WO2004038019A2 - Dnazyme - Google Patents

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WO2004038019A2
WO2004038019A2 PCT/GB2003/004614 GB0304614W WO2004038019A2 WO 2004038019 A2 WO2004038019 A2 WO 2004038019A2 GB 0304614 W GB0304614 W GB 0304614W WO 2004038019 A2 WO2004038019 A2 WO 2004038019A2
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dnazyme
site
helix
mutation
cleavage
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WO2004038019A3 (fr
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David Beeson
Matthew Wood
Amr Abdelgany
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Isis Innovation Limited
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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
    • 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
    • C12N15/1138—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 receptors or cell surface proteins
    • 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
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • 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
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • the present invention relates to the production and use of DNAzymes (RNA-clearing DNA enzymes). More particularly, the present invention relates to the design of DNAzymes that can be used to target specific mutant alleles and thereby achieve a therapeutic effect.
  • DNAzymes RNA-clearing DNA enzymes
  • Genetic disorders are often both progressive and incurable.
  • the pathogenic agent may be expressed from a mutant allele throughout life and its effects are not ameliorated by expression from a normal allele.
  • One therapeutic approach for genetic disorders would be to suppress expression from the mutant allele, by targeting the mutant RNA transcripts.
  • DNAzymes are short oligonucleotides that have the capacity to cleave
  • RNA molecules in an enzymatic fashion. They consist of a central catalytic motif . (helix II) flanked by two arms (helices I and III) that can bind to a target RNA molecule on a Watson-Crick basis (see Fig 1 ).
  • the catalytic activity of the core structure is dependent on the presence of divalent metal cations. The cleavage occurs by breaking the phosphodiester bond between purine and pynmidine nucleotides, with one of the nucleotides being bound (hybridised) to the DNAzyme and the other nucleotide remaining unbound.
  • RNAzymes that cleave mutated forms of RNA.
  • the target RNA comprises a mutation at the site of cleavage and this enables discrimination between the mutant and the wild-type form.
  • the cleavage site must comprise the purine-pyrimidine sequence that acts as the catalytic substrate for the DNAzyme. A difficulty with this is that mutants will not always form a catalytic substrate. Accordingly, for certain mutants, it is not possible to use the DNAzymes described in Santoro et al to cleave the mutant RNA. Summary of the Invention
  • the present invention is based on the realisation that mutants can be targeted using the flanking regions (helices I and III) of a DNAzyme, the
  • DNAzyme being designed to cleave its substrate at a putative cleavage site that is remote from the site of mutation. This permits DNAzymes to be designed to target a wide variety of mutations to ameliorate associated disorders.
  • a DNAzyme that selectively cleaves a mutant polynucleotide by cleaving at a site remote from the mutation site.
  • a DNAzyme of the invention is used in therapy.
  • a DNAzyme of the invention is used in the manufacture of a medicament for the treatment of a dominantly inherited disorder.
  • a pharmaceutical composition comprises a DNAzyme as defined above and a pharmaceutically acceptable carrier.
  • a DNAzyme is used in the manufacture of a medicament for the treatment of a disorder associated with the over-expression of a gene, wherein the DNAzyme comprises a central catalytic motif and two flanking regions that bind to the target gene or its expressed product, and wherein at least one flanking region comprises a mismatch sequence with respect to the target sequence.
  • a method of producing a DNAzyme as defined above comprises the steps of: identifying a putative cleavage site remote from the site of mutation on the mutant allele; designing a DNAzyme with a flanking region that is complementary to the mutant sequence or that of its expressed product such that the DNAzyme can bind selectively to the mutant sequence and position a catalytic region at the cleavage site; and synthesising the DNAzyme.
  • Figure 1 shows the structure of a DNAzyme with a "10-23" catalytic region"; the top strand is an RNA substrate with a RY cleavage site, where R is A or G and Y is U or C and Helix I and III are antisense binding-arms.
  • the present invention provides DNAzymes which can be designed to target specific mutant alleles or their expressed products resulting in selective cleavage.
  • the DNAzymes are designed to hybridise with the mutant at the site of mutation, with cleavage occurring at a site remote from the mutation site.
  • a DNAzyme according to the invention binds selectively to a mutant allele or its expressed product and comprises a central catalytic motif
  • Helix II and two flanking regions (Helix I and III) wherein at least one of the flanking regions has a polynucleotide sequence complementary to a region that includes the mutation in the mutant allele or to that of its expressed product.
  • DNAzyme is intended to refer to a DNA molecule that has specificity for and cleaves a target nucleic acid sequence, which may be either DNA or RNA.
  • mutant allele refers to a genetic sequence that is a variant of a wild-type sequence. The mutant may vary from the wild-type by a single nucleotide (a single nucleotide polymorphism) which may result from a replacement of one nucleotide by another different polynucleotide, or from insertion or deletion of a single nucleotide.
  • expressed product is intended to refer to the RNA transcribed by the mutant allele, i.e. mRNA.
  • DNAzymes of the invention have the capability of being designed to hybridise and cleave DNA or RNA, it is preferred that the DNAzymes are designed to target and cleave RNA (e.g. mRNA).
  • DNAzymes are known in the art and comprise typically a catalytic region and two flanking regions that have specificity for the target nucleic acid (substrate). The DNAzyme is a single-stranded DNA molecule.
  • the catalytic region may be referred to as Helix II and the two flanking substrate-recognition regions may be referred to as Helix I (for that region at the 5'terminus) and Helix III (for that region at the 3'terminus).
  • the catalytic region may be of any suitable sequence that exerts a cleavage activity on contact with a suitable substrate. Examples of suitable catalytic domains are described in Santoro and Joyce, PNAS (USA), 1997; 94(9): 4262-4266 and US patent No. 5807718.
  • the catalytic region may be that referred to as "10-23" or "8-17” according to the nomenclature adopted by Santoro et al., 1998, Supra.
  • the sequence for the "10- 23" sequence is GGCTAGCTACAACGA (SEQ. ID. NO. 1 ) and that for the "8-17" catalytic region is AGCAGGCCGAGCCT (SEQ. ID. No. 2).
  • the 10-23 and 8-17 catalytic regions require a purine-pyrimidine sequence for cleavage.
  • the 10-23 catalytic region can cleave the following purine-pyrimidine combinations: AC, AU, GC and GU.
  • the catalytic region exhibits greatest cleavage activity against AU sites, followed by, in order, GU, GC and AC sites.
  • the 8-17 catalytic domain cleaves only at AG substrate sites.
  • flanking regions may comprise any suitable sequence that enables selective binding to the target sequence. At least one of the flanking regions will comprise a sequence that is complementary to the mutated region on the mutant allele or its expressed product (i.e. the flanking region will contain a mismatch with respect to the wild-type sequence). In a preferred embodiment, both flanking regions are complementary to mutated regions. This provides further specificity compared to the wild-type allele.
  • the flanking regions are designed so that they hybridise to the target at the site of mutation and position the catalytic region in correct proximity to the appropriate cleavage site. The position of the mutation with respect to the cleavage site is therefore determined by the sequence of the mutant.
  • the DNAzymes it is preferable to design the DNAzymes to hybridise at sites where the mutation is close to the cleavage site, preferably 1 -5 nucleotides upstream or downstream of the cleavage site, more preferably 2 or 3 nucleotides.
  • it is Helix I that hybridises to the site of mutation.
  • it is both Helix I and Helix III that hybridise at sites of mutation.
  • the size of Helix I and Helix III may be any suitable size that is sufficient to achieve selectivity between the mutant and the wild-type sequences and which also enables sufficient cleavage to occur.
  • the two flanking regions may comprise the same number or a different number of nucleotides. Increasing the number of nucleotides in the flanking regions will usually increase the binding affinity of the DNAzyme but will diminish the selectivity for the mutant sequence. It is therefore preferable for the flanking regions to comprise between 5 and 25 nucleotides, mere preferably 7 to 21 nucleotides, more preferably 8 to 15 nucleotides and most preferably 9 nucleotides.
  • the DNAzymes are designed with a shorter Helix I compared to Helix III.
  • Helix I comprises 9 nucleotides and Helix III comprises 13 nucleotides. Variations on this will be evident to the skilled person depending on the degree of selectivity and cleavage required in any particular instance.
  • the DNAzymes may be modified to protect against degradation in vivo.
  • Methods for protecting DNAzymes against degradation are known in the art. Examples include incorporating a 3'-3'inversion at one or more terminae of the DNAzyme. This entails modifying the 3'terminal nucleotide so that covalent phosphate bonding occurs between the 3'carbons of the terminal nucleotide and its adjacent nucleotide.
  • the DNAzymes may contain modified nucleotides or nucleotide linkages, for example N3'- P ⁇ 'phosphoramidate linkages, 2'-O-methyl substitutions and peptide-nucleic acid linkages.
  • An alternative strategy for stabilising the DNAzymes is to employ a stem-loop structure at one or each terminus, as described in Gavin and Gupta, J. Biol. Chem., 1997; 272: 1451-1472.
  • the present invention provides a general method for the design of DNAzymes that can be used to target any mutant allele.
  • a method for designing suitable DNAzymes comprises identifying a putative cleavage site remote from the site of mutation on the target mutant, designing a DNAzyme flanking region that is complementary to the mutant sequence such that the DNAzyme can bind selectively to the mutant sequence and position a catalytic motif at the cleavage site, and synthesising the DNAzyme.
  • the DNAzymes are designed to cleave at either an AU site or GU site. More preferably, the DNAzymes cleave on an AU site.
  • the DNAzymes of the present invention have wide utility in the treatment of genetic diseases associated with a mutant allele, in particular dominantly inherited disorders.
  • a DNAzyme of the invention is used to treat a dominantly inherited disorder selected from the group consisting of Achondroplasia, ALS with SOD1 mutation, Marfan syndrome, Hypercholesterolaemia, osteogenesis imperfecta and SCCMS.
  • the dominantly inherited disorder is SCCMS.
  • Achondroplasia is the most common form of dwarfism in humans.
  • a recurrent glycine-to-arginine mutation at codon 380 (G380R) of the transmembrane domain of fibroblast growth factor receptor-3 (FGFR-3) was identified in the majority of Western and Japanese patients, which is uncommon in other autosomal dominant genetic diseases.
  • the single point mutation at codon 380 replaces G with either A or C.
  • a DNAzyme incorporating this sequence has a mismatch at position 1.2 in helix I (i.e. 2 nucleotides from the cleavage site; see Fig. 1 ).
  • a DNAzyme incorporating this sequence has a mismatch at position 1.2 in helix I.
  • Missense mutations in the human Cu/Zn superoxides disutase gene cause many cases of autosomal dominant familial amyotrophic lateral sclerosis (FALS).
  • FALS familial amyotrophic lateral sclerosis
  • FALS patients may carry a missense mutation which is a G12R substitution in exon 1 , and a F45C substitution in exon 2, respectively, (i) For the G12 R mutation
  • DNAzyme sequence G GC GAC C GC CCA GTG (SEQ.ID.No. 10) A DNAzyme incorporating this sequence has a mismatch at position 1.5 in helix I. (ii) For the F45C mutation
  • a DNAzyme targeting this mutation can comprise the sequence:
  • D1155N in exon 27 was identified in the fibrillin-1 gene which affected the cellular processing.
  • a DNAzyme that has a mismatch with the wild-type at a position close to the cleavage site and in helix I will be suitable for this mutation. 4 - Hypercholersterolaemia
  • Familial hypercholesterolaemia is an autosomal dominant disorder of lipid metabolism characterised by elevated low-density lipoprotein (LDL), the formation of tendon and skin xanthomata and the development of premature coronary atherosclerosis. It is caused by a defect in the receptor-mediated hepatic uptake of LDL due to mutations in the LDL receptor.
  • LDL low-density lipoprotein
  • V623M Two missense mutations (V623M, R645Q) in the regulatory domain have been identified in sterol regulatory element binding protein (SREBP-2).
  • SREBP-2 sterol regulatory element binding protein
  • a suitable DNAzyme targeting this mutation will create a mismatch with the wild- type at position 1.3 in helix I. 5 - Osteogenesis imperfecta
  • Osteogenesis imperfecta is an inheritable disease of bone characterised by low bone mass and bone fragility.
  • Six different types of Ol have been described to date, based on clinical phenotype and histological findings.
  • the genetic defect in many patients with Ol types l-IV is due to mutations in the genes encoding type I collagen, while patients with Ol types V and VI show no evidence of mutations in the COL1A1/COL1A2 genes.
  • the DNAzymes may be administered to a patient using any of the conventional methods used in DNAzymes treatments. Administration may be carried out, for example, intravenously, orally, via implant, transmucosally, transdermally, topically, intramuscularly or subcutaneously.
  • the DNAzymes will usually be administered in conjunction with a physiologically acceptable excipient, buffer or diluent.
  • the DNAzymes are incorporated into liposomes which can help prevent degradation on administration. This will all be evident to the skilled person.
  • the amount of DNAzymes to be administered will be determined based on the extent of target specificity and cleavage detivity. This can be determined by the skilled person prior to administration.
  • DNAzymes of the present invention are intended primarily for selective gene knockdown of a mutant sequence, there may be circumstances where it is desirable to achieve regulated partial gene knockdown of either the mutant or wild-type sequence. This can be accomplished by varying the distance between the cleavage site and the mismatched base on the flanking region of the DNAzyme. Varying the position of the mismatch is shown in the Examples to vary the degree of selectivity and the extent of cleavage. It is therefore possible to design DNAzymes that control the extent of cleavage, permitting regulated gene knockdown. For example, it is possible to reduce
  • DNAzyme selectivity by varying the position of the mismatch on a flanking region, thereby permitting controlled cleavage of the wild-type sequence. This can be used to decrease protein production in a patient, regulating a disorder caused by over expression of a gene.
  • the administered dose of DNAzyme is between 0.1 mg and 1 g, more preferably between 1 mg and 60 mg.
  • a single therapeutic dose of the DNAzyme can be administered over time as a plurality of smaller doses.
  • two or more different DNAzymes are administered.
  • the DNAzymes target the same mutant allele (or its expressed product) but at different sites of mutation and/or cleavage. This has the benefit of increasing the extent of cleavage for any target.
  • Type II DNAzymes bearing the 10-23 catalytic motif were designed with different binding-arm (helices I and III) lengths and were synthesised as standard oligonucleotides. To facilitate DNAzymes nomenclature, we will refer to them as,
  • 0.269HIII5 13+9, where ⁇ 269 is the substrate name, Hill is helix III, 5 is the distance of the mismatch (compared to wild-type) from the cleavage site, and (13+9) is the lengths of helix III and helix I respectively.
  • cRNA substrate preparation cDNAs encoding the human AChR ⁇ and ⁇ subunits were subcloned into pcDNA3.1hygro (Invitrogen). Missense mutations that underlie slow channel congenital myasthenic syndromes were introduced by the SculptorTM in vitro mutagenesis system (Amersham Pharmacia Biotech).
  • Plasmids harbouring the mutant cDNA were checked by DNA 32 sequencing.
  • 32 P-labelled cRNA substrates containing the full coding sequence of mutant and wild-type subunits were synthesised using the MegascriptTMT7 in vitro transcription kit (Ambion Biosciences).
  • target cRNA and DNAzymes were incubated either in 10 mM MgCI 2 , 50 mM Tris pH 7.5 or under simulated physiological conditions (2 mM MgCI 2 , 150 mM KCI, 50 mM Tris pH 7.5, 37°C) in a volume of 30 ⁇ l for four hours.
  • the reactions were carried out under single turn-over conditions (ie. with excess enzymes) using a molar ratio for enzyme: substrate of 10: 1.
  • the reaction was stopped by adding 90 ⁇ l stop buffer (95% formamide, 0.025% xylene cyanol, 0.025% bromophenol blue, 18 mM EDTA and 0.025% SDS).
  • the percentage of cleavage was calculated: (P1+P2/ P1+P2+S ) x 100, where P1 and P2 are the 5' and 3'products, and S is the substrate.
  • the degree of selectivity was calculated, (100 - mismatched activity). For example, the 1561-11115(10+10) enzyme cleaved 45% whereas its matched counterpart cleaved 72% of the substrate.
  • DNAzymes were designed to target mutations that do not introduce cleavage sites. Putative cleavage sites were identified near the mutation and DNAzymes designed with arms that perfectly match the mutant sequence but have a mismatch in the binding arm for the wild-type counterpart.
  • Both wild-type and mutant cRNA have the same DNAzyme cleavage site, but differ through the binding arm mismatch with the wild type sequence. Thus, selectivity would be obtained through the effects of mismatch on cleavage activity.
  • 32 P-labelled cRNA substrates were used and the cleaved products signal quantitated by phosphoimaging. In the these experiments the efficacy of the DNAzyme was measured in terms of selectivity rather than cleavage efficiency.
  • DNAzyme activity is calculated by normalising the percent cleavage of DNAzymes with mismatched arms to percent cleavage obtained for the equivalent DNAzyme with matched arms. The degree of selectivity is then defined as the difference in cleavage activity of the equivalent DNAzymes with perfectly matched or mismatched binding arms (see methods).
  • Missense mutation ⁇ S269l which creates an AU cleavage site, has two additional GU cleavage sites in close proximity. After showing 100% selectivity of targeting the mutation site, we examined the binding arm mismatch approach by targeting the two cleavage sites either side of the mutation. Two asymmetric DNAzymes were designed to target these sites. ⁇ 269HI5(9+13) targets the cleavage site five nucleotides upstream of the mutation creating a mismatch with wild-type in helix I at position 1.5 (according to ribozymes nomenclature of Hertel et al., Nucleic Acids Res., 1992; 20(12):3252. The second cleavage site was targeted by the 269HIII5(13+9) DNAzyme creating the mismatch with the wild-type in helix III at 16.5.
  • the ⁇ 269H 15(9+13) enzyme cleaved 43% of the mismatched target (wild type) compared with 72% of its matched one (mutant) (57% degree of selectivity).
  • the ⁇ 269HIII5 (13+9) DNAzyme showed a dramatic reduction for the wild-type target, cleaving only 3%, which was at least 20 fold less than its matched mutant counterpart which showed 95% selectivity.
  • the SCCMS mutant ⁇ V156M is due to a G 466 to A nucleotide substitution, which does not create a putative cleavage site. However, there are two putative cleavage sites in close proximity to the mutation. DNAzymes were designed to target these two cleavage sites. DNAzyme 156HI3 (10+10), was designed to target the cleavage site three nucleotides upstream of the mutation site , creating a mismatch with the wild-type cRNA is in helix I at position 1.3. Similarly, DNAzyme ⁇ 156HIII6 (10+10) created a mismatch in helix III at 16.6.
  • a set of DNAzymes were designed to target the n472AU site of ⁇ VI56M.
  • Symmetrical (10+10) DNAzymes were designed with sequential mismatches in the binding arms for the mutant target, helix I (1.1, 1.2, 1.3, 1.4, 1.5; and helix III (16.1 , 16.2, 16.3, 16.4, 16.5).
  • helix I 1.1, 1.2, 1.3, 1.4, 1.5
  • helix III (16.1 , 16.2, 16.3, 16.4, 16.5
  • helix I For helix I, a mismatch at position 1.1 showed no cleavage. This is expected since it is a required position for cleavage. A mismatch at position 1.2 gave very low cleavage activity (15%). As the mismatch distance of the mismatch from the cleavage site increases, the enzymes become more tolerant. Position 1.3 mismatch showed 44% activity, which is 2 fold less than the matched substrate. Positions 1.4 and 1.5 reduced the cleavage by 2.3 and 2 folds respectively. By contrast, except for position 16.2, mismatches in helix III have less effect on catalytic activity. In general a mismatch in helix I had a far more pronounced effect than a mismatch in helix III suggesting a vital role for this helix in establishing the enzyme activity.
  • n472HIII5 (10+10) DNAzyme which targets ⁇ V156M at n472AU site, was redesigned with asymmetric arms (13+9).
  • n472HIII5 (13+9) showed more tolerance to the mismatch by cleaving 62% of the wild type substrate compared to 45% for the (10+10) enzyme.
  • the degree of selectivity for n472HIII5 (10+10) was calculated to be 37% and dropped to 18% for n472HIII5 (13+9) which has a longer binding-arm.
  • n694GT Another site (n694GT) is located near a fourth SCCMS mutation in the AChR ⁇ subunit, ⁇ S226F.
  • a DNAzyme with asymmetric arms (13+9) cut with high efficiency and cleaved 85% of the substrate.
  • the 8-17 catalytic motif cleaves the phosphodiester bond between A and G 5'- 3', where G forms a wobble pair with U at the first base pair.
  • Symmetrical (10+10) DNAzymes were designed for each motif, targeting two adjacent cleavage sites in the ⁇ subunit full-length cRNA.
  • the cleavage site for the 10-23 motif was GU. Both sites were shown to be in an un-folded region by the computer prediction M-fold.
  • In vitro cleavage reaction was carried out under simulated physiological conditions for four hours. The percentage of cleavage was calculated; the 8-17 cleaved 44% of the substrate, whereas the 10-23 cleaved 62%. ii.
  • the 10-23 motif showed more catalytic efficiency than the 8-17, it was further investigated under different conditions. Unlike the 8-17, which has only one cleavage site, the RY rule gives the 10-23 four cleavage site possibilities: GU, GC, AU, and AC. The cleavage efficiency for enzymes targeting each of these sites was compared. Three different mutation regions among the ⁇ -subunit gene were targeted. In order to minimize the effect of differences in folding among the RNAs, the cleavage sites under comparison were selected to be as close as possible to each other. Three adjacent cleavage sites, GU, AU, and AC, around the ⁇ V156M mutation, were targeted by three symmetric enzymes (10+10).
  • the cleavage reactions were carried out in three different Mg 2+ concentrations, 50, 10, and 2mM (simulated physiological conditions).
  • the GU site was cleaved by 79%, 66% and 60% in 50, 10 mM Mg 2+ and physiological conditions respectively.
  • the AU site was also cleaved at high efficiency, but with 10% more efficiency than the GU one under all the examined conditions.
  • the AC site was cleaved at very low efficiency, 35%, 25% and 10% under the same conditions.
  • Another region near the ⁇ S226T was targeted using two asymmetric DNAzymes (13+9) at226GU and226AC sites. The overall behaviour of both GU and AC-cleaving enzymes were similar to that observed in targeting the ⁇ VI56M region.
  • the examined sites were then compared with respect to the incubation time under physiological conditions.
  • Each of the three DNAzymes targeting the ⁇ V156M region, 156AU (10+10), 156GU (10+10) and 156AC (10+10) was incubated with the substrate for different times (15 m, 30 m, 1h, 2h, 3h, 4h, 8h).
  • the AU and GU substrates were cleaved by 88% and 76% respectively after eight hours.
  • the AC site was cleaved by only 32%.
  • the AU enzyme cleaved 50% of the substrate after 30 minutes and the GU enzyme cleaved 50% after 1 hour.
  • Both DNAzymes targeted the 269 region, 269GU (13+9) and 269GC (13+9) and both cleaved their substrate by 50% after 3 hours, in contrast, the 269AC ( 13+9) cleaved only 16% after eight hours.
  • the 269AU site was targeted by a symmetrical (10+10) DNAzyme which cleaved 55% of the substrate; lengthening the arms to (13+13) increased the efficiency to 76%.
  • the enzyme-substrate concentrations also play a role in cleavage efficiency. All previous experiments were done under single turn-over with an enzyme-substrate molar ratio (100: 10) pmol. The cleavage efficiency was tested under different enzyme concentrations. 10 pmols of the substrate were incubated with different concentrations (3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 400, 800, 1600, 3200 pmols) of the 156AU (10+10) DNAzyme. Interestingly, it was noticed that as little as 3 pmol were able to show a detectable cleavage (14%). Also, 50% of the substrate was cleaved with 25 pmol. At 100 pmol the activity was 73% and started to show a steady stage of activity by increasing the concentration up to 3200 pmol, suggesting the saturation of the reaction. ii) Mixed enzymes
  • Full-length 32 P-labelled cRNA was incubated with 100 pmol 156AU (10+10) alone and 100 pmol 156GU (10+10) alone.
  • the DNAzymes were mixed (50 pmol each) and incubated with the substrate. Both DNAzymes target the same mutation region and compete for the same target sequence because they share most of the arm sequence.
  • the two DNAzymes targeting 156AU (10+10) and 156GU (10+10) cleaved 75% and 61 % respectively after four hours under simulated physiological conditions. In contrast mixing the two enzymes cleaved 83% of the substrate.
  • the third region was targeted at two sites, AC and GC, incubated separately and mixed with the substrate.
  • the AC was cleaved by less than 26% whereas the GC was cleaved by 57%.
  • mixing the two enzymes cleaved the substrate as poor as the AC-cleaving enzyme alone (25%), in contrast the GC-enzyme alone cleaved 55%.

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des DNAzymes conçus pour le ciblage d'allèles mutants et le clivage en un site distant du site de mutation. Les DNAzymes se lient sélectivement avec un allèle mutant et comprennent un motif catalytique central ainsi que deux régions flanquantes. Au moins une de ces régions présente une séquence polynucléotidique complémentaire d'une région qui comporte la mutation dans l'allèle mutant.
PCT/GB2003/004614 2002-10-23 2003-10-23 Dnazyme WO2004038019A2 (fr)

Priority Applications (1)

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AU2003274382A AU2003274382A1 (en) 2002-10-23 2003-10-23 Dnazyme cleaving mutant polynucleotides

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GB0224663A GB0224663D0 (en) 2002-10-23 2002-10-23 DNAzyme
GB0224663.5 2002-10-23

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WO2004038019A2 true WO2004038019A2 (fr) 2004-05-06
WO2004038019A3 WO2004038019A3 (fr) 2004-08-19

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WO (1) WO2004038019A2 (fr)

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Publication number Priority date Publication date Assignee Title
CN105264084A (zh) * 2013-01-14 2016-01-20 中央研究院 静默egfr表达的脱氧核糖核酸酶

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WO1997037013A1 (fr) * 1996-04-02 1997-10-09 Commonwealth Scientific And Industrial Research Organisation Ribozymes asymetriques en forme de tete de marteau

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Publication number Priority date Publication date Assignee Title
WO1997037013A1 (fr) * 1996-04-02 1997-10-09 Commonwealth Scientific And Industrial Research Organisation Ribozymes asymetriques en forme de tete de marteau

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PHYLACTOU L A ET AL: "HAMMERHEAD RIBOZYMES TARGETED TO THE FBN1 MRNA CAN DISCRIMINATE A SINGLE BASE MISMATCH BETWEEN RIBOZYME AND TARGET" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 249, no. 3, 1998, pages 804-810, XP000914845 ISSN: 0006-291X *
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WOOD M J ET AL: "RIBOZYMES AND SIRNA FOR THE TREATMENT OF DISEASES OF THE NERVOUS SYSTEM" CURRENT OPINION IN MOLECULAR THERAPEUTICS, vol. 5, no. 4, 2003, pages 383-388, XP009029174 ISSN: 1464-8431 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105264084A (zh) * 2013-01-14 2016-01-20 中央研究院 静默egfr表达的脱氧核糖核酸酶
EP2943578B1 (fr) * 2013-01-14 2019-04-10 National Taiwan University Adn à activité catalytique servant à supprimer l'expression de l'egfr

Also Published As

Publication number Publication date
AU2003274382A8 (en) 2004-05-13
AU2003274382A1 (en) 2004-05-13
GB0224663D0 (en) 2002-12-04
WO2004038019A3 (fr) 2004-08-19

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