention of vascular restenosis following angioplasty or other insult to a blood vessel, and efforts toward preventing restenosis, or at least ameliorating the condition, have focused on methods for inhibiting smooth muscle cell proliferation.
  • PDGF platelet-derived growth factor
  • a monomeric form of PDGF is provided as a therapeutic compound in excess over the naturally occurring dimeric molecule.
  • the therapeutic compound In order to be effect against restenosis, the therapeutic compound must successfully compete with dimeric PDGF for binding sites (PDGF receptors) on smooth muscle cells that are necessary for cell growth to be stimulated by PDGF.
  • PDGF receptors binding sites
  • antisense interactions involve hybridization of complementary oligonucleotides (hence, the term "antisense”) to their selected nucleic acid target (e . g. , viral RNA or other undesired genetic messages) in a sequence specific manner such that the complex thus formed, either alone or in combination with other agent(s) (e . g. , enzymes, such as RNAse) can no longer function as a template for the translation of genetic information into proteins.
  • agent(s) e . g. , enzymes, such as RNAse
  • An antisense oligonucleotide may be designed to interfere, for example, with the expression of foreign genes (e . g.
  • viral genes such as HIV
  • endogenous genes e.g., a normal gene that is aberrantly expressed as a mutated oncogene
  • endogenous genes e.g., a normal gene that is aberrantly expressed as a mutated oncogene
  • endogenous genes e.g., a normal gene that is aberrantly expressed as a mutated oncogene
  • endogenous genes whose response to specific stimuli may be undesired in certain instances, such as in the case of restenosis where smooth muscle cell proliferation is undesired at the site of angioplasty or by-pass surgery.
  • antisense-based nucleic acid therapeutics raise the possibility of therapeutic arrest of restenosis at the early replication and expression stage, rather than attacking the resulting protein at a later stage of smooth muscle cell proliferation (as in the case of therapeutic products requiring direct interaction with the protein)
  • antisense oligonucleotides have been actively pursued as the premier therapeutic compounds for use in the prevention or amelioration of restenosis.
  • previous studies have reportedly demonstrated inhibition of restenosis in in vitro and in vivo models following the application of antisense oligonucleotides targeting the c-myb and c-myc genes, among others, which are believed to be involved in the proliferation of smooth muscle cells. See, e . g. , International Application No.
  • nucleic acid therapeutic compounds have sequences that may or may not be complementary to a target sequence, but can, nevertheless, prevent an undesired result by interfering with either: (1) the expression (e. g. , replication and/or translation) of the undesired genetic material; or (2) the required interaction(s) of proteins involved in the cellular pathway.
  • the use of oligonucleotides having multiple contiguous guanine residues has been suggested for use in inhibiting the activity of viruses, in addition to inhibiting the activity of phospholipase A2 and modulating the telomere length of chromosomes. See, International Patent Application No.
  • PCT/US93/09297 wherein: (1) at least one series of four contiguous guanine residues or two series of three contiguous guanine residues were reported to inhibit herpes simplex virus (HSV) or phospholipase A2 activity; and (2) at least one series of four contiguous guanine residues was reported to inhibit cytomegalovirus, influenza virus or human immunodeficiency virus (HIV) .
  • HSV herpes simplex virus
  • HAV phospholipase A2 activity
  • guanine-rich oligonucleotides are not expected to be effective against restenosis other than in the case of a coincidental appearance of a region of contiguous guanine residues as a natural consequence of composition of the target sequence.
  • nucleic acid therapeutic compound that is capable of inhibiting restenosis.
  • oligonucleotides that are able to potently inhibit smooth muscle cell proliferation.
  • the present invention provides multi-G oligonucleotides that are capable of inhibiting smooth muscle cell proliferation.
  • Pharmaceutical compositions containing the multi-G oligonucleotides of the present invention are also provided. These pharmaceutical compositions are useful in the method of the present invention, wherein multi-G oligonucleotides are used as nucleic acid therapeutic compounds to inhibit smooth muscle cell proliferation in arteries ex vivo, and more particularly against restenosis.
  • the present invention further provides a method for screening oligonucleotides for their ability to inhibit smooth muscle cell proliferation.
  • Figure 1 is a table of modified oligonucleotide sequences used in the examples.
  • Figure 2A is a graph showing the percent suppression of rabbit smooth muscle cell proliferation by various oligonucleotide sequences at a concentration of 10 ⁇ M.
  • Figure 2B is a graph showing the percent suppression of rabbit smooth muscle cell proliferation by various oligonucleotide sequences at a concentration of 30 ⁇ M.
  • Figure 2C is a graph showing the percent suppression of rabbit smooth muscle cell proliferation by various oligonucleotide sequences at a concentration of 60 ⁇ M.
  • Figure 2D is a graph showing the percent suppression of pig smooth muscle cell proliferation by various oligonucleotide sequences at a concentration of 15 ⁇ M.
  • Figure 3 is a graph showing the percent suppression of rabbit smooth muscle cells by multi-G modified oligonucleotides (versus control) at a concentration of 15 ⁇ M.
  • Figure 4 is a graph showing the percent suppression of rabbit smooth muscle cells by inosine- substituted modified oligonucleotides at a concentration of 30 ⁇ M.
  • Figure 5 is a graph showing the percent suppression of BrdU incorporation into rabbit smooth muscle cells by modified multi-G oligonucleotides vs . control at a concentration of 30 ⁇ M.
  • Figure 6 is a graph showing the growth response of rabbit smooth muscle cells following removal of modified multi-G oligonucleotides vs . control at a concentration of 30 ⁇ M.
  • Figures 7A, 7B and 7C are graphs showing BrdU incorporation by smooth muscle cells in rabbit ear arteries as determined by measuring the number of BrdU- labeled nuclei per mm2.
  • the present invention provides multi-G oligonucleotides that are capable of inhibiting smooth muscle cell proliferation.
  • the multi-G oligonucleotides of the present invention are comparable in length to true antisense oligonucleotides, but have multiple contiguous guanine residues at one or more locations in the oligonucleotide.
  • These multi-G oligonucleotides can be used as nucleic acid therapeutic compounds to inhibit smooth muscle cell proliferation in vivo, and more particularly to inhibit vascular restenosis following injury or insult to a blood vessel.
  • a method for screening oligonucleotides for their ability to inhibit smooth muscle cell proliferation in vivo is also provided.
  • Oligonucleotide refers to a polymer of at least two nucleoside units, wherein each of the individual nucleoside units is covalently linked to at least one other nucleoside unit through a single phosphorus moiety. In the case of naturally occurring oligonucleotides, the covalent linkage between nucleoside units is a phosphodiester bond.
  • oligonucleotide includes oligonucleotides that are modified (as compared to naturally occurring oligonucleotides) with respect to any one or more of the following: (1) the phosphodiester bond between nucleoside units; (2) the individual nucleoside units themselves; and/or (3) the ribose, or sugar, moiety of the nucleoside units.
  • base refers to a purine or pyrimidine, such as adenine, guanine, cytosine, thymine, inosine and uracil as well as modified forms of these bases, such as 5-methylcytosine and 5-propynyl pyrimidines.
  • residue refers to a particular base or nucleobase as well as the sugar moiety to which it is attached and the phosphate moiety (or corresponding moiety in the case of a modified oligonucleotide) as it exists in an oligonucleotide.
  • contiguous means next to or adjacent.
  • contiguous nucleic acid residues refers to nucleic acid residues that are adjacent each other in the oligonucleotide sequence.
  • multi-G oligonucleotide refers to an oligonucleotide having at least four contiguous guanine residues or at least ' two series of at least three contiguous guanine residues.
  • poly-G oligonucleotide refers to an oligonucleotide having only guanine residues.
  • Nucleoside refers to an individual monomeric nucleoside unit consisting of a base covalently bonded to the 1'-position of a 5-carbon sugar.
  • the 5-carbon sugar will typically be a naturally occurring sugar such as deoxyribose, ribose or arabinose, but can be any
  • 5-carbon sugar or modified form thereof including but not limited to, 2'-fluoro-2'-deoxyribose or even carbocyclic sugars (where a carbon function is substituted for the oxygen atom in the sugar ring; i . e. , 6-carbon analog) or thiol sugars (where a sulfur atom is substituted for the oxygen atom in the sugar ring) .
  • the base will be linked to the sugar moiety at conventional positions, such as N9 of adenine, guanine and other purines or Nl of cytosine, thymine, uracil and other pyrimidines.
  • Nucleotide refers to a monomeric nucleoside unit further having a phosphorus moiety covalently bonded to the sugar moiety of the nucleoside at either the 3 1 - or 5*-position of the sugar.
  • modified internucleotide linkage refers to any modification of the phosphodiester bond joining individual nucleoside units in naturally occurring oligonucleotides that improves activity of the oligonucleotide, such as by imparting nuclease resistance to the internucleotide linkage.
  • modified oligonucleotide specifically refers to an oligonucleotide having at least one modified internucleotide linkage.
  • partially modified oligonucleotide means a modified oligonucleotide wherein at least one but fewer than all internucleotide linkages are modified.
  • oligonucleotide means a modified oligonucleotide wherein all of the internucleotide linkages are modified.
  • Target sequence refers to the nucleotide sequence to which an oligonucleotide or a modified oligonucleotide is designed to hybridize.
  • the "target sequence” may be, but is not necessarily limited to, a naturally occurring messenger RNA coding for a viral protein, cancer related protein or other proteins involved in disease states or other undesired conditions.
  • the multi-G oligonucleotides of the present invention strongly inhibit the proliferation of primary smooth muscle cells both in cell culture in vitro and in arteries ex vivo .
  • the anti-proliferative effect of the multi-G oligonucleotides of the present invention is sequence specific, it does not operate according to a true antisense mechanism.
  • the multi-G oligonucleotides of the present invention act as inhibitors of smooth muscle cell proliferation by interacting directly with one or more of the proteins involved in the proliferative process. This is in marked contrast to the interaction at a genetic level (i . e .
  • oligonucleotide/oligonucleotide that is required in the case of true antisense oligonucleotides and has been alluded to even in the instance of antiproliferative effects that have been observed in the case of (antisense) oligonucleotides that coincidentally contain multiple contiguous guanine residues. See, Yaswen et al . , Antisense Res . and Dev. , 3, 67-77 (1993) .
  • the multi-G oligonucleotides of the present invention appear to inhibit smooth muscle cell proliferation by blocking cells from re-entering the cell cycle after stimulation with growth factors such as by the addition of serum. Furthermore, it is clear that, at least at lower concentrations, the inhibitory effect of the multi-G oligonucleotides of the present invention against smooth muscle cell proliferation is not due to non-specific toxicity. Indeed, smooth muscle cells in culture look healthy and appear to undergo a normal proliferative response if the multi-G oligonucleotide is removed and fresh media is added.
  • the multi-G oligonucleotides of the present invention are designed to have at least one series of at least four contiguous guanine residues or at least two series of at least three contiguous guanine residues.
  • the multi-G oligonucleotides of the present invention have at least two series of at least four contiguous guanine residues, it being understood that the two series of contiguous guanine residues may occur adjacent to each other in the oligonucleotide such that, in the preferred case of at least two series of a least four contiguous guanine residues, a series of at least eight contiguous guanine residues results.
  • the multi-G oligonucleotides of the present invention have at least two series of at least six contiguous guanine residues, it again being understood that these two series of contiguous guanine residues may be placed adjacent each other in the oligonucleotide, yielding a series of at least twelve contiguous guanine residues.
  • the multi-G oligonucleotides of the present invention have at least about twelve contiguous guanine residues.
  • the multi-G oligonucleotides of the present invention have at least one base other than a guanine on each of the 3'- and 5'- ends of the oligonucleotide.
  • the lengths of the multi-G oligonucleotides of the present invention may vary to some degree, but will generally be from about 10 to about 30 nucleotides in length. It is preferred that the multi-G oligonucleotide be from about 14 to about 20 bases long, as longer oligonucleotides within the 10 to 30 nucleotide range may hybridize nonspecifically to other non-target sequences if the oligonucleotide is too long (i . e . , substantially longer than 20 nucleotides). It is more preferred that the multi-G oligonucleotide be an
  • an oligonucleotide length of about 12 to about 25 bases represents an oligonucleotide length that is consistent with other oligonucleotides designed for nucleic acid therapeutics and is a manageable length from a practical standpoint. It is understood, however, that shorter oligonucleotides are more commercially feasible. Thus, it will almost always be preferred to employ the shortest oligonucleotide exhibiting the maximum efficacy.
  • poly-G oligonucleotides are less preferred than other multi-G oligonucleotides according to the present invention, because of handling difficulties inherent with these oligonucleotides.
  • poly-G oligonucleotides exhibit a tendency toward self-aggregation, likely due to the formation of tetrads, wherein four-stranded structures are formed as a result of intra-oligonucleotide hydrogen bonding of guanine residues.
  • tetramer formation has been suggested as the key phenomenon involved in the observed inhibitory action of multi-G oligonucleotides against the HIV virus (Wyatt et al, Proc. Natl . Acad. Sci . USA, 91, 1356-1360 (1994)), these tetrameric structures are not believed to be necessary for inhibition of smooth muscle cell proliferation according to the present invention.
  • the multi-G oligonucleotides of the present invention be modified oligonucleotides in order to enhance activity of the oligonucleotide.
  • specific activity of the multi-G oligonucleotide will be increased over time where the oligonucleotide has been modified to resist the various nucleases that are endogenous to a human or animal body.
  • the multi-G oligonucleotides of the present invention be modified by having sufficient phosphorothioate, phosphorodithioate or 3'-carbon modified linkages to impart nuclease resistance.
  • phosphorothioate and phosphorodithioate linkages are more preferred, it is important to note that sulfur-containing oligonucleotides such as these are known to bind to proteins, resulting in a level of non-specific activity, that may have an undesired non-specific level of inhibition at higher concentrations of these oligonucleotides.
  • the multi-G oligonucleotides of the present invention can be made according to any one of a number of methods routinely known in the art. Generally, oligonucleotides are synthesized using automated synthetic protocols requiring large pieces of equipment such as the DNA synthesizers sold by Applied Biosystems
  • a method for screening oligonucleotides for potential therapeutic use against smooth muscle cell proliferation is also provided in accordance with the present invention.
  • the screening method of the present invention simulates the occurrence of restenosis in vitro and initially requires the arrest of smooth muscle cells in Go growth phase. After the smooth muscle cells have been arrested, proliferation of the smooth muscle cells is initiated by serum addition in the presence of multi-G oligonucleotides.
  • Multi-G oligonucleotides demonstrating greater than 50% inhibition at a concentration of less than or equal to 10 ⁇ m are believed to be effective for use against smooth muscle cell proliferation in arteries ex vivo, with oligonucleotides demonstrating greater than 50% inhibition at or below 2 ⁇ m concentrations being particularly effective.
  • smooth muscle cells it is preferred to attach the smooth muscle cells to a solid support in conducting the screening method. It is also preferred to arrest the smooth muscle cells in Go growth phase by placing the smooth muscle cells in a starvation media after which proliferation of said smooth muscle cells is initiated by replacing said starvation media with normal growth media containing serum.
  • the present invention further contemplates a method for inhibiting smooth muscle cell proliferation comprising the administration of multi-G oligonucleotides to a human or animal subject.
  • Preferred multi-G oligonucleotides for delivery to such human or animal subject include multi-G oligonucleotides having four contiguous guanine residues and multi-G oligonucleotides having two series of three contiguous guanine residues. In the latter case, it is preferred that the two series of three contiguous guanine residues be separated by a single residue other than a guanine residue.
  • Other preferred multi-G oligonucleotides for use in the method of the present invention include the preferred multi-G oligonucleotides previously described.
  • the multi-G oligonucleotides of the present invention are preferably delivered locally to a site of insult or injury to a blood vessel that is, by virtue of such injury or insult, at risk of restenosis.
  • Localized delivery of the multi-G oligonucleotides can be accomplished via direct injection at the desired site or can be delivered using an infusion pump.
  • the multi-G oligonucleotides will be combined with a pharmaceutically acceptable carrier and delivered by way of a catheter, stent, or other implantable device. In this way, the delivery of the multi-G oligonucleotides can be incorporated into a device used during the particular medical intervention procedure (e . g. , angioplasty) which necessarily renders a blood vessel site at risk of restenosis.
  • One preferred type of pharmaceutically acceptable carrier is a polymeric material, such as a hydrogel, that does not cause an adverse or undesired reaction such as inflammation.
  • a polymeric material such as a hydrogel
  • Many such materials are known, including those made from both natural and synthetic polymers.
  • Many of these polymeric materials are available commercially and have varying properties such as different melting temperatures and biodegradeability profiles, such that a particular pharmaceutically acceptable carrier can be selected to accommodate the specific type of method selected to deliver the multi-G oligonucleotide of the present invention.
  • the multi-G oligonucleotides are admixed into the selected polymeric material in a liquid state. The resulting mixture is applied onto the surface to be treated, e. g. , by spraying or painting during surgery or using a catheter or endoscopic procedures.
  • lipids, liposomes, microcapsules, implantable devices and cell surface-directed delivery vehicles including those which operate by membrane insertion.
  • cell surface-directed device which operates by way of membrane insertion includes a long chain alkyl function (linker) which may be covalently linked to a selected multi-G oligonucleotide in either a bio- releasable or non-releasable form.
  • linker arm of the resulting conjugate embeds itself in the membrane of living cells, thereby situating the multi-G oligonucleotide in close proximity to the targeted cell.
  • Multi-G oligonucleotides according to the present invention can also be incorporated into implants made of biodegradable materials such as polyanhydrides, polyorthoesters, polylactic acid and polyglycolic acid and copolymers thereof, collagen, and protein polymers, or non-biodegradable materials such as ethylenevinyl acetate, polyvinyl acetate, ethylene vinyl alcohol, and derivatives thereof as the implant material is polymerized or solidified or mechanically mixed with the material.
  • biodegradable materials such as polyanhydrides, polyorthoesters, polylactic acid and polyglycolic acid and copolymers thereof, collagen, and protein polymers, or non-biodegradable materials such as ethylenevinyl acetate, polyvinyl acetate, ethylene vinyl alcohol, and derivatives thereof as the implant material is polymerized or solidified or mechanically mixed with the material.
  • the oligonucleotides are mixed into or applied onto coatings (which may include, e.g., cell-surface directed delivery vehicles or liposomes) for implantable devices such as dextran-coated silica beads, stents, or catheters.
  • coatings which may include, e.g., cell-surface directed delivery vehicles or liposomes
  • implantable devices such as dextran-coated silica beads, stents, or catheters.
  • oligonucleotides used in the following examples were synthesized using an Applied Biosystems Inc. (Foster City, California) Model 394 DNA synthesizer. Phosphorothioate modified linkages were introduced by oxidizing the phosphite linkage formed during oligonucleotides synthesis with 3H-1,2- benzodithiole-3-1,1-dioxide (Beaucage reagent. Glen Research, Sterling Virginia)- instead of the standard iodine oxidation.
  • the four common nucleoside phosphoramidites i.e., adenine, thymine, guanine and cytosine
  • deoxyinosine phosphoramidite purchased from Applied Biosystems Inc. All unmodified phosphodiester and modified thioate-containing oligonucleotides were deprotected by treatment with concentrated ammonia at 55°C for 12 hours. The oligonucleotides were purified by gel exclusion chromatography and ethanol precipitation and lyophilized to dryness.
  • homopolymers of guanosine are not soluble in 1 ml of water after concentration.
  • the purified homopolymers must first be dissolved by adding 3 ml of formamide to the aqueous suspension, after which 0.4 ml of 3 M sodium acetate is added, followed by 12 ml of isopropanol. These samples must be stored at -20°C overnight. The resulting precipitate can be collected by centrifugation and dried in vacuo.
  • polyguanosine oligonucleotides sodium salt form
  • oligonucleotides used in the following examples were all fully modified with phosphorothioate linkages and are summarized in Figure 1.
  • All 18-mer phosphorothioate-modified oligonucleotides were based on the sequence of c-myb codon numbers two through seven. Nucleotides shown in bold were switched to generate either: (1) mismatch controls for the rabbit antisense sequence (Set A) ; (2) various permutations of the human c-myb scrambled (G4) control (Set B) ; or (3) guanine-containing oligonucleotides having inosine substitutions (Set C) .
  • the contiguous guanine residues discussed herein are underlined and are indicated parenthetically with the identifying name assigned to the particular oligonucleotide.
  • Oligonucleotides A ⁇ ainst Rabbit and Pi ⁇ Smooth Muscle
  • This example demonstrates a comparison of the inhibitory action of potential antisense oligonucleotides and multi-G oligonucleotides against smooth muscle cell proliferation in an in vitro assay.
  • the antisense sequences were designed using the c-myb gene as the antisense target, and were compared against multi-G oligonucleotides, scrambled oligonucleotides and control oligonucleotides. All of the oligonucleotides in this example were fully modified with phosphorothioate linkages.
  • one set of oligonucleotides was based on an antisense sequence against the c-myb target, but contained two strategically placed mismatched bases (Set A, Figure 1) . It was expected that these mismatches would significantly disrupt hybridization of the oligonucleotide to the target gene/mRNA, thus blocking any anti-proliferative effect caused by a true antisense mechanism. One of the mismatch sequences also disrupts the four contiguous guanine residues that also coincidentally occur in the antisense sequence. In addition, two control sequences were designed which have no known antisense targets.
  • rb SCR random
  • rb AS random
  • the second control oligonucleotide, rb SCR (G4) maintained the position and length of the multi-G region (4 Gs within an 18 mer oligonucleotide) , but the nucleotides on both the 5'- and 3'-side of the multi-G region were completely scrambled.
  • a second set of oligonucleotides (Set B, Figure 1) was based on a scrambled multi-G oligonucleotide sequence having four contiguous guanine residues, but with the remainder of the sequence being "scrambled", designated hu SCR (G4) .
  • This control sequence had the same base composition as the human anti-c-m b antisense sequence. However, the positions of two residues within this sequence were further altered to either retain or disrupt the four contiguous guanine residues, in an effort to further investigate the role of multi-G oligonucleotides in inhibition smooth muscle cell growth.
  • a third set of oligonucleotides (Set C, Figure 1) was designed based upon the hu SCR (G4) control sequence, wherein the guanine residues in the sequence were replaced by either two or four of the structurally related base inosine (I) .
  • the c- yb sense sequence was included in the oligonucleotide series for the purpose of serving as an additional control for determining non-specific effects on proliferation.
  • the Set A oligonucleotides shown in Figure 1 were assayed for anti-proliferative effect against smooth muscle cells in culture according to the following assay.
  • An in vitro smooth muscle cell proliferation assay was performed as follows. Primary rabbit smooth muscle cells were isolated by an explant procedure from the aorta of New Zealand White Rabbits. The cells were expanded in culture (using a growth media of DME Dulbeco's Modified Eagle's medium (Gibco) + 10% FBS fetal bovine serum (HyClone) , Penicillin/Streptomycin (Gibco) and glutamine) for 2 passages. Passage 2 (“P2”) cells were frozen. The frozen cells were grown up and confirmed to be smooth muscle cells using antibodies to SMC-specific isoforms of actin and myosin. Essentially all of the cells stained positively for both proteins.
  • Pen/Strep and glutamine for 3 or 4 days to arrest the cells in Go of the cell cycle.
  • the starvation media was removed and replaced with normal growth media (10% FBS) with or without completely modified (phosphorothioate-containing) oligonucleotides at the designated concentrations.
  • Triplicate wells for every sample were analyzed. Zero counts were performed at the initiation of proliferation (i.e., on starved cells) to confirm that the cells were growth arrested. (BrdU-labeling experiments also confirmed that the cells were not cycling under these conditions, as described below) .
  • the cells were trypsinized and counted with a Coulter Counter. The percent suppression was calculated according to the following-formula:
  • Zc zero count
  • F c final count
  • Figures 2A, 2B and 2C show the percent suppression of rabbit smooth muscle cells at 10, 30 and 60 ⁇ M oligonucleotide concentrations, respectively, for each of the above-described oligonucleotides in Set A.
  • the results of the in vitro tests demonstrated that the anti-proliferative effect observed with respect to smooth muscle cells is not due to a true antisense mechanism.
  • All of the multi-G oligonucleotides were significantly more inhibitory than the three sequences (designated with an "*" in Figures 2A, 2B and 2C) lacking the four contiguous guanine residues.
  • the most interesting result observed was the decreased anti-proliferative effect of the rb AS 6/12 oligonucleotide sequence, wherein the four contiguous guanine residues were disrupted. Similar mismatch sequences rb AS 4/12 (G4) and rb AS 2/18 (G4) inhibited proliferation to an extent similar to the antisense and other multi-G oligonucleotides.
  • the anti-proliferative effect of the multi-G oligonucleotides became less important at higher oligonucleotide concentrations, which was probably due to a general anti-proliferative effect caused by the high concentration of sulfur- containing modified oligonucleotides.
  • oligonucleotides were analyzed for inhibition of smooth muscle cell proliferation according to the in vitro assay described in Example 2.
  • disruption of the four contiguous guanine residues [hu SCR (random) and hu SCR 8/15 (2xG2) ] was observed to abrogate the inhibition of smooth muscle cell proliferation.
  • This example demonstrates the decrease in anti-proliferative effect that is observed when a series contiguous guanine residues in a multi-G oligonucleotide is disrupted. Because it was desired to change as little as possible with respect to base composition of the oligonucleotides, inosine was selected to create the desired disruption in the series of contiguous guanine residues.
  • inosine is an extremely rare base that only appears in naturally occurring DNA or RNA at a rate of approximately one in every 1,000 bases. Unlike guanine, however, inosine cannot participate to the same degree in hydrogen bonding, because it lacks the 2-amino functionality which is present in guanine. Because the results from Example 2 suggested a relationship between the observed anti-proliferative effect and the four contiguous guanine residues in the multi-G oligonucleotides, three additional oligonucleotides were designed in which either 2 or 4 of the guanine residues in the series of contiguous guanine residues were replaced with inosine nucleotides. The sequence of the hu SCR (G4) multi-G oligonucleotide was otherwise maintained. The particular sequences used in this example, set forth in Table II below (see, Set C in
  • the "G4 pools” were generated by using the following 18-mer multi-G sequence as a template from which to build randomized oligonucleotides:
  • Oligonucleotide 10 ⁇ M 5 ⁇ M 2 ⁇ M 1 ⁇ M
  • G4/A Pool 44 19 1 G4/C Pool 57 30 -3 G4/T Pool 57 20 0 G4/G Pool 76 63 33 12
  • the G4/G pool having five contiguous guanine residues, demonstrated the greatest amount of inhibition against smooth muscle cell proliferation, particularly at the 5 ⁇ M level, indicating that these five contiguous guanine residues may be preferred over four contiguous guanine residues for inhibition of smooth muscle cell growth in certain situations.
  • Multi-G oligonucleotide pools were also generated for oligonucleotides having: (1) five contiguous guanine residues; and (2) six contiguous guanine residues.
  • the pools in this example were generated in a manner similar to the pools described above for multi-G oligonucleotides having four contiguous guanine residues, except that the pools for multi-G oligonucleotides having five contiguous guanine residues were made with a 3' defined base, according to the formula: (N) G5X(N) ⁇ G; and the pools for multi-G oligonucleotides having six contiguous guanine residues were made with a 5* defined base, according to the formula: (N) 3XG6 (N) ⁇ G .
  • Oligonucleotide 10 ⁇ M 5 ⁇ M 2 ⁇ M 1 ⁇ M
  • the G5/G pool having six contiguous guanine residues, demonstrated the greatest amount of inhibition against smooth muscle cell proliferation, similar to the G4 pool results, except that the difference in inhibition of the different G5 pools at the level was not nearly as marked as was observed in the case of the G4 pools.
  • Oligonucleotide I Sequence SEO ID NO.
  • A2-G4-A6-G4-A2 AAG GGG AAA AAA GGG GAA 32
  • A-G4-A8-G4-A AGG GGA AAA AAA AGG GGA 34
  • Smooth muscle celis were treated according to the standard in vitro proliferation assay described in Example 2, with the following exceptions: (1) the cells were plated onto Nunc culture slides; and (2) following the starvation period, 10% serum and BrdU were added simultaneously and left in contact with the cells for approximately 24 hours. The level of BrdU incorporation was detected using a horseradish peroxidase (HRP)-linked secondary antibody to BrdU, followed by an HRP-dependent staining reaction. Total nuclei were labeled by incubating fixed cells with propidium iodide. An image analysis program was developed to automatically count the BrdU labeled nuclei and the total nuclei based on their different staining.
  • HRP horseradish peroxidase
  • Figure 5 shows the results of the BrdU incorporation assay.
  • the pattern of inhibition seen in the BrdU experiment is remarkably similar to the results obtained using the cell count assay (Examples 2 and 3) . This confirms that the multi-G oligonucleotides of the present invention are preventing entry into the cell cycle, rather than exerting a toxic effect on smooth muscle cells.
  • Smooth muscle cells were subjected to treatment with multi-G oligonucleotides at 30 ⁇ M concentrations. At the end of 3 days, one set of wells was counted to determine the percent suppression (as measured in a typical assay) . Identically treated wells were washed once with PBS and then fed with normal growth media. One set of wells was counted on each subsequent day for 3 days to determine if the smooth muscle cells could recover from treatment with oligonucleotides and proliferate normally following removal of the oligonucleotide.
  • the rabbits were deeply anesthetized, after which 12 mm acrylic discs were centered over the central vascular bundle (which contains the central artery) of both sides of each ear and secured with Kelly clamp forceps. After 30 minutes, the forceps were released, the discs removed, and the rabbit terminated by lethal injection. The length of vascular bundle under the discs was dissected free of surrounding tissue, rinsed in phosphate buffered saline and removed to culture. A total of 72 artery segments were obtained from 36 rabbits for use in these experiments.
  • the rabbit ear arteries were incubated in wells of a 24-well culture dish containing DMEM + 5% FBS and the appropriate treatment. A total of seven different oligonucleotides were screened in three separate experiments using groups of 12 rabbits per experiment. The 24 artery segments from the 12 rabbits were randomized into one of three oligonucleotides treatments (at 100 ⁇ M) or control per experiment.
  • the oligonucleotides sequences screened were as follows:
  • BrdU was added to each of the culture wells at a final concentration of 10 ⁇ g/ml, and the vascular bundle segments incubated for a further 4 hours.
  • the media was removed, the samples rinsed in PBS and then fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 4 hours.
  • the samples were then dehydrated through a series of alcohols and processed into paraffin blocks. The segments were serial sectioned and the sections stained with antibodies to BrdU .
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:l: GTGCCGGGGT CTCCGGGC 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:2: CGCCGTCGCG GCGGTTGG 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:3: GCCCGGAGAC CCCGGCAC 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:4: GTGTCGGGGT CTCCGGGC 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:5: GCTGCGGGGC GGCTCCTG 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:6: GCGCCGGGGT CTCCGGGT 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:7: GTGTCGGGGT CCCCGGGC 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:8: GTGCCTGGGT CGCCGGGC 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:9: GTGCCGGGGT CTTCGGGC 18 (11) INFORMATION FOR SEQ ID NO:10:
  • SEQUENCE CHARACTERISTICS SEQUENCE CHARACTERISTICS:
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO: 10: TGCCTGCGCG GCGGTTGG 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:ll: GCTGTGGGGC GGCTCCTG 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:12: GCTGTGGGGT GGCTCCCG 18 (14) INFORMATION FOR SEQ ID NO:13:
  • SEQUENCE CHARACTERISTICS SEQ ID NO:12: GCTGTGGGGT GGCTCCCG 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:13: GCTGTGGGGG GGCTCCTC 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:14: GCTGTCGGGC GGGTCCTG 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:15: GCTGTGGCGC GGCTGCTG 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:16: GCTGTIIGGC GGCTCCTG 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:17: GCTGTGGIIC GGCTCCTG 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:18: GCTGTIIIIC GGCTCCTG 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:19: NNNNNGGGGT NNNNNNNG 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:20: NNNNNGGGGC NNNNNNNG 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:2l: NNNNNGGGGA NNNNNNNG 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:22: NNNNNGGGGG NNNNNNNG 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:23: TTTTTTTGGG GTTTTTTT 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:24: TTTTTGGGGG GGGTTTTT 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:25: TTTGGGGGGG GGGGGTTT 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:26: TGGGGGGGGG GGGGGGGT 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:28: AAAAGGGGAA GGGGAAAA 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:29: TTTGGGGTTT TGGGGTTT 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:31: TTGGGGTTTT TTGGGGTT 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:32: AAGGGGAAAA AAGGGGAA 18
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:34: AGGGGAAAAA AAAGGGGA 18

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Abstract

L'invention concerne des oligonucléotides multi-G capables d'inhiber la prolifération de cellules musculaires lisses. Les oligonucléotides multi-G de l'invention peuvent être utilisés en tant que composés thérapeutiques d'acides nucléiques afin d'inhiber la prolifération de cellules musculaires lisses dans des artères ex vivo, et plus particulièrement contre la resténose. L'invention concerne également des compositions pharmaceutiques contenant les oligonucléotides multi-G. En outre, l'invention concerne un procédé de triage d'oligonucléotides en fonction de leur aptitude à inhiber la prolifération de cellules musculaires lisses ex vivo.
PCT/US1995/012770 1994-10-05 1995-10-03 Procede d'inhibition de la proliferation des cellules musculaires lisses et oligonucleotides utilises dans celui-ci WO1996011266A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU38895/95A AU3889595A (en) 1994-10-05 1995-10-03 Method for inhibiting smooth muscle cell proliferation and oligonucleotides for use therein

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US31845894A 1994-10-05 1994-10-05
US08/318,458 1994-10-05

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WO1996011266A2 true WO1996011266A2 (fr) 1996-04-18
WO1996011266A3 WO1996011266A3 (fr) 1996-12-12

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WO2024229397A1 (fr) 2023-05-03 2024-11-07 Sanegene Bio Usa Inc. Conjugués à base de lipides pour système nerveux central systémique, système nerveux périphérique et administration oculaire
WO2025024523A1 (fr) 2023-07-24 2025-01-30 Sanegene Bio Usa Inc. Agent d'amélioration à base de lipides pour l'administration d'arn et une thérapie à arn
WO2025052278A1 (fr) 2023-09-05 2025-03-13 Genevant Sciences Gmbh Lipides cationiques à base de pyrrolidine pour administration de nanoparticules lipidiques d'agents thérapeutiques à des cellules stellaires hépatiques
WO2025133951A1 (fr) 2023-12-21 2025-06-26 Genevant Sciences Gmbh Lipides ionisables appropriés pour des nanoparticules lipidiques

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