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WO2000009673A1 - Dnazymes et methodes de traitement de troubles en rapport avec le papillomavirus - Google Patents

Dnazymes et methodes de traitement de troubles en rapport avec le papillomavirus Download PDF

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Publication number
WO2000009673A1
WO2000009673A1 PCT/IB1999/001486 IB9901486W WO0009673A1 WO 2000009673 A1 WO2000009673 A1 WO 2000009673A1 IB 9901486 W IB9901486 W IB 9901486W WO 0009673 A1 WO0009673 A1 WO 0009673A1
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hpv
dnazyme
mrna
dnazymes
pharmaceutical composition
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PCT/IB1999/001486
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English (en)
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Lun-Quan Sun
Murray J. Cairns
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Johnson & Johnson Research Pty. Limited
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Priority to AU52986/99A priority Critical patent/AU5298699A/en
Publication of WO2000009673A1 publication Critical patent/WO2000009673A1/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
    • C12N15/1131—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 viruses
    • 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
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes

Definitions

  • This invention relates to using DNAzymes to treat and inhibit the onset of human papilloma virus-related disorders.
  • the DNAzymes accomplish this end by cleaving human papilloma viral mRNA, whose expression in human host cells is required for related disorders to occur.
  • HPV Human papilloma viruses
  • HPV Human papilloma viruses
  • Genital HPV infection first results in the formation of genital warts.
  • the Center for Disease Control and Prevention has estimated that nearly 1 million new cases of HPV-mediated genital warts are diagnosed every year in the United States alone.
  • HPV infection is often co-existent with disorders such as syphilis, gonorrhea, chlamydia, herpes simplex virus (“HSV”) , and human immunodeficiency virus (“HIV”).
  • HPV infection is common among all races and socioeconomic groups, and is prevalent throughout the world among sexually active people. As mentioned already, HPV infection first manifests itself in the form of genital warts. Unfortunately, however, the infection can later result in additional and far more severe disorders.
  • HPV is currently the central risk factor for cervical neoplasia and cervical cancer. Cervical cancer remains the second most common cancer among women worldwide.
  • HPV 16, 18, 31 and 45 combined account for over two-thirds of the viral types identified in cervical cancer specimens.
  • the risk of disease progression following HPV infection seems to be related to the persistence of the infection, HPV viral type and viral load.
  • cervical dysplasia and carcinoma are associated with HPV infection, the increasing prevalence of genital warts in young women may portend a future increase in the rate of resulting cervical dysplasia and carcinoma.
  • HPV s are small naked viruses with an icosahedral symmetry, 72 capsomers, and a double- stranded circular DNA genome.
  • the viral DNA is complexed with low-molecular weight histones of cellular origin.
  • the genome consists of a double- stranded DNA molecule of approximately 8,000 base pairs, having a molecular weight of 5 x 10 6 daltons. All of the genome's open-reading frames ("ORF's”) are located on one strand.
  • HPV types are numerous (Phelps; Shillitoe) , and examples of predominant types are provided in the Examples section below. All HPV types have a similar genetic organization ( Figure 1).
  • the viral genome is divided into three regions: (a) an early region
  • HPV gene expression is controlled by both viral and cellular factors.
  • the regulatory region in the HPV genome is called the upstream regulatory region ("URR") .
  • Transcriptional enhancers are present in the central part of the URR (Shillitoe) . These enhancers are composed of a number of sequence modules of low or moderate activity. These modules act cooperatively as a single, strong enhancer whose activity depends on interactions with cellular transcriptional factors such as j un/fos, NF-1 (nuclear factor-1), Oct-1 (octamer-binding factor-1), and TEF (transcriptional enhancing factor) .
  • a similar set of binding sites for these cellular proteins is generally found in the center of the URR of genital HPV s . Both API (activator protein-1) and NF1 recognition sites are consistent features of all enhancers.
  • the main enhancer in HPV s is keratinocyte-specific, which may help explain the tissue tropism of these viruses (Dollard) .
  • HPV genomes encode three transforming proteins: E7, E6 and E5. It was known early on that cervical carcinomas contain HPV genomes which, although disrupted and rearranged as a consequence of integration, uniformly express the E6 and E7 genes. This uniform expression is in contrast to the expression of several other early viral genes such as El, E2, E4 and E5, which are often separated from the viral promoter, deleted, or significantly rearranged. The consistent retention and expression of the E6 and E7 genes in tumors suggest that these genes are also required for the maintenance of the malignant state, and not merely for its development.
  • HPV E7 protein is an acidic phosphoprotein consisting of approximately 100 amino acid residues. While initial studies localized the E7 protein predominantly to the cytoplasm, subsequent immunofluorescence and cell fractional assays have revealed that E7 also resides within the nuclear compartment (Sato) .
  • the E7 protein binds to the retinoblastoma- susceptibility gene product ("RB") (Dyson) .
  • RB retinoblastoma- susceptibility gene product
  • the RB protein is a phosphoprotein which, in its under- phosphorylated form, appears to negatively regulate entry into the S-phase of the cell cycle. Initiation of the S-phase is accompanied by the specific phosphorylation of the RB protein via cyclin- dependent kinases (“cdks”) . When cells enter mitosis, the RB protein is dephosphorylated and thus returned to its growth-inhibitory form.
  • the E7 protein binds with the highest affinity to the under-phosphorylated form of RB, thereby interfering with a critical growth regulatory step in the cell cycle. Since the RB protein associates not only with HPV E7 protein but with several cell proteins as well (including the E2F transcriptional factor, cdk p34 cdc2, and phosphatase 1A) , it is likely that E7 also modulates several of these interactions. Indeed, the binding of E7 to RB leads to dissociation of an E2F/RB complex. This complex, in turn, appears to inhibit transcription of genes possessing E2F-binding motifs (Morris) .
  • HPV E6 protein contains approximately 150 amino acids. From immunofluorescence microscopy, it is known that E6 proteins from HPV 16 and 18 reside in the nuclear matrix as well as in non-nuclear membrane fractions.
  • HPV E6 gene also possesses its own independent transforming activities, in addition to its role in augmenting E7-mediated transformation of human genital keratinocytes, .
  • the E6 genes of both high- and low-risk HPV types are able to facilitate cellular immortalization, although the high-risk E6 proteins appear to be the most efficient at doing so (Crook) .
  • antisense nucleic acid technology has been one of the major tools of choice to inactivate genes whose expression causes disease and is thus undesirable.
  • the anti-sense approach employs a nucleic acid molecule that is complementary to, and thereby hybridizes with, an mRNA molecule encoding an undesirable gene. Such hybridization leads to the inhibition of gene expression.
  • Anti-sense technology suffers from certain drawbacks.
  • Anti-sense hybridization results in the formation of a DNA/target mRNA heteroduplex.
  • This heteroduplex serves as a substrate for RNAse H-mediated degradation of the target mRNA component.
  • the DNA anti-sense molecule serves in a passive manner, in that it merely facilitates the required cleavage by endogenous RNAse H enzyme.
  • This dependence on RNAse H confers limitations on the design of anti-sense molecules regarding their chemistry and ability to form stable heteroduplexes with their target mRNA' s .
  • Anti- sense DNA molecules also suffer from problems associated with non-specific activity and, at higher concentrations, even toxicity.
  • catalytic nucleic acid molecules As an alternative to anti-sense molecules, catalytic nucleic acid molecules have shown promise as therapeutic agents for suppressing gene expression, and are widely discussed in the literature (Haseloff; Breaker (1994) ; Koizumi; Otsuka; Kashani-Sabet; Raillard; and Carmi) .
  • a catalytic nucleic acid molecule functions by actually cleaving its target mRNA molecule instead of merely binding to it.
  • Catalytic nucleic acid molecules can only cleave a target nucleic acid sequence if that target sequence meets certain minimum requirements.
  • the target sequence must be complementary to the hybridizing regions of the catalytic nucleic acid, and the target must contain a specific sequence at the site of cleavage.
  • RNA molecules Catalytic RNA molecules (“ribozymes”) are well documented (Haseloff; Symonds; and Sun), and have been shown to be capable of cleaving both RNA
  • Ribozymes are highly susceptible to enzymatic hydrolysis within the cells where they are intended to perform their function. This in turn limits their pharmaceutical applications.
  • DNAzymes are single-stranded, and cleave both RNA (Breaker (1994); Santoro) and DNA (Carmi).
  • a general model for the DNAzyme has been proposed, and is known as the "10-23" model.
  • DNAzymes following the "10-23” model also referred to simply as “10-23 DNAzymes” have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate- recognition domains of seven to nine deoxyribonucleotides each. In vitro analyses show that this type of DNAzyme can effectively cleave its substrate RNA at purine :pyrimidine junctions under physiological conditions (Santoro) .
  • DNAzymes show promise as therapeutic agents. However, DNAzyme success against a disease caused by the presence of a known mRNA molecule is not predictable. This unpredictability is due, in part, to two factors. First, certain mRNA secondary structures can impede a DNAzyme' s ability to bind to and cleave its target mRNA. Second, the uptake of a DNAzyme by cells expressing the target mRNA may not be efficient enough to permit therapeutically meaningful results. For these reasons, merely knowing of a disease and its causative target mRNA sequence does not alone allow one to reasonably predict the therapeutic success of a DNAzyme against that target mRNA, absent an inventive step. Summary of the Invention
  • This invention provides a DNAzyme which specifically cleaves HPV mRNA, comprising
  • a catalytic domain that has the nucleotide sequence GGCTAGCTACAACGA and cleaves mRNA at any purine :pyrimidine cleavage site at which it is directed, (b) a binding domain contiguous with the 5' end of the catalytic domain, and (c) another binding domain contiguous with the 3' end of the catalytic domain, wherein the binding domains are complementary to, and therefore hybridize with, the two regions immediately flanking the purine residue of the cleavage site within the HPV mRNA, respectively, at which DNAzyme-catalyzed cleavage is desired, and wherein each binding domain is at least six nucleotides in length, and both binding domains have a combined total length of at least 14 nucleotides .
  • This invention also provides a pharmaceutical composition for treating or inhibiting the onset of an HPV-related disorder, which comprises the instant DNAzyme and a pharmaceutically acceptable carrier suitable for topical administration.
  • This invention further provides a method for inhibiting the onset of an HPV-related disorder in a subject at risk of contracting the disorder, which comprises topically administering to the subject a prophylactically effective dose of the instant pharmaceutical composition.
  • this invention provides a method for treating an HPV-related disorder in a subject, which comprises topically administering to the subject a therapeutically effective dose of the instant pharmaceutical composition.
  • FIG 1 shows the HPV 16 genomic structure and related targeting strategy.
  • "10-23" model-based DNAzymes were designed against the HPV 16 E6 gene at the AUG start codon region.
  • Figure 2 shows a kinetics study of anti-HPV 16 E6 DNAzymes.
  • a short synthetic RNA substrate oligonucleotide (final concentration l ⁇ M) was end- labeled with [ ⁇ - 32 P]ATP at 37°C for 30 minutes and
  • Figure 3 shows the cleavage activity of modified anti-E6 DNAzymes.
  • DNAzymes were chemically modified as indicated. Cleavage reactions were performed in the presence of 10 mM MgCl , 50 mM Tris.Cl, pH 7.5, at 37°C for 60 minutes.
  • Figure 4 shows the stability of the modified DNAzymes in human serum. 150 mM DNAzyme was incubated in 100% human serum (Sigma) . 5 ⁇ l of sample were withdrawn at various time points, and the DNAzyme was labeled with [ ⁇ - 3 P]ATP and assayed on a 16% PAGE.
  • Figure 5 shows a schematic description of a transient assay for DNAzyme-mediated down-regulation of the E6 gene.
  • Figure 6 shows the inhibition of E6 gene expression in a transient assay. After co-transfection of 10 ⁇ M
  • RNA samples were prepared from the cells, blotted onto membranes and hybridized with 32 P- labelled E6 probe. The signals were analyzed using a Phosphorlmager .
  • This invention is directed to using DNAzymes to treat and inhibit the onset of human papilloma virus- related disorders.
  • human papilloma virus, or HPV infects a subject, this infection manifests itself as genital warts. These warts often precede more serious HPV-related disorders such as cervical dysplasia and carcinoma.
  • HPV genes via mRNA synthesis, is vital to HPV s ability to infect and propagate disease in its human host.
  • Such mRNA expression is the "Achilles heel" at which this invention is directed, in that the invention employs DNAzymes which specifically cleave HPV mRNA and thereby prevent HPV gene expression.
  • this application provides a DNAzyme which specifically cleaves HPV mRNA, comprising
  • binding domain means a DNA molecule that specifically recognizes and cleaves a distinct target nucleic acid sequence, which can be either DNA or RNA.
  • the instant DNAzyme cleaves RNA molecules, and is of the "10-23" model, as shown in Figure 1, named so for historical reasons. This type of DNAzyme is described in Santoro.
  • the RNA target sequence requirement for the 10-23 DNAzyme is any RNA sequence consisting of NNNNNR*YNNNNNN, NNNNNNNNR*YNNNNN or NNNNNNR*YNNNNNNN, where R*Y is the cleavage site, R is A or G, Y is U or C, and N is any of G, U, C, or A.
  • the binding domain lengths can be of any permutation, and can be the same or different. Various permutations such as 7+7, 8+8 and 9+9 are envisioned, and are exemplified more fully in the Examples that follow. It is well established that the greater the binding domain length, the more tightly it will bind to its complementary mRNA sequence. Accordingly, in the preferred embodiment, each binding domain is nine nucleotides in length. In one embodiment, the instant DNAzyme has the sequence TCCCGAAAGGCTAGCTACAACGAATTGCAGT ( Figure 1) .
  • a 3' -3' inversion means the covalent phosphate bonding between the 3' carbons of the 3' terminal nucleotide and its adjacent nucleotide.
  • the instant DNAzymes can contain modified nucleotides.
  • Modified nucleotides include, for example, N3'-P5' phosphoramidate linkages, and peptide-nucleic acid linkages. These are well known in the art (Wagner) .
  • any contiguous purine: pyrimidine nucleotide pair within the HPV mRNA can serve as a cleavage site.
  • purine:uracil is the desired purine:pyrimidine cleavage site.
  • the HPV mRNA region containing the cleavage site can be any region.
  • the location within the mRNA at which DNAzyme-catalyzed cleavage is desired can be the translation initiation site, a splice recognition site, the 5' untranslated region, or the 3' untranslated region.
  • the cleavage site is located at the translation initiation site.
  • HPV mRNA means any mRNA sequence encoded by an HPV strain, such as HPV 16.
  • HPV 16 proteins E5, E6 and E7 examples include those encoding HPV 16 proteins E5, E6 and E7.
  • HPV mRNA to which the instant DNAzyme is directed encodes proteins E6 or E7.
  • HPV mRNA's include both mature as well as immature mRNA's.
  • This invention also provides a pharmaceutical composition for treating or inhibiting the onset of an HPV-related disorder, which comprises the instant DNAzyme and a pharmaceutically acceptable carrier suitable for topical administration.
  • HPV-related disorder means any disease or physiological abnormality resulting from HPV infection.
  • the HPV-related disorder is cervical carcinoma, cervical dysplasia or genital warts.
  • Other HPV-related disorders are exemplified in the Examples section below.
  • topically administering the instant pharmaceutical compositions can be effected or performed using any of the various methods and delivery systems known to those skilled in the art.
  • the topical administration can be performed, for example, transdermally, orally, transmucosally, and via topical injection.
  • compositions for topical administration are well known in the art, as are methods for combining same with active agents to be delivered.
  • topical carriers and their uses are well known in the art (Ramchandani; Barry; Wenniger; Martindale's Parmacopoeia; U.S. Pharmacopeoia) .
  • the following delivery systems, which employ a number of routinely used carriers, are only representative of the many embodiments envisioned for administering the instant composition.
  • Dermal delivery systems include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microe ulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone) .
  • the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer.
  • Transmucosal delivery systems include tablets, suppositories, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids) , and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid) .
  • solubilizers and enhancers e.g., propylene glycol, bile salts and amino acids
  • other vehicles e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid
  • Injectable drug delivery systems include solutions, suspensions, gels, microspheres and polymeric injectables, and can comprise excipients such as solubility-altering agents (e.g., ethanol, propylene glycol and sucrose) and polymers (e.g., polycaprylactones and PLGA's).
  • solubility-altering agents e.g., ethanol, propylene glycol and sucrose
  • polymers e.g., polycaprylactones and PLGA's.
  • Oral delivery systems include tablets and capsules. These can contain excipients such as binders (e.g., hydroxypropyl ethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch) , diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.g., stearates and talc) .
  • excipients such as binders (e.g., hydroxypropyl ethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch) , diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellul
  • inhibiting the onset of an HPV- related disorder means either lessening the severity of the disorder once HPV infection occurs, or preventing the onset of the HPV-related disorder entirely. In the preferred embodiment, inhibiting the onset of an HPV- related disorder means preventing its onset entirely.
  • Treating an HPV-related disorder means either slowing, stopping or reversing the disorder's progression. In the preferred embodiment, “treating" an HPV-related disorder means reversing the disorder's progression, ideally to the point of eliminating the disorder itself.
  • This invention further provides a method for inhibiting the onset of an HPV-related disorder in a subject at risk of contracting the disorder, which comprises topically administering to the subject a prophylactically effective dose of the instant pharmaceutical composition.
  • Subjects at risk of contracting HPV-related disorders include, for example, (a) those infected with high-risk HPV types; (b) those having abnormal PAP Smear results; (c) those afflicted with cervical cancer; and (d) those who are immuno- compromised.
  • the prophylactically effective dose contains between about 0.1 mg and about 1 g of the instant DNAzyme. In another embodiment, the prophylactically effective dose contains between about 1 mg and about 100 mg of the instant DNAzyme. In a further embodiment, the prophylactically effective dose contains between about 10 mg and about 50 mg of the instant DNAzyme. In yet a further embodiment, the prophylactically effective dose contains about 25 mg of the instant DNAzyme.
  • this invention provides a method for treating an HPV-related disorder in a subject, which comprises topically administering to the subject a therapeutically effective dose of the instant pharmaceutical composition.
  • the therapeutically effective dose contains between about 0.1 mg and about 1 g of the instant DNAzyme. In another embodiment, the therapeutically effective dose contains between about 1 mg and about 100 mg of the instant DNAzyme. In a further embodiment, the therapeutically effective dose contains between about 10 mg and about 50 mg of the instant DNAzyme. In yet a further embodiment, the therapeutically effective dose contains about 25 mg of the instant DNAzyme.
  • HPV HPV-containing carcinomas and cell lines
  • E6 and E7 The oncogenic function of HPV has been assigned primarily to two early genes, E6 and E7. As discussed previously, these transforming genes are expressed in HPV-containing carcinomas and cell lines, and have transforming potential both in vitro and in vivo. Inhibition of E6 and E7 can be achieved by using antisense transcripts and oligonucleotides. This inhibition leads to reduced growth rates and loss of the transformed phenotype of cervical and oral carcinoma cell lines (Cowsert; Hu; von Knebel) .
  • the HPV 16 E6 gene has been chosen as a target for DNAzyme action. Based on previous data, the region around the translation initiation site (AUG) of the E6 gene was chosen as the target sequence (Sun (1995a); Sun (1995b)) ( Figure 1).
  • DNAzymes that were designed against the E6 gene are based on the "10-23" model (Santoro) .
  • a range of DNAzymes containing the 10-23 catalytic domain targeted against the translation initiation region of the HPV E6 gene were synthesized with various modifications and arm lengths as shown in Figure 1.
  • the product and unreacted substrate were resolved by electrophoresis on a denaturing 16% polyacrylamide sequencing gel.
  • the gel was then exposed to a phosphor-storage screen for scanning in a Molecular Dynamics Phosphorlmager .
  • the extent of reaction for each time point was determined by densitometry using Image Quant software (Molecular Dynamics) .
  • the percentage of band intensity volume in the cleavage product was compared to that of the combined substrate, and product intensity was determined. The results were then analyzed graphically in a plot against time.
  • %P %P ⁇ - C-exp[- kt]
  • %P the percentage product
  • k the first order rate constant.
  • the first order rate constant (k ⁇ bs) was used to compare the rates of different DNAzymes.
  • a range of DNAzymes containing the 10-23 catalytic domain targeted to the HPV 16 E6 translation initiation site were synthesized with various chemical modifications and binding arm lengths. These DNAzymes were then compared by analyzing their single turnover kinetics on a short synthetic RNA substrate ( Figure 2) .
  • the most active DNAzymes are those with 8-base arms, either unmodified or modified at the 3' terminal by an amine group. These molecules have observed first order rate constants of 0.23 and 0.21 min -1 , respectively. The activity of the amine-modified DNAzyme was not affected by the extension to the terminal phosphate.
  • a range of chemically modified oligonucleotides were synthesized and tested in an in vitro cleavage assay.
  • the DNAzyme with 8/8 arms targeting the E6 start codon was separately modified by (i) a 3 '-3'- terminal base inversion, (ii) a 3 ' -phosphorpropylamine group, and (iii) 1-5 bp phosphorothioate linkages (i.e. "phosphorothioate cap”) in the DNAzyme arms. All of these modifications maintained DNAzyme activity to varying degrees, with the 3 '-inversion showing the greatest ability to preserve activity, followed by the phosphorpropylamine modification, and the single base phosphorothioate cap (Figure 3).
  • DNAzyme stability was tested in 100% human serum. The experiment was done using 10 ⁇ M unlabelled DNAzyme in 1 ml 100% human serum incubated at 37 °C. Duplicate 5 ⁇ l samples were removed at the time points of 0, 2, 8, 24, 48, and 96 hours for DNAzymes 41 (7/7 arms unmodified), 42 (7/7 arms, 3' -inversion) , 23 (7/7 arms, 3' -amine), 24 (7/7 arms, 1 ' -phosphorothioate caps), 26 (7/7 arms, 3' -phosphorothioate caps) and 28 (7/7 arms, 5' -phosphorothioate caps). Immediately upon sampling, 295 ⁇ l TE were added to the 5 ⁇ l aliquot, and phenol/chloroform extraction was performed.
  • a transient assay system was used for anti-HPV DNAzyme testing ( Figure 5) .
  • This assay is based on co- transfection of an HPV 16 E6 expression vector and DNAzymes into 3T3 cells to measure potential DNAzyme- mediated suppression of target RNA.
  • 3T3 cells were plated at 3xl0 5 cells per well in 6-well plates. The following day, transfections were performed using DOTAP (N-[l-(2,3- dioleoyloxy) -N, N, N-trimethyl-ammoniummethylsulfate) mixed with 2-5 ⁇ g of plasmid DNA (pCDNA3.E6) and 10 mM DNAzyme or control oligonucleotides. After 17 hours of incubation, the cells were washed, trypsinised and resuspended. RNA was then extracted using a Qiagen RNeasy kit (Qiagen) . Typically, approximately 15 ⁇ g of RNA was obtained per sample.
  • DOTAP N-[l-(2,3- dioleoyloxy) -N, N, N-trimethyl-ammoniummethylsulfate
  • pCDNA3.E6 plasmid DNA
  • 10 mM DNAzyme or control oligonucleotides After
  • RNA (2 ⁇ g) was then added to three parts of formamide/formaldehyde buffer and heated to 65°C for 5 minutes. The mixture was placed on ice, and an equal part of 20x SSC buffer was added. The RNA samples were then blotted on to a Hybond membrane pre-soaked in lOx SSC with a Bio-Rad dot-blot apparatus. The blot was then hybridized with radio-labeled E6 probe, and analyzed using a Phosphorlmager .
  • DNAzymes 41 and 42 proved effective at inhibiting the expression of E6, when compared with DNAzyme 49.

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Abstract

La présente invention concerne un DNAzyme qui clive spécifiquement l'ARNm du papillomavirus humain et qui comprend un domaine catalytique à 15 nucléotides et deux domaines de liaison, ces deux domaines de liaison étant contigus l'un avec l'extrémité 5' du domaine catalytique, l'autre avec l'extrémité 3' du domaine catalytique. Cette invention porte également sur une composition pharmaceutique conçue pour empêcher l'apparition d'un trouble en rapport avec le papillomavirus, ou pour traiter cette pathologie, et comprenant le DNAzyme susmentionné ainsi qu'un vecteur pharmaceutiquement acceptable pour administration locale. Enfin, cette invention concerne des méthodes d'utilisation de ladite composition pharmaceutique pour enrayer l'apparition de troubles en rapport avec le papillomavirus ou pour traiter cette pathologie.
PCT/IB1999/001486 1998-08-13 1999-08-11 Dnazymes et methodes de traitement de troubles en rapport avec le papillomavirus WO2000009673A1 (fr)

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001011023A1 (fr) * 1999-08-04 2001-02-15 Johnson & Johnson Research Pty Ltd Traitement d'affections inflammatoires et malignes
WO2004002416A2 (fr) * 2002-06-26 2004-01-08 The Penn State Research Foundation Procedes et substances pour traiter des infections au virus du papillome humain
EP1397489A2 (fr) * 2000-04-13 2004-03-17 Medical University of South Carolina Agents toxiques, riboyzmes, andyzmes et oligonucleotides antisens specifiques des tissus et des pathogenes et procede d'utilisation associe
EP1611231A2 (fr) * 2003-02-21 2006-01-04 The Penn State Research Foundation Compositions interferant avec l'arn, et procedes correspondants
EP1702983A2 (fr) 2000-04-13 2006-09-20 Medical University of South Carolina Agents toxiques, ribozymes, ADNzymes et oligonucléotides antisens spécifiques des tissus et des pathogènes et procédé d'utilisation associé
EP2533763A2 (fr) * 2010-02-10 2012-12-19 Sterna Biologicals GmbH & Co. Kg Composition pharmaceutique dermatologique adaptée aux oligonucléotides
CN103013953A (zh) * 2012-10-19 2013-04-03 中国科学院成都生物研究所 一种剪切dna的dna酶
CN103525819A (zh) * 2013-10-22 2014-01-22 上海市农业科学院 一种用于抑制prrsv感染的脱氧核酶
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US9724404B2 (en) 2009-04-13 2017-08-08 INSERM (Institut National de la Santé et de la Recherche Médicale) HPV particles and uses thereof
US10117947B2 (en) 2013-09-18 2018-11-06 Aura Biosciences, Inc. Virus-like particle conjugates for diagnosis and treatment of tumors
US11207339B2 (en) 2015-10-30 2021-12-28 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Targeted cancer therapy

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EP1702983A3 (fr) * 2000-04-13 2007-01-10 Medical University of South Carolina Agents toxiques, ribozymes, ADNzymes et oligonucléotides antisens spécifiques des tissus et des pathogènes et procédé d'utilisation associé
EP1397489A2 (fr) * 2000-04-13 2004-03-17 Medical University of South Carolina Agents toxiques, riboyzmes, andyzmes et oligonucleotides antisens specifiques des tissus et des pathogenes et procede d'utilisation associe
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WO2004002416A3 (fr) * 2002-06-26 2005-02-03 Penn State Res Found Procedes et substances pour traiter des infections au virus du papillome humain
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