WO2000055374A1 - Méthodes et compositions servant à inhiber l'épissage de l'arn - Google Patents

Méthodes et compositions servant à inhiber l'épissage de l'arn Download PDF

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WO2000055374A1
WO2000055374A1 PCT/US2000/007045 US0007045W WO0055374A1 WO 2000055374 A1 WO2000055374 A1 WO 2000055374A1 US 0007045 W US0007045 W US 0007045W WO 0055374 A1 WO0055374 A1 WO 0055374A1
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seq
oligonucleotide
precursor
intron
rna
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PCT/US2000/007045
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English (en)
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Stephen M. Testa
Matthew D. Disney
Sergei M. Gryaznov
Douglas H. Turner
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Geron Corporation
University Of Rochester
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Priority to US09/936,146 priority Critical patent/US6958215B1/en
Priority to AU38927/00A priority patent/AU3892700A/en
Publication of WO2000055374A1 publication Critical patent/WO2000055374A1/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
    • C12N15/1137—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 enzymes
    • 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
    • C12N2310/10—Type of nucleic acid
    • C12N2310/13—Decoys
    • 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
    • C12N2310/314—Phosphoramidates
    • C12N2310/3145—Phosphoramidates with the nitrogen in 3' or 5'-position

Definitions

  • the present invention relates to methods and compositions for inhibiting the self-splicing of Group I introns and, particularly, to methods and compositions for inhibiting the growth of certain pathogenic organisms containing such Group I introns and the treatment of diseases or conditions caused by such pathogenic organisms.
  • the first generation of such therapeutics consists of antisense nucleic acids that bind mRNA through Watson-Crick base-pairing and thereby regulate translation
  • Nucleic acids used for antisense therapeutics typically between 15-20 nucleotides long, suffer from a number of disadvantages including high cost of synthesis (Wagner et al., Nature Biotechnology 14:840-844 (1996)), lack of specificity (Herschlag, Proc. Natl. Acad. Sci. U.S.A. 88:6921-6925 (1991)) and instability in vivo.
  • oligonucleotides in which the phosphodiester moiety is replaced by a more stable linking group.
  • RNA elements that can serve as molecular targets for pharmacological intervention.
  • Group I introns are one example of such an RNA element.
  • Many pathogenic fungi have Group I introns in critical structural RNAs, for example, in ribosomal RNAs (rRNA).
  • RNAs containing Group I introns undergo a process of self-splicing to remove the intron to produce a functional RNA.
  • This self-splicing process of Group I introns is well known.
  • Cech "Self-Splicing of Group I Introns", Ann. Rev. Biochem. 59:543 (1990).
  • Group I introns contain a guanosine binding site and catalyze a reaction in which a guanosine (or a guanosine nucleotide) attacks the 5' residue of the intron to produce 5' exon and guano sine-intron-3' exon intermediates, which then further react to yield linear guano sine-intron and the spliced 5' exon-3' exon product.
  • a region of the RNA at the 3' end of the 5' exon is thought to pair with a complementary sequence within the intron (the internal guide sequence or IGS) to align the 5' splice site for reaction.
  • FIGURE 2 (right side panel Al through Cl) depicts this pairing and subsequent guanosine attack and cleavage followed by joining of the exons.
  • ribosomal RNA (rRNA) precursor contains a Group I self-splicing intron (Testa et al., Biochemistry 36:15303-15314 (1997); Liu et al, Nucleic Acids Res. 20:3763-3772 (1992)) that provides a potential therapeutic target (Liu et al., (1992); Mei et al., Bioorg. Med. Chem.
  • oligonucleotide hexamer having a sequence that "mimics" the sequence of the putative 5' exon guide sequence of a P. carinii ribosomal RNA Group I intron can tightly bind to a derived ribozyme through base- pairing and tertiary interaction.
  • the ability of the mimic to compete for binding to the IGS with the endogenous 5' exon guide sequence was not shown because the derived ribozyme used for this work did not contain the 5' exon guide sequence that is endogenous to the P. carinii rRNA precursor.
  • the present invention provides methods and compositions for inhibiting the self-splicing reaction of Group I introns using oligonucleotides having a 3' terminal ribonucleoside that mimic the 5' exon guide sequence. More specifically, a suicide inhibitor of a Group I intron self-splicing reaction is provided that has an inhibitor oligonucleotide having a polynucleotide sequence that binds to a 5' internal guide sequence of a precursor RNA containing a Group I intron, or to a portion thereof.
  • the inhibitor oligonucleotide is capable of binding with the 5' internal guide sequence of the precursor RNA wherein the inhibitor oligonucleotide is trcws-spliced to the 3' exon of the precursor RNA.
  • the oligonucleotides have modified internucleosidal linkages, such as, for example phosphoramidate or thiophosphoramidate linkages.
  • the present invention provides a method for inhibiting the growth of an organism containing a Group I intron.
  • the growth inhibition method involves contacting a precursor RNA containing a Group I intron with an inhibitor oligonucleotide, wherein said inhibitor oligonucleotide is trt y-spliced to a 3' exon sequence of the precursor RNA.
  • a method for designing a suicide inhibitior of Group I intron splicing whereby a nucleotide sequence that is essentially identical to a 5' exon guide sequence and is present in the 5' exon flanking a Group I intron is chosen.
  • An inhibitor oligonucleotide is then made based upon homology to the 5' exon guide sequence in the precursor RNA.
  • the inhibitor oligonucleotide is made to have has a 3' terminal ribonucleotide.
  • the present invention provides a method for treating a disease or condition caused by an organism containing a Group I intron.
  • a patient suffering from the disease or condition is administered a therapeutically effective amount of a suicide inhibitor oligonucleotide of the present invention.
  • the present invention provides a method for treating a disease or condition caused by certain pathogenic fungi, including Pneumocystis carinii, Candida albicans and Aspergillus nidulans.
  • Pharmaceutical compositions comprising an inhibitor oligonucleotide are also provided.
  • FIGURE 1 is a schematic representation of the self-splicing (c/s-splicing) and tr ⁇ r ⁇ s-splicing reactions of a Group I intron.
  • the self-splicing reaction follows the pathway A— »A1— »B1— »C1.
  • the trc s-splicing reaction follows the pathway A + augacuon ->B2 ⁇ C2.
  • Steps B— >C are essentially irreversible due to the low concentration of the spliced products.
  • the upper case letters and intervening line represent the Group I intron; the lower case letters and terminal lines represent the 5' and 3' exons; the italicized lower case letters represent the exogenous N3' — >P5' phosphoramidate hexanucleotide; filled circles represent tertiary interactions with the intron's catalytic core.
  • the internal guide sequence (IGS) is shown as 5' -GGUCAU-3* .
  • FIGURE 2 shows graphical representations of the magnesium dependence of the formation of splice products from internally radiolabeled precursor. Reactions were run for 1 h in HxMg buffer, consisting of 50 mM Hepes (25 mM Na + ) at pH 7.5, 135 mM KC1, and x mM MgCl 2 , where x is listed below the plots. Each plot is the average of two independently run self-splice gel assays, and the error of each point is typically ⁇ 6% of the average value. Circles represent the 5' exon-intron product generated by either tr «s-splicing or intron-3' exon junction hydrolysis.
  • FIGURE 3 shows graphical representations of the magnesium dependence of the tr s-splicing reaction with 4 nM 5' end-labeled
  • FIGURE 4 Oligonucleotide concentration dependence of self-splicing (cis- splicing) and traws-splicing. Reactions consisted of approximately 6 nM 3' end radiolabeled precursor, 1 mM pG, H4Mg buffer, and various concentrations of (dA)n(dT)n(dG)n(dA)n(dC)n(rU) [Seq. ID. No:l] (listed in nM below the graph). Circles represent the hexamer-3' exon t/'tws'-spliced product and squares represent the 5' exon-3' exon c/s-spliced product.
  • fractions of the trims-spliced and cts-spliced products are 0.74 and 0.01 at 30 ⁇ M (dA)n(dT)n(dG)n(dA)n(dC)n(rU) [Seq. LD. No:l].
  • FIGURE 5 shows graphical representations of the magnesium dependence of the formation of spliced products from internally radiolabeled C. albicans precursor. Reactions were run for 1 h in HxMg buffer, consisting of 50 mM Hepes (25 mM Na + ) at pH 7.5, 135 mM KC1, and x mM MgCl , where x is listed below the plots. Each plot is the average of two independently run self-splice gel assays, and the error of each point is typically ⁇ 6% of the average value. Circles represent the 5' exon- 3' exon product generated by c/s-splicing. Triangles represent the hexamer-3' exon product formed by trans-splicing.
  • Results are shown (A) in the presence of 1 mM pG and 30 ⁇ M (dG)n(dC)n(dC)n(dT)n(dC)n(rU) [Seq. ID. No:2], and (B) in the absence of added hexamer.
  • FIGURE 6 shows graphical representations of the magnesium dependence of the formation of spliced products from internally radiolabeled C. albicans precursor. Reactions were run for 1 h in HxMg buffer, consisting of 50 mM Hepes (25 mM Na + ) at pH 7.5, 135 mM KC1, and x mM MgCl 2 , where x is listed below the plots. Each plot is the average of two independently run self-splice gel assays, and the error of each point is typically ⁇ 6% of the average value. Circles represent the 5' exon- 3' exon product generated by c/s-splicing.
  • FIGURE 7 shows graphical representations of the magnesium dependence of the formation of spliced products from internally radiolabeled C. albicans precursor. Reactions were run for 1 h in HxMg buffer, consisting of 50 mM Hepes (25 mM Na + ) at pH 7.5, 135 mM KC1, and x mM MgCl 2 , where x is listed below the plots. Each plot is the average of two independently run self-splice gel assays, and the error of each point is typically ⁇ 6% of the average value. Circles represent the 5' exon- 3' exon product generated by cJs-splicing. Triangles represent the hexamer-3' exon product formed by trans-splicing.
  • Results are shown (A) in the presence of 1 mM pG and 30 ⁇ M (rG)n(rA)n(rC)n(rT)n(rC)n(rU) [Seq. ID. No:4], and (B) in the absence of added hexamer.
  • a short oligonucleotide having a sequence that is essentially complementary to the IGS of a Group I intron and containing phosphoramidate and/or thiophosphoramidate linkages in place of phosphodiester linkages can inhibit the self-splicing reaction of Group I intron- containing RNAs, particularly the rRNA of P. carinii, thereby preventing the formation of functional, spliced RNA products.
  • IO inhibitor oligonucleotides
  • the IO thus function as "suicide inhibitors" of the Group I intron self-splicing reaction.
  • These inhibitor oligonucleotides can be used in a method to inhibit the self-splicing reaction and thereby inhibit or impair the growth of the organism containing the Group I intron.
  • Inhibitor oligonucleotides of the present invention are therefore useful in a method to treat any pathogenic condition resulting from the opportunistic growth of organisms containing Group I introns, particularly P. carinii, C. albicans and A. nidulans.
  • Nucleotide residues joined by a N3' — »P5' phosphoramidate linkage will be written with "n" between, for example, (dA)n(dT)n(rU) indicates a trinucleotide having the sequence "ATU” where the A and T residues are deoxyribonucleotides, the U is a ribonucleotide and the residues are all joined by N3' — P5' phosphoramidate linkages.
  • Phosphodiester linkages are indicated by the absence of any notation between the nucleotide residues, for example, (dA)(dT)(rU) indicates a trinucleotide identical to the one just described but having phosphodiester linkages rather than phosphoramidate.
  • the oligonucleotides may be written with parentheses enclosing any consecutive string of deoxyribonucleotides or of ribonucleotides with a "d" or "r" preceding the parenthesis.
  • the trinucleotides indicated above as (dA)n(dT)n(rU) and (dA)(dT)(rU) could alternatively be written as d(AnTn)rU and d(AT)rU.
  • the first system of notation will preferably be used herein.
  • the inhibitor oligonucleotides of the present invention may comprise deoxyribonucleotides or ribonucleotides, or modified forms thereof, or combinations of the foregoing.
  • the inhibitor oligonucleotides will comprise primarily deoxyribonucleotides except for the 3' terminal nucleotide.
  • the 3' terminal nucleotide will be a ribonucleotide or similar moiety containing a 2',3' cis-diol or 2'- hydroxy, 3' -amino or 2'-amino, 3' -hydroxy (where 2' and 3' here refer conventionally to positions on the ribose ring moiety).
  • the inhibitor oligonucleotides will preferably contain those bases that are normally found in DNN or R ⁇ A, that is, adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U), and may contain other bases such as 2,6 diaminopurine.
  • the inhibitor oligonucleotides of the present invention will contain ⁇ 3' ⁇ P5' phosphoramidate or N3' — >P5' thiophosphoramidate linkages between adjacent nucleotide residues rather than the phosphodiester linkages typical of naturally occurring DNA and RNN although some phophodiester linkages may be present. At least one ⁇ 3' — >P5' phosphoramidate linkage or N3' - P5' phosphoramidate linkage will be present, typically more than one and more usually all linkages between adjacent nucleotide residues will be N3' — P5' phosphoramidate linkages.
  • N3' — »P5' phosphoramidate oligonucleotides is well known in the art and has been disclosed, inter alia, in U.S. Patent No. 5,837,835 and Gryaznov et al. (J. Amer. Chem. Soc, 116:3143 (1994)).
  • the synthesis of oligonucleotides containing N3' — >P5' thiophosphoramidate linkages is also known and described in Pongracz et al. (Tetrahedron Let. 40:7661-7664 (1999)).
  • the inhibitor oligonucleotides of the present invention may also be made with other types of modified internucleoside linkages such as, for example, methyl phosphonate linkages as described by U.S. Patent No. 5,936,080.
  • the inhibitor oligonucleotides of the present invention may range in size from
  • the size of the inhibitor oligonucleotide will be determined, at least in part, by the size of the IGS as described below.
  • the sequence of the inhibitor oligonucleotide is chosen to be essentially complementary to the internal guide sequence (IGS) of the Group I intron, the splicing of which is to be inhibited.
  • IGS internal guide sequence
  • the internal guide sequences for a number of Group I introns have been described, for example, Mercure et al., Nucleic Acids Res. 21:6020-6027 (1993); Netzker et al, Nucleic Acids Res.
  • IGS internal guide sequences useful in the practice of the present invention may be determined by methods that are well known in the art, for example, by inspection of the sequence of the RNA or by use of the algorithm of Lisacek et al. (J. Mol. Biol. 235: 1206 (1994).
  • the IGS will be within the Group I intron, near the 5' end of the intron and will have a sequence that is essentially complementary to a region in the flanking 5' exon, which region is referred to herein as the exon guide sequence or EGS.
  • the EGS region is also known as the 5' side of PI (see, Cech (1990)).
  • the inhibitor oligonucleotide of the present invention is designed to be complementary to the IGS sequence.
  • the inhibitor oligonucleotide will therefore be a "mimic" of the EGS, that is, the inhibitor oligonucleotide will have essentially the same sequence as the EGS since the EGS is itself complementary to the IGS.
  • the term "complementary” is intended to have the usual meaning in the art, that is, that the bases of the inhibitor oligonucleotide will pair, in anti-parallel fashion, with the bases of the IGS to form a double stranded region.
  • the base pairs formed will preferably be typical of those observed in naturally occurring DNAs and RNAs and include A with T or U, and G with C or U, and less preferably, C with A. Other base pairing is possible provided that a double stranded region with the IGS will result.
  • the inhibitor oligonucleotide of the present invention may differ in sequence from the EGS in that the EGS, for example, may contain a U in some position whereas the IO may contain a T in the corresponding position and still maintain the requisite pairing with an A in the complementary position in the IGS. Likewise the IO may contain a C in the position corresponding to a U in the EGS, and so forth.
  • the EGS may contain ribonucleotides whereas the IO may contain deoxyribonucleotides or ribonucleotides, or modified forms thereof, particularly phosphoramidate forms.
  • the IO of the present invention will have a U at the 3' end, preferably a rU.
  • the IO for any particular Group I intron will be designed based on the sequences of the IGS and the EGS for that particular Group I intron.
  • the IO will be selected to have a sequence that is identical to the EGS, or a portion of the EGS.
  • the IO may differ in sequence from the EGS but in most cases will be selected to maintain the ability to pair with the IGS.
  • the IO will have the sequence of only a portion of the EGS, as where, for example, the EGS is longer than approximately 6 bases, the IO may be selected to have the sequence of 6 consecutive bases in the EGS. Thus the IO may be shorter than the EGS sequence.
  • the IO may contain additional bases flanking the sequence of the EGS and thus the IO will be longer than the EGS.
  • the IO may contain additional nucleotides at the 5' and/or 3' ends to enhance its ability to bind to the IGS or to improve the specificity of the binding (that is, to eliminate or decrease binding to regions within the intron other than the IGS or to other RNAs or DNAs). Optimization of the sequence for any particular IO will be readily within the skill of one of ordinary skill in the art using the teachings disclosed herein. The present inventors have found that an IO of formula I below is particularly useful for inhibition of self-splicing of a Group I intron within a rRNA of P. carinii.
  • an IO of formula II below is of use in inhibiting self-splicing of a Group I intron within a rRNA of C. albicans.
  • any particular IO to inhibit the self-splicing of a Group I intron can be tested in any of a variety of ways that are well known in the art, for example, in an in vitro splicing system as described herein in Example 1 for the rRNA of P. carinii. See for example, U.S. Patent No. 5,849,484.
  • the IO will be assayed for splicing inhibitory activity in vitro using as a substrate for the self- splicing reaction an appropriate precursor RNA containing a Group I intron flanked by the 5' and 3' exons to be joined by the c/s-splicing reaction.
  • the entire 5' exon or 3' exon need not be present as long as there is an amount sufficient to function as a cJs-splicing substrate.
  • the appearance of c/s-splicing reaction products may be monitored on a denaturing polyacrylamide gel using standard protocols with radioactive substrate.
  • the self-splicing reaction and inhibition assay is typically carried out in 50 mM Hepes (25 mM Na + ), 135 mM KC1, and MgCl 2 from 0 to 15 mM, preferably l-3mM.
  • the splicing substrate RNA is annealed at approximately 55°C for 5 min, then slow cooled to 37°C.
  • pG is added to approximately 1 mM and IO from approximately 0-100 ⁇ M.
  • reaction is carried out at 37°C for about 1 hr and stopped by addition of a stop buffer and the reaction products separated on a denaturing polyacrylamide gel.
  • the present invention provides a method for inhibiting the growth of an organism having a Group I intron in its RNN by contacting the organism with a composition comprising the IO.
  • a composition comprising the IO One of ordinary skill in the art may readily identify those organisms that will be appropriate for application of the method of the present invention, as numerous organisms containing Group I introns have been identified (Damberger and Gutell (1994); Lisacek et al. (1994); Cech, T.R., in The RNA World, 1 st edition, Gesteland and Atkins, eds., pp. 239-263 (1993) Cold Spring Harbor Press).
  • the organisms most susceptible to the IO will be those in which the splicing of the Group I intron is critical to the function of some essential R ⁇ A species, for example, one of the ribosomal R ⁇ A subunits.
  • the method of the present invention may be used to inhibit the self-splicing of the rR ⁇ A ofE. carinii.
  • An IO of formula I is particularly useful in the practice of this method.
  • the method of the present invention may be used to inhibit the self- splicing of the rR ⁇ A of C. albicans.
  • An IO of formula II is particularly useful in the practice of this method.
  • the present invention provides a method for the treatment of a disease or condition resulting from the opportunistic growth of a pathogenic organism containing a Group I intron in its R ⁇ A, by administering to an individual suffering from such disease or condition, a therapeutically effective amount of a pharmaceutical composition comprising an IO.
  • a therapeutically effective amount is meant an amount that relieves (to some extent) one or more symptoms of the disease or condition in the individual.
  • therapeutically effective amount is meant an amount that returns to normal, either partially or completely, physiological or biochemical parameters associated with or causative of such a disease or condition.
  • it is an amount between about 1 nmole and 1 mmole of the molecule, depending on the potency of the IO as determined in an in vitro self-splicing assay such as shown herein, and on other factors, such as, the age, size, and disease associated with the patient.
  • compositions and appropriate treatment regimes will vary depending upon the particular disease or condition, the particular pathogenic organism and the particular IO selected.
  • One of ordinary skill in the art will be competent to select appropriate formulations, dosages, treatment regimes given the general knowledge of such in the art and the specific disclosures herein. Protocols for testing the ability of compounds to inhibit the growth of pathogenic organism like P. carinii are well known in the art, see for example, U.S. Patent Nos. 5,668,166 and 5,302,598.
  • Pharmaceutical Compositions are well known in the art, see for example, U.S. Patent Nos. 5,668,166 and 5,302,598.
  • the IO are utilized in a manner appropriate for antisense therapy in general.
  • the IO of the invention can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA.
  • compositions for aerosol and enteral, especially oral, and for parenteral administration are especially preferred.
  • the compositions comprise an inhibitor oligonucleotide alone or, preferably, together with a pharmaceutically acceptable carrier.
  • the dosage of the IO depends upon the disease to be treated and upon the species, age, weight and condition of the individual to be treated, and also upon the mode of administration.
  • a medical condition described herein for example P. carinii pneumonia or C. albicans
  • IO described herein or a salt thereof when salt-forming groups are present, in an amount effective for the inhibition of the splicing of the Group I intron, together with at least one pharmaceutically acceptable carrier.
  • Suitable pharmaceutical formulations and methods for delivery of therapeutic nucleic acids have been abundantly described, see for example, U.S. Patent Nos. 5,908,635, 5,855,911, 5,858,987, 5,593,974 and 5,294,533.
  • the compounds of the invention are formulated for pulmonary administration.
  • One such method of administration involves the aerosolization of a solution containing, preferably, an aqueous-soluble compound of the invention.
  • Aerosol compositions can alternatively include the active compound packaged in reverse micelles or Hposomes.
  • Pharmaceutical compositions suitable for such a method of administration can additionally include aerosol propellants and a surfactant. Examples of small compounds administered by this method can be found in U.S. Patent Nos. 5,364,615, 5,292,499, and 5,238,683. Both phospholipid and nonconventional Hposomes are rapidly becoming accepted as pharmaceutical agents which improve the therapeutic value of a wide variety of compounds (Cancer Res. 43:4730 (1983)) and can be applied to inhibitor oligonucleotides identified by methods of the present invention.
  • solubilizing agents such as ionic surfactants, cholates, polyethylene glycol (PEG), ethanol, or other agents which may have undesirable effects when used for inhalation.
  • a treatment requiring successful delivery into alveoli of the lower pulmonary region may preclude from the formulation the use of certain irritants such as chlorofluorocarbons and should involve a minimum number of required doses.
  • Hposomes or hydrophobic particles can be used.
  • An inhalation formulation providing for a sustained release of such a compound using aerosol droplet particles approximately 1-2.1 ⁇ in size, preferably less than 1 ⁇ , would satisfy these special needs.
  • Small particle aerosol Hposomes and liposome-nucleic acid combinations for medical use are known in the art, see, for example, U.S. Patent No. 5,858,784.
  • Conventional liposomal formulations sometimes have an uncontrollable and fast release rate and frequently have larger particle sizes than are useful for directing a drug to alveoli.
  • nonconventional Hposomes which are formed solely by cholesterol and cholesterol derivatives, or alternatively amphipathic lipid components, have been used successfully with controllable sustained release, improved solubility, high encapsulation, absence of need for multiple dosing, and extended stability.
  • Suitable liposomal formulations for sustained release of such compounds include sodium cholesterol sulfate: cholesterol :compound% molar ratios of 55:40:5; 50:40:10; 53:37:9, and most preferably 50:40: 10 (U.S. Patent No. 5,049,389).
  • the pharmaceutical compositions comprise from approximately 5% to approximately 95% active ingredient, dosage forms in single dose form preferably comprising from approximately 20% to approximately 90% active ingredient and dosage forms that are not in single dose form preferably comprising from approximately 5% to approximately 20% active ingredient
  • Unit dose forms, such as dragees, tablets or capsules comprise from approximately 0.01 mg to approximately 10.0 mg of active ingredient.
  • compositions of this invention are prepared in a manner known per se, for example by means of conventional mixing, granulating, confectioning, dissolving or lyophilizing processes.
  • pharmaceutical compositions for oral use can be obtained by combining the active ingredient with one or more solid carriers, optionally granulating a resulting mixture, and, if desired, processing the mixture or granules, if appropriate with the addition of additional excipients, to form tablets or dragee cores.
  • Suitable carriers include fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, also binders, such as starches, for example corn, wheat, rice or potato starch, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, such as the above-mentioned starches, also carboxymethyl starch, cross-linked polyvinylpyrrolidone, alginic acid or a salt thereof, such as sodium alginate.
  • Additional excipients include flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol, or derivatives thereof.
  • Dragee cores can be provided with suitable, optionally enteric, coatings, there being used inter alia concentrated sugar solutions which may contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents or solvent mixtures, or, for the production of enteric coatings, solutions of suitable cellulose preparations, such as acetyl-cellulose phthalate or hydroxypropylmethylcellulose phthalate. Colourings or pigments may be added to the tablets or dragee coatings, for example for identification purposes or to indicate different doses of active ingredient.
  • suitable, optionally enteric, coatings there being used inter alia concentrated sugar solutions which may contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents or solvent mixtures, or, for the production of enteric coatings, solutions of suitable cellulose preparations, such as acetyl-cellulose phthalate or hydroxypropylmethylcellulose phthal
  • Orally administrable pharmaceutical compositions also include dry-filled capsules consisting of gelatin, and also soft, scaled capsules consisting of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the dry-filled capsules may contain the active ingredient in the form of granules, for example in admixture with fillers, such as corn starch, binders and/or gildants, such as talc or magnesium stearate, and optionally stabilizers.
  • the active ingredient is preferably dissolved or suspended in suitable liquid excipients, such as fatty oils, paraffin oil, liquid polyethylene glycols or fatty acid esters of ethylene or propylene glycol, to which stabilizers and detergents, for example of the polyoxethylen-sorbitan fatty acid ester type, may also be added.
  • suitable liquid excipients such as fatty oils, paraffin oil, liquid polyethylene glycols or fatty acid esters of ethylene or propylene glycol, to which stabilizers and detergents, for example of the polyoxethylen-sorbitan fatty acid ester type, may also be added.
  • oral dosage forms are, for example, syrups prepared in customary manner which comprise the active ingredient, for example, in suspended form and in a concentration of about 5% to 20%, preferably about 10%, or in a similar concentration that provides a suitable single dose, for example, when administered in measures of 5 or 10 ml.
  • suitable are, for example, powdered or liquid concentrates for the preparation of shakes, for example in milk. Such concentrates may also be packaged in single dose quantities.
  • compositions include suitable aqueous solutions of an active ingredient in water-soluble form, for example in the form of a water- soluble salt, or aqueous injection suspensions that contain viscosity-increasing substances, for example sodium carboxymethylcellulose, sorbitol and/or dextran, and, if desired, stabilizers.
  • the active ingredient optionally together with excipients, can also be in the form of a lyophilisate and can be made into a solution prior to parenteral administration by the addition of suitable solvents. Solutions such as those that are used, for example, for parenteral administration can also be used as infusion solutions.
  • the IO of the invention are formulated into ointments, salves, gels, or creams as generally known in the art EXAMPLES
  • the P-h rRNA precursor was 3' end radiolabeled by incubating 1 ⁇ M [5' - 32 P] pCp, 440 nM P-h RNA transcript, 10 mM MgCl 2 , 5 ⁇ M ATP, 3 mM DTT, 250 ng BSN 50 mM Hepes (pH 8.3), and 30 units T4 R ⁇ A ligase in a total volume of 25 ⁇ L for 5 h at 22 °C.
  • the reaction mixture was passed through a chromaspin G 100 size- exclusion spin column (CLO ⁇ TECH Laboratories Inc., Palo Alto, CA) to remove unincorporated [5' - 32 P] pCp, and then added to 12.5 ⁇ L of 2X stop buffer (10 M urea, 3.1 mM EDTN 10 mM Tris, and 9 mM Boric Acid at pH 8.4) and 2 ⁇ L glycerol.
  • 2X stop buffer (10 M urea, 3.1 mM EDTN 10 mM Tris, and 9 mM Boric Acid at pH 8.4
  • 2X stop buffer 10 M urea, 3.1 mM EDTN 10 mM Tris, and 9 mM Boric Acid at pH 8.4
  • 2X stop buffer 10 M urea, 3.1 mM EDTN 10 mM Tris, and 9 mM Boric Acid at pH 8.4
  • 2X stop buffer 10 M urea, 3.1 mM EDTN 10 mM
  • the self-splicing reaction was conducted with the control oligonucleotide (dC)n(dA)n(dG)n(dT)n(dA)n(rU) [Seq. ID No: 5] as above using H2Mg buffer and 1 mM pG, conditions that maximize production of the 5' exon- intron band with (dA)n(dT)n(dG)n(dA)n(dC)n(rU) [Seq. ID No: l].
  • Gels were dried under vacuum and the bands quantified on a Molecular Dynamics phosphorimager. The intensity of each band was corrected for the number of adenines in each sequence.
  • FIGURE 2B shows results in the presence of 1 mM pG and absence of (dA)n(dT)n(dG)n(dA)n(dC)n(rU) [Seq. ID No: l].
  • [Mg 2+ ] > 4 mM the fraction of 5' exon-intron band is the same in the presence (FIGURE 2A) and absence (FIGURE 2B) of (dA)n(dT)n(dG)n(dA)n(dC)n(rU) [Seq. ID No: l].
  • [Mg 2+ ] > 4 mM the 5' exon-intron band is likely due to hydrolysis at the intron- 3' exon junction.
  • FIGURE 3 shows that the 5' exon mimic, (dA)n(dT)n(dG)n(dA)n(dC)n(rU) [Seq. ID No:l], is incorporated into two products; one is 33 nucleotides in length corresponding to the expected trans- spliced product, and one is approximately 350 nucleotides (the origin of this product is unknown at present).
  • the trans-spliced product plateaus at about 500 nM (dA)n(dT)n(dG)n(dA)n(dC)n(rU) [Seq. ID No:l], and the amount of tr ⁇ ns-spliced product is greater than the amount of properly spliced product at concentrations of (dA)n(dT)n(dG)n(dA)n(dC)n(rU) [Seq. ID N ⁇ : l] as low as 200 nM (FIGURE 4). Since the K d for (dA)n(dT)n(dG)n(dA)n(dC)n(rU) [Seq.
  • Assays were run in HxMg buffer, consisting of 50 mM Hepes (25 mM Na + ) at pH 7.5, 135 mM KC1, and x mM MgCl . Each reported value is the average of two independent assays.
  • the dissociation constant, Kd was determined by direct band-shift gel electrophoresis.
  • ⁇ d was determined by competition band-shift gel electrophoresis (Testa et al., Biochemistry 37:9379-9385 (1998)).
  • the fraction of precursor RNA that is tr ⁇ ns-spliced is half maximal at 200 nM (dA)n(dT)n(dG)n(dA)n(dC)n(rU) [Seq. ID No: l] in 4 mM Mg 2+ .
  • (dA)n(dT)n(dG)n(dA)n(dC)n(rU) [Seq. ID No: l] effectively competes with the intramolecular 5' exon sequence for binding in the catalytic pocket. This is surprising since intramolecular binding has a considerable effective concentration advantage over bimolecular binding (Jencks, Catalysis in Chemistry and Enzymology (1987), Dover Publications, New York).
  • thermophilia in 5 mM Mg 2+ at 30 °C in the presence of pG, the 3' terminal end of the 5' exon of the Group I intron from T. thermophilia can base-pair with upstream exon sequences, allowing exogenous 5' exon mimics to bind the internal guide sequence and act as tr ⁇ ns-splicing substrates (Woodson et al., Biochemistry 30:2042-2050 (1991)). The 3' end of the P.
  • carinii 5' exon can also form such an upstream structure, but the predicted thermodynamics suggest that it is significantly weaker than the structure with the 3' end of the 5' exon base-pairing to the internal guide sequence (-7 vs -3 kcal/mol).
  • the completely excised intron product predominates in the presence of pG and (dA)n(dT)n(dG)n(dA)n(dC)n(rU) [Seq. ID No: l], suggesting that under these conditions formation of the intramolecular 5' exon - internal guide sequence helix is favored (FIGURES 2 and 3).
  • the internal guide sequence may be more accessible at low Mg 2+ concentrations because the intron may not be completely or properly folded.
  • Intracellular Mg 2+ concentrations are often less than 2 mM (Hamson et al., (1980) In Metals in Biochemistry, Chapman and Hall., New York. pp. 8-9; Maquire, (1990) In Sigel. H. and Sigel. A. (eds), Metals in Biological Systems 26, Marcel Dekkar Inc., New York, pp. 135-153) and cases are known where proteins are required to stabilize or catalyze proper folding of Group I introns (Weeks et al., Cell 82:221-230 (1995)).
  • C-10/lx is a Candida albicans rRNA Group I ribozyme Dissociation constants for 5' exon oligonucleotide mimics which bind to the C-10/lx ribozyme were measured by competitive band-shift native electrophoresis using methods described in Testa et al., Biochemistry 37 9379-9385 (1998) The results for a variety of oligonucleotides complementary or containing a one base mismatch to the C-10/lx 5' exon guide sequence are shown in Table 2
  • ⁇ ⁇ 15Mg buffer consists of 50 mM Hepes (25mM Na + ), 15 mM MgCl 2 , and 135 mM
  • BETI tertiary interactions
  • FIGURE 5A shows that in the presence of (dG)n(dC)n(dC)n(dT)n(dC)n(rU) [Seq. ID No:2] and pG, the hexamer- 3' exonproduct (tr ⁇ ns-splicing) reaches a maximum at 1 mM Mg 2+ , where it is about 5-fold more prevalent than the completely excised intron product (c/s-splicing).
  • FIGURE 5B shows the amounts of cis- and tr ⁇ ns-splicing products formed in the presence of 1 mM pG and no (dG)n(dC)n(dC)n(dT)n(dC)n(rU) [Seq. ID No:2] oligonucleotide.
  • suicide splicing Example 2
  • increasing the Mg 2+ concentration just a little results in a predominance of 5' exon-3' exon product (c/s-splicing) (FIGURE 5 A).
  • FIGURES 6 and 7 show analogous results for the Mg 2+ dependence of splicing of a C. albicans model precusor rRNA in the presence of 30 ⁇ m of ribophosphoramidate oligonucleotide (rG)n(rC)n(rC)n(rU)n(rC)n(rU) [Seq. ID No:3] and internal guide sequence mismatch ribophosphoramidate oligonucleotide (rG)n(rA)n(rC)n(rU)n(rC)n(rU) [Seq. ID No:4], respectively.
  • FIGURE 6A show that in the presence of (rG)n(rC)n(rC)n(rU)n(rC)n(rU) [Seq. ID No:3] and pG, the 5' exon-intron product (tr ⁇ ns-splicing) reaches a maximum at 2 mM Mg + , where it is about 5-fold more prevalent than the 5' exon-3' exon product (cis-splicing).
  • the 5' exon guide sequence one base mismatch ribophosphoramidate oligonucleotide (rG)n(rA)n(rC)n(rU)n(rC)n(rU) [Seq.
  • oligonucleotide concentration dependence of trans- verses cis-splicing at 1 mM Mg 2+ and 2 mM Mg 2+ was measure for (rG)n(rC)n(rC)n(rT)n(rC)n(rU) [Seq. LD No: 3].
  • Trans-splicing of the C. albicans Group I precursor RNA was found to predominate at oligonucleotide concentration greater than about 100 nM at 1 mM Mg 2+ and at about 1000 nM at 2 mM Mg 2+ (data not shown).

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Abstract

L'invention concerne une méthode servant à inhiber l'auto-epissage d'un intron du Groupe I. L'invention concerne l'utilisation d'un oligonucléotide contenant une séquence pratiquement identique à une séquence guide trouvée dans l'exon 5' et se terminant par un ribonucléotide 3'. En général, l'oligonucléotide présente les liaisons N3'→P5' phosphoramidate ou N3'→P5' thiophosphoramidate. L'invention concerne également une méthode destinée à inhiber la croissance d'organismes présentant un intron du Groupe I, en particulier, certains champignons pathogènes parmi lesquels P. carinii, C. albicans, et A. nidulans, à l'aide dudit oligonucléotide.
PCT/US2000/007045 1999-03-15 2000-03-15 Méthodes et compositions servant à inhiber l'épissage de l'arn WO2000055374A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5849484A (en) * 1992-07-30 1998-12-15 University Of Medicine & Dentistry Of Nj In vitro assay for inhibitors of the intron self-splicing reaction in Pneumocystis carinii

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5849484A (en) * 1992-07-30 1998-12-15 University Of Medicine & Dentistry Of Nj In vitro assay for inhibitors of the intron self-splicing reaction in Pneumocystis carinii

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIU, Y. ET. AL.: "Sequence and Variability of the 5.8S and 26S rRNA Genes of Pneumocystis Carinii", NUCLEIC ACIDS RESEARCH, vol. 20, no. 14, January 1992 (1992-01-01), pages 3763 - 3772, XP002929993 *
TESTA, S. M. ET. AL.: "In Vitro Suicide Inhibitors of Self-Splicing of a Group I Intron from Pneumocystis Carinii by an N3'-P5' Phosphoramidate Hexanucleotide", PROC. NATL. ACAD. SCI. USA., vol. 96, 16 March 1999 (1999-03-16), pages 2734 - 2739, XP002929992 *

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