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WO2013153139A1 - Méthodes pour le traitement et le diagnostic de la leucémie aiguë - Google Patents

Méthodes pour le traitement et le diagnostic de la leucémie aiguë Download PDF

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Publication number
WO2013153139A1
WO2013153139A1 PCT/EP2013/057530 EP2013057530W WO2013153139A1 WO 2013153139 A1 WO2013153139 A1 WO 2013153139A1 EP 2013057530 W EP2013057530 W EP 2013057530W WO 2013153139 A1 WO2013153139 A1 WO 2013153139A1
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WIPO (PCT)
Prior art keywords
compound
snorna
snord113
expression
snornas
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PCT/EP2013/057530
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English (en)
Inventor
Pierre BROUSSET
Wilfried VALLERON
Laure BERQUET
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Université Paul Sabatier (Toulouse III)
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Publication of WO2013153139A1 publication Critical patent/WO2013153139A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to a method of identifying a patient having or at risk of having or developing an acute leukemia, comprising measuring in a sample obtained from said patient, at least one snoRNAs of the DLK1-DI03 locus.
  • the invention relates also to a compound which inhibits the expression of a snoRNA of the DLK1-DI03 locus for use in the treatment of acute leukemia in a patient in need thereof.
  • ncRNAs non-coding RNAs
  • small nucleolar RNAs are 60 to 300 nucleo tide- long ncRNAs that are excised from the intron regions of pre-mRNAs, usually those encoding fundamental housekeeping proteins (Kiss T., 2002).
  • snoRNAs There are two structurally distinct classes of snoRNAs, called the box C/D and H/ACA snoRNAs, which function mainly as guide RNAs in the site-specific 2 '-O-methylation and pseudouridylation of rRNAs, respectively (Kiss T., 2002).
  • C/D the box C/D
  • H/ACA snoRNAs which function mainly as guide RNAs in the site-specific 2 '-O-methylation and pseudouridylation of rRNAs, respectively.
  • SNORD1 12 to SNORD1 16 are encoded by multiple, tandemly- arranged intronic genes that display high sequence similarity (Cavaille J. et al, 2000).
  • the invention relates to The present invention relates to a method of identifying a patient having or at risk of having or developing an acute leukemia, comprising measuring in a sample obtained from said patient, at least one snoR As of the DLK1-DI03 locus.
  • the invention relates also to a compound which inhibits the expression of a snoR A of the DLK1-DI03 locus for use in the treatment of acute leukemia in a patient in need thereof.
  • snoRNAs for "Small nucleolar RNAs" denotes a class of small RNA molecules that primarily guide chemical modifications of other RNAs, mainly ribosomal RNAs, transfer RNAs and small nuclear RNAs.
  • snoRNAs There are two main classes of snoRNA, the C/D box snoRNAs which are associated with methylation, and the H/ACA box snoRNAs which are associated with pseudouridylation.
  • snoRNAs are commonly referred to as guide RNAs but should not be confused with the guide RNAs that direct RNA editing in trypanosomes.
  • snoRNAs pertaining to the invention are known per se and sequences of them are publicly available from the data bases http ://www-snorna.biotoul. fr/ index.php and http://www.ensembl.org.
  • the snoRNAs of the invention are listed in Table A: snoRNAs snoRNAs Accession
  • Table A list of the snoRNAs according to the invention
  • a patient denotes a mammal.
  • a patient according to the invention refers to any patient (preferably human) afflicted with or susceptible to be afflicted with acute leukemia.
  • a first aspect of the invention relates to a method of identifying a patient having or at risk of having or developing an acute leukemia, comprising measuring in a sample obtained from said patient, at least one snoRNAs of the DLK1-DI03 locus.
  • the invention relates to a method of identifying a patient having or at risk of having or developing an acute leukemia, comprising a step of measuring in a sample obtained from said patient the expression level of at least one snoRNAs selected from the groups consisting of SNORD112, SNORD113-4, SNORD113-6, SNORD113-7, SNORD113- 8, SNORD113-9, SNORD114-1, SNORD114-3, SNORD114-9, SNORD114-12, SNORD114-14, SNORD114-17, SNORD114-22 or SNORD114-26.
  • snoRNAs selected from the groups consisting of SNORD112, SNORD113-4, SNORD113-6, SNORD113-7, SNORD113- 8, SNORD113-9, SNORD114-1, SNORD114-3, SNORD114-9, SNORD114-12, SNORD114-14, SNORD114-17, SNORD114-22 or SNORD114-26.
  • the invention relates to a method of identifying a patient having or at risk of having or developing an acute leukemia, comprising a step of measuring in a sample obtained from said patient the expression level of all snoR As of the group consisting of SNORD112, SNORD113-6, SNORD113-7, SNORD113-8, SNORD113-9, SNORD 114-1.
  • the invention in another embodiment, relates to a method of identifying a patient having or at risk of having or developing an acute leukemia, comprising a step of measuring in a sample obtained from said patient the expression level of all the snoRNAs SNORD114-1.
  • the acute leukemia may be an acute myeloblastic, an acute lymphoblastic or acute promyelocytic leukemia.
  • the method of the invention may further comprise a step consisting of comparing the expression level of at least one snoRNAs in the sample with a control, wherein detecting differential in the expression level of the snoRNAs between the sample and the control is indicative of having or a risk of having or developing an acute leukemia.
  • the control may consist in sample associated with a healthy patient not afflicted with acute leukemia or in a sample associated with a patient afflicted with acute leukemia.
  • hight expression level of at least one snoRNAs of the DLK1- DI03 locus is indicative of patient having or at risk of having or developing an acute leukemia.
  • measuring the expression level of the snoRNAs of the invention in the sample obtained from the patient can be performed by a variety of techniques.
  • the nucleic acid contained in the samples is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions.
  • the extracted snoRNAs is then detected by hybridization (e. g., Northern blot analysis) and/or amplification (e.g., RT-PCR).
  • hybridization e. g., Northern blot analysis
  • amplification e.g., RT-PCR
  • RT-PCR e.g., RT-PCR
  • RT-PCR e.g., Northern blot analysis
  • RT-PCR e.g., RT-PCR
  • RT-PCR e.g., RT-PCR
  • RT-PCR e.g., RT-PCR
  • RT-PCR e.g., Northern blot analysis
  • RT-PCR e.g., RT-PCR
  • RT-PCR e.g., RT-
  • the determination comprises contacting the sample with selective reagents such as probes or primers and thereby detecting the presence, or measuring the amount of snoRNAs originally in the sample.
  • Contacting may be performed in any suitable device, such as a plate, microtiter dish, test tube, well, glass, column, and so forth
  • the contacting is performed on a substrate coated with the reagent, such as a snoRNAs array.
  • the substrate may be a solid or semi-solid substrate such as any suitable support comprising glass, plastic, nylon, paper, metal, polymers and the like.
  • the substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc.
  • the contacting may be made under any condition suitable for a detectable complex, such as a snoRNAs hybrid, to be formed between the reagent and the snoRNAs of the sample.
  • Nucleic acids exhibiting sequence complementarity or homology to the snoRNAs of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin).
  • the probes and primers are "specific" to the snoRNAs they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC.
  • Tm melting temperature
  • SCC is a 0.15 M NaCl, 0.015 M Na-citrate
  • snoRNAs arrays or snoRNAs probe arrays which are macroarrays or microarrays of nucleic acid molecules (probes) that are fully or nearly complementary or identical to a plurality of snoRNAs molecules positioned on a support or support material in a spatially separated organization.
  • Macroarrays are typically sheets of nitrocellulose or nylon upon which probes have been spotted.
  • Microarrays position the nucleic acid probes more densely such that up to 10,000 nucleic acid molecules can be fit into a region typically 1 to 4 square centimeters.
  • Microarrays can be fabricated by spotting nucleic acid molecules, e.g., genes, oligonucleotides, etc., onto substrates or fabricating oligonucleotide sequences in situ on a substrate. Spotted or fabricated nucleic acid molecules can be applied in a high density matrix pattern of up to about 30 non-identical nucleic acid molecules per square centimeter or higher, e.g. up to about 100 or even 1000 per square centimeter. Microarrays typically use coated glass as the solid support, in contrast to the nitrocellulose-based material of filter arrays. By having an ordered array of snoR As-complementing nucleic acid samples, the position of each sample can be tracked and linked to the original sample.
  • nucleic acid molecules e.g., genes, oligonucleotides, etc.
  • array devices in which a plurality of distinct nucleic acid probes are stably associated with the surface of a solid support are known to those of skill in the art.
  • Useful substrates for arrays include nylon, glass, metal, plastic, latex, and silicon.
  • Such arrays may vary in a number of different ways, including average probe length, sequence or types of probes, nature of bond between the probe and the array surface, e.g. covalent or non-covalent, and the like.
  • the population of target nucleic acids is contacted with the array or probes under hybridization conditions, where such conditions can be adjusted, as desired, to provide for an optimum level of specificity in view of the particular assay being performed.
  • Suitable hybridization conditions are well known to those of skill in the art and reviewed in Sambrook et al. (2001). Of particular interest in many embodiments is the use of stringent conditions during hybridization. Stringent conditions are known to those of skill in the art.
  • snoRNAs quantification method may be performed by using stem-loop primers for reverse transcription (RT) followed by a real-time TaqMan® probe.
  • said method comprises a first step wherein the stem-loop primers are annealed to snoRNAs targets and extended in the presence of reverse transcriptase. Then snoRNAs-specific forward primer, TaqMan® probe, and reverse primer are used for PCR reactions. Quantitation of snoRNAs is estimated based on measured CT values.
  • Expression level of a snoRNA may be expressed as absolute expression level or normalized expression level.
  • expression levels are normalized by correcting the absolute expression level of a snoRNA by comparing its expression to the expression of a mRNA that is not a relevant for determining patient having or at risk of having or developing an acute leukemia, e.g., a housekeeping mRNA that is constitutively expressed.
  • Suitable mRNA for normalization includes housekeeping mRNAs such as the 5S rRNA. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources.
  • a further aspect of the invention relates to a method for monitoring the efficacy of a treatment for acute leukemia.
  • Methods of the invention can be applied for monitoring the treatment (e.g., drug compounds) of the patient.
  • the effectiveness of an agent to affect the expression level of the snoR As (as herein after described) according to the invention can be monitored during treatments of patients receiving acute leukemia treatments.
  • acute leukemia treatment relates to any type of acute leukemia therapy undergone by the acute leukemia patients previously to collecting the cancerous tissue samples, including anticancer treatment.
  • the present invention relates to a method for monitoring the treatment of patient affected with an acute leukemia, said method comprising the steps consisting of:
  • the invention relates to a method for diagnosis acute leukemia in a patient comprising a step consisting of determining the expression level of at least one snoRNAs of the DLK1-DI03 locus.
  • the invention relates to a method for diagnosis acute leukemia in a patient comprising a step consisting of determining the expression level of at least one snoRNAs selected from the group consisting of SNORD112, SNORD113-4, SNORD113-6, SNORD113-7, SNORD113-8, SNORD113-9, SNORD 114-1, SNORD114-3, SNORD114-9, SNORD114-12, SNORD114-14, SNORD114-17, SNORD114-22 or SNORD 114-26.in a sample obtained from said patient.
  • the acute leukemia may be an acute myeloblastic, an acute lymphoblastic or acute promyelocytic leukemia.
  • the sample according to the invention may be a blood, plasma, serum, lymph or bone marrow.
  • said sample is blood or bone marrow.
  • snoRNAS 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 snoRNAS are measured.
  • the term "detecting” as used above includes qualitative and/or quantitative detection (measuring levels) with or without reference to a control.
  • snoR As expression level may be measured for example by RT-PCR performed on the sample.
  • kits for performing the methods of the invention comprising means for measuring the expression level of the snoRNAs clusters of the invention in the sample obtained from the patient.
  • the kits may include probes, primers macroarrays or microarrays as above described.
  • the kit may comprise a set of snoRNAs probes as above defined, usually made of DNA, and that may be pre-labelled. Alternatively, probes may be unlabelled and the ingredients for labelling may be included in the kit in separate containers.
  • the kit may further comprise hybridization reagents or other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards.
  • the kit of the invention may comprise amplification primers (e.g. stem- loop primers) that may be pre-labelled or may contain an affinity purification or attachment moiety.
  • the kit may further comprise amplification reagents and also other suitably packaged reagents and materials needed for the particular amplification protocol.
  • the kit of the invention relates to a kit for identifying whether a patient has or is at risk of having or developing an acute leukemia, comprising means for measuring, in a sample obtained from said patient, at least one snoRNAs of the DLK1-DI03 locus.
  • the kit of the invention relates to a kit for identifying whether a patient has or is at risk of having or developing an acute leukemia, comprising means for measuring, in a sample obtained from said patient, at least one snoRNAs selected from the group consisting of SNORD112, SNORD113-4, SNORD113-6, SNORD113-7, SNORD113-8, SNORD113-9, SNORD114-1, SNORD114-3, SNORD114-9, SNORD114-12, SNORD114-14, SNORD114-17, SNORD114-22 or SNORD114-26.
  • snoRNAs selected from the group consisting of SNORD112, SNORD113-4, SNORD113-6, SNORD113-7, SNORD113-8, SNORD113-9, SNORD114-1, SNORD114-3, SNORD114-9, SNORD114-12, SNORD114-14, SNORD114-17, SNORD114-22 or SNORD114-26.
  • the kit of the invention relates to a kit which further comprise means for comparing the expression level of the snoRNAs in the sample with a control, wherein detecting differential in the expression level of the snoRNAs between the sample and the control is indicative of having, or a risk of having or developing an acute leukemia.
  • the control may consist in sample associated with a healthy patient not afflicted with acute leukemia or in a sample associated with a patient afflicted with acute leukemia.
  • a second aspect of the invention relates to a compound which inhibits the expression of a snoRNA of the DLK1-DI03 locus for use in the treatment of acute leukemia in a patient in need thereof.
  • the snoRNA to inhibit is selected from the group consisting of
  • the snoRNA to inhibit is the SNORD 114-1.
  • a compound which inhibits the expression of a snoRNA may be identifying by high throughput screening.
  • the term "compound which inhibits the expression of a snoRNA” refers to any nucleotidic compound able to prevent the action of the selected snoRNA.
  • the compound of the present invention is a compound that inhibits or reduces the activity of the selected snoRNA for example by inhibiting the production of the snoRNA selected. In this case, the production of selected the snoRNA in the organism after treatment is less than the amount produced prior to treatment.
  • One skilled in the art can readily determine whether the selected snoRNA expression has been inhibited in the organism, using for example the techniques for determining snoRNA transcript level.
  • Suitable compounds according to the invention include double-stranded RNA (such as short- or small-interfering RNA or "siRNA”), antagosnoRNAs, antisense nucleic acids, and enzymatic RNA molecules such as ribozymes.
  • siRNA short- or small-interfering RNA or "siRNA”
  • antagosnoRNAs antisense nucleic acids
  • enzymatic RNA molecules such as ribozymes.
  • RNA molecules such as ribozymes.
  • RNA molecules such as ribozymes.
  • dsRNA isolated double-stranded RNA
  • the dsRNA molecule is a "short or small interfering RNA” or "siRNA".
  • siRNA useful in the present methods comprise short double-stranded RNA from about 17 nucleotides to about 29 nucleotides in length, preferably from about 19 to about 25 nucleotides in length.
  • the siRNA comprise a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson-Crick base-pairing interactions (hereinafter "base-paired").
  • the sense strand comprises a nucleic acid sequence which is substantially identical to a nucleic acid sequence contained within the target snoRNA.
  • a nucleic acid sequence in a siRNA which is "substantially identical" to a target sequence contained within the target mRNA is a nucleic acid sequence that is identical to the target sequence, or that differs from the target sequence by one or two nucleotides.
  • the sense and antisense strands of the siRNA can comprise two complementary, single-stranded RNA molecules, or can comprise a single molecule in which two complementary portions are base-paired and are covalently linked by a single-stranded "hairpin" area.
  • the siRNA can also be altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, or modifications that make the siRNA resistant to nuclease digestion, or the substitution of one or more nucleotides in the siRNA with deoxyribonucleotides.
  • One or both strands of the siRNA can also comprise a 3' overhang.
  • a 3' overhang As used herein, a
  • the siRNA comprises at least one 3' overhang of 1 to about 6 nucleotides (which includes ribonucleotides or deoxyribonucleotides) in length, preferably from 1 to about 5 nucleotides in length, more preferably from 1 to about 4 nucleotides in length, and particularly preferably from about 2 to about 4 nucleotides in length.
  • the 3' overhang is present on both strands of the siRNA, and is 2 nucleotides in length.
  • each strand of the siRNA can comprise 3' overhangs of dithymidylic acid ("TT") or diuridylic acid ("uu”).
  • the siRNA can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as described above. Exemplary methods for producing and testing dsRNA or siRNA molecules are described in U.S. published patent application 2002/0173478 to Gewirtz and in U.S. published patent application 2004/0018176 to Reich et al, the entire disclosures of which are herein incorporated by reference.
  • an antisense nucleic acid refers to a nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-peptide nucleic acid interactions, which alters the activity of the target RNA.
  • Antisense nucleic acids suitable for use in the present methods are single-stranded nucleic acids (e.g., RNA, DNA, RNA-DNA chimeras, PNA) that generally comprise a nucleic acid sequence complementary to a contiguous nucleic acid sequence in a snoRNA.
  • the antisense nucleic acid comprises a nucleic acid sequence that is 50-100% complementary, more preferably 75-100% complementary, and most preferably 95-100% complementary to a contiguous nucleic acid sequence in an snoRNA.
  • Nucleic acid sequences for the snoRNAs are provided in Table A. Without wishing to be bound by any theory, it is believed that the antisense nucleic acids activate RNase H or some other cellular nuclease that digests the snoRNA/antisense nucleic acid duplex.
  • the inhibitor is an antagosnoRNA and/or an antisense oligonucleotide.
  • RNA as used herein refers to a chemically engineered small
  • the antagosnoRNA is complementary to the specific snoRNA target with either mis-pairing or some sort of base modification. AntagosnoRNAs may also include some sort of modification to make them more resistant to degradation. In one embodiment the antagosnoRNA is a chemically engineered cholesterol- conjugated single-stranded RNA analogue.
  • Inhibition of snoRNAs can also be achieved with antisense 2'-0-methyl (2'-0-Me) oligoribonucleotides, 2'-0-methoxyethyl (2'-0-MOE), phosphorothioates, locked nucleic acid (LNA), morpholino oligomers or by use of lentivirally or adenovirally expressed antagomirs (Stenvang and Kauppinen (2008), Expert Opin. Biol. Ther. 8(1):59-81).
  • MOE (2'-0-methoxyethyl phosphorothioate) or LNA (locked nucleic acid (LNA) phosphorothioate chemistry)-modification of single-stranded RNA analogous can be used to inhibit snoRNA activity.
  • LNA locked nucleic acid
  • Antisense nucleic acids can also contain modifications of the nucleic acid backbone or of the sugar and base moieties (or their equivalent) to enhance target specificity, nuclease resistance, delivery or other properties related to efficacy of the molecule. Such modifications include cholesterol moieties, duplex intercalators such as acridine or the inclusion of one or more nuclease-resistant groups.
  • Antisense nucleic acids can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as described below. Exemplary methods for producing and testing are within the skill in the art; see, e.g., Stein and Cheng (1993), Science 261 : 1004 and U.S. Pat. No. 5,849,902 to Woolf et al, the entire disclosures of which are herein incorporated by reference.
  • an "enzymatic nucleic acid” refers to a nucleic acid comprising a substrate binding region that has complementarity to a contiguous nucleic acid sequence of a snoRNA, and which is able to specifically cleave the snoRNA.
  • the enzymatic nucleic acid substrate binding region is 50-100% complementary, more preferably 75-100% complementary, and most preferably 95-100% complementary to a contiguous nucleic acid sequence in a snoRNA.
  • the enzymatic nucleic acids can also comprise modifications at the base, sugar, and/or phosphate groups.
  • An exemplary enzymatic nucleic acid for use in the present methods is a ribozyme.
  • the enzymatic nucleic acids can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as described below.
  • Exemplary methods for producing and testing dsRNA or siRNA molecules are described in Werner and Uhlenbeck (1995), Nucl. Acids Res. 23:2092-96; Hammann et al. (1999), Antisense and Nucleic Acid Drug Dev. 9:25-31; and U.S. Pat. No. 4,987,071 to Cech et al, the entire disclosures of which are herein incorporated by reference.
  • the compound of the invention can be obtained using a number of standard techniques.
  • the compound of the invention can be chemically synthesized or recombinantly produced using methods known in the art.
  • the compound of the invention are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
  • Commercial suppliers of synthetic RNA molecules or synthesis reagents include, e.g., Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, 111., USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA) and Cruachem (Glasgow, UK).
  • a synthetic compound of the invention contains one or more design elements. These design elements include, but are not limited to: (i) a replacement group for the phosphate or hydroxyl of the nucleotide at the 5' terminus of the complementary region; (ii) one or more sugar modifications.
  • a synthetic compound of the invention has a nucleotide at its 5' end of the complementary region in which the phosphate and/or hydroxyl group has been replaced with another chemical group (referred to as the "replacement design"). In some cases, the phosphate group is replaced, while in others, the hydroxyl group has been replaced.
  • the replacement group is biotin, an amine group, a lower alkylamine group, an acetyl group, 2'0-Me (2'oxygen-methyl), DMTO (4,4'-dimethoxytrityl with oxygen), fluorescein, a thiol, or acridine, though other replacement groups are well known to those of skill in the art and can be used as well.
  • the sugar modification is a 2'0-Me modification.
  • the compound of the invention is resistant to degradation by nucleases.
  • nucleases One skilled in the art can readily synthesize nucleic acids which are nuclease resistant, for example by incorporating one or more ribonucleotides that are modified at the 2'-position into the snoRNAs. Suitable 2'-modified ribonucleotides include those modified at the 2'-position with fluoro, amino, alkyl, alkoxy, and O-allyl.
  • the present invention also relates to a vector comprising a compound according to the invention for use in the treatment of acute leukemia.
  • a vector may be a plasmid or a viral vector which express a compound like a siRNA.
  • the vector denotes a DNA structure in which a specific sequence; like a sequence encoding a siRNA, is contained.
  • the compound of the invention can be expressed from recombinant circular or linear DNA plasmids using any suitable promoter.
  • suitable promoters for expressing RNA from a plasmid include, e.g., the U6 or HI RNA pol III promoter sequences, or the cytomegalovirus promoters. Selection of other suitable promoters is within the skill in the art.
  • the recombinant plasmids of the invention can also comprise inducible or regulatable promoters for expression of the compound of the invention in the organism.
  • the compound of the invention that is expressed from recombinant plasmids can be isolated from cultured cell expression systems by standard techniques.
  • the compound of the invention which is expressed from recombinant plasmids can also be delivered to, and expressed directly in, the organism. The use of recombinant plasmids to deliver the compound of the invention to organism is discussed in more detail below.
  • the compound of the invention can be expressed from a separate recombinant plasmid, or can be expressed from a unique recombinant plasmid.
  • the compound of the invention is expressed as the nucleic acid precursor molecules from a single plasmid, and the precursor molecules are processed into the functional compound by a suitable processing system.
  • suitable processing systems include, e.g., the in vitro Drosophila cell lysate system as described in U.S. published application 2002/0086356 to Tuschl et al. and the E. coli RNAse III system described in U. S . published patent application 2004/0014113 to Yang et al, the entire disclosures of which are herein incorporated by reference.
  • plasmids suitable for expressing the compound of the invention are within the skill in the art. See, for example, Zeng et al. (2002), Molecular Cell 9: 1327-1333; Tuschl (2002), Nat. Biotechnol, 20:446-448; Brummelkamp et al. (2002), Science 296:550-553; Miyagishi et al. (2002), Nat. Biotechnol. 20:497-500; Paddison et al. (2002), Genes Dev. 16:948-958; Lee et al. (2002), Nat. Biotechnol. 20:500-505; and Paul et al. (2002), Nat. Biotechnol. 20:505-508, the entire disclosures of which are herein incorporated by reference.
  • a plasmid expressing the compound of the invention comprises a sequence encoding a compound precursor under the control of the CMV intermediate early promoter.
  • "under the control" of a promoter means that the nucleic acid sequences are located 3' of the promoter, so that the promoter can initiate transcription of the compound coding sequences.
  • the compound of the invention can also be expressed from recombinant viral vectors. It is contemplated that the compound of the invention can be expressed from separate recombinant viral vectors, or from a unique viral vector.
  • the compound expressed from the recombinant viral vectors can either be isolated from cultured cell expression systems by standard techniques, or can be expressed directly in organism. The use of recombinant viral vectors to deliver the compound to organism is discussed in more detail below.
  • the recombinant viral vectors of the invention comprise sequences encoding the compound of the invention and any suitable promoter for expressing the compound sequences. Suitable promoters include, for example, the U6 or HI RNA pol III promoter sequences, or the cytomegalovirus promoters. Selection of other suitable promoters is within the skill in the art.
  • the recombinant viral vectors of the invention can also comprise inducible or regulatable promoters for expression of the compound in organism.
  • Any viral vector capable of accepting the coding sequences for the compound of the invention can be used; for example, vectors derived from adenovirus (AV); adeno associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like.
  • the tropism of the viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.
  • lentiviral vectors of the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like.
  • AAV vectors of the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes.
  • an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2.
  • This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector.
  • Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J. E. et al. (2002), J Virol 76:791801, the entire disclosure of which is herein incorporated by reference.
  • Preferred viral vectors are those derived from AV and AAV.
  • a suitable AV vector for expressing the compound of the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells are described in Xia et al. (2002), Nat. Biotech. 20: 1006-1010, the entire disclosure of which is herein incorporated by reference.
  • Suitable AAV vectors for expressing the compound of the invention, methods for constructing the recombinant AAV vector, and methods for delivering the vectors into target cells are described in Samulski et al. (1987), J. Virol. 61 :3096-3101 ; Fisher et al. (1996), J.
  • the compound of the invention is expressed from a single recombinant AAV vector comprising the CMV intermediate early promoter.
  • a recombinant AAV viral vector of the invention comprises a nucleic acid sequence encoding a compound precursor in operable connection with a polyT termination sequence under the control of a human U6 RNA promoter.
  • operable connection with a polyT termination sequence means that the nucleic acid sequences encoding the sense or antisense strands are immediately adjacent to the polyT termination signal in the 5' direction.
  • the polyT termination signals act to terminate transcription.
  • the compound according to the invention can be administered to a patient by any means suitable for delivering these compounds to the patient.
  • the compound can be administered by methods suitable to transfect cells of the patient with these compounds, or with nucleic acids comprising sequences encoding these compounds.
  • the cells are transfected with a plasmid or viral vector comprising sequences encoding at least one compound.
  • Suitable enteral administration routes for the present methods include, e.g., oral, rectal, or intranasal delivery.
  • Suitable parenteral administration routes include, e.g., intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra- arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., intra-retinal injection, or subretinal injection); subcutaneous injection or deposition, including subcutaneous infusion (such as by osmotic pumps); direct application to the tissue of interest, for example by a catheter or other placement device (e.g., a retinal pellet or a suppository or an implant comprising a porous, non-porous, or gelatinous material); and inhalation.
  • intravascular administration e.g., intravenous bolus injection, intravenous infusion, intra- arterial bolus injection, intra-arterial infusion
  • a compound in the present methods, can be administered to the patient either as naked RNA, in combination with a delivery reagent, or as a nucleic acid (e.g., a recombinant plasmid or viral vector) comprising sequences that express the compound.
  • Suitable delivery reagents include, e.g, the Minis Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine), and liposomes.
  • Recombinant plasmids and viral vectors comprising sequences that express the compound, and techniques for delivering such plasmids and vectors to organism, are discussed above.
  • liposomes are used to deliver a compound (or nucleic acids comprising sequences encoding them) to a patient.
  • Liposomes can also increase the blood half-life of the gene products or nucleic acids.
  • Liposomes suitable for use in the invention can be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half- life of the liposomes in the blood stream.
  • liposomes for example, as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng. 9:467; and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, the entire disclosures of which are herein incorporated by reference.
  • the liposomes for use in the present methods can comprise a ligand molecule that targets the liposome to organism.
  • Ligands which bind to receptors prevalent in organism such as monoclonal antibodies that bind to organism antigens, are preferred.
  • the liposomes for use in the present methods can also be modified so as to avoid clearance by the mononuclear macrophage system ("MMS") and reticuloendothelial system ("RES").
  • MMS mononuclear macrophage system
  • RES reticuloendothelial system
  • modified liposomes have opsonization-inhibition moieties on the surface or incorporated into the liposome structure.
  • a liposome of the invention can comprise both opsonization-inhibition moieties and a ligand.
  • Opsonization-inhibiting moieties for use in preparing the liposomes of the invention are typically large hydrophilic polymers that are bound to the liposome membrane.
  • an opsonization inhibiting moiety is "bound" to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids.
  • These opsonization-inhibiting hydrophilic polymers form a protective surface layer that significantly decreases the uptake of the liposomes by the MMS and RES; e.g., as described in U.S. Pat. No. 4,920,016, the entire disclosure of which is herein incorporated by reference.
  • Opsonization inhibiting moieties suitable for modifying liposomes are preferably water- soluble polymers with a number-average molecular weight from about 500 to about 40,000 daltons, and more preferably from about 2,000 to about 20,000 daltons.
  • Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamide or poly N- vinyl pyrrolidone; linear, branched, or dendrimeric polyamido amines; polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylic or amino groups are chemically linked, as well as gangliosides, such as ganglioside GM1.
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • synthetic polymers such as polyacrylamide or poly N- vinyl
  • Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable.
  • the opsonization inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide.
  • the opsonization inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; animated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups.
  • the opsonization-inhibiting moiety is a PEG, PPG, or derivatives thereof. Liposomes modified with PEG or PEG-derivatives are sometimes called "PEGylated liposomes".
  • the opsonization inhibiting moiety can be bound to the liposome membrane by any one of numerous well-known techniques.
  • an N-hydroxysuccinimide ester of PEG can be bound to a phosphatidyl- ethanolamine lipid-soluble anchor, and then bound to a membrane.
  • a dextran polymer can be derivatized with a stearylamine lipid-soluble anchor via reductive animation using Na(CN)BH3 and a solvent mixture, such as tetrahydrofuran and water in a 30: 12 ratio at 60° C.
  • Liposomes modified with opsonization-inhibition moieties remain in the circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes called "stealth” liposomes. Stealth liposomes are known to accumulate in tissues fed by porous or "leaky” micro vasculature. Thus, tissue characterized by such microvasculature defects will efficiently accumulate these liposomes; see Gabizon, et al. (1988), Proc. Natl. Acad. Sci., USA, 18:6949-53. In addition, the reduced uptake by the RES lowers the toxicity of stealth liposomes by preventing significant accumulation of the liposomes in the liver and spleen.
  • liposomes that are modified with opsonization-inhibition moieties are particularly suited to deliver the compound (or nucleic acids comprising sequences encoding them) to the organism.
  • a therapeutically effective amount of said compound to be administered to a given patient by taking into account factors such as the size and weight of the patient; the extent of disease penetration; the age, health and sex of the patient; the route of administration; and whether the administration is regional or systemic.
  • An effective amount of said compound can be based on the approximate or estimated body weight of a patient to be treated.
  • such effective amounts are administered parenterally or enterally, as described herein.
  • an effective amount of the compound is administered to a patient can range from about 5-10000 micrograms/kg of body weight, and is preferably between about 5-3000 micrograms/kg of body weight, and is preferably between about 700-1000 micrograms/kg of body weight, and is more preferably greater than about 1000 micrograms/kg of body weight.
  • an appropriate dosage regimen for the administration of the compound to a given patient can be determined.
  • the compound can be administered to the patient once (e.g., as a single injection or deposition).
  • Another object of the invention relates to a method for treating acute leukemia comprising administering to a subject in need thereof a therapeutically effective amount of a compound as described above.
  • treating denotes reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of the disorder or condition to which such term applies.
  • the acute leukemia may be an acute myeloblastic, an acute lymphoblastic or acute promyelocytic leukemia.
  • the compound of the invention may be used or prepared in a pharmaceutical composition.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound as described above and a pharmaceutical acceptable carrier for use in the treatment of acute leukemia in a patient in need thereof.
  • the compounds of the invention are preferably formulated as pharmaceutical compositions, prior to administering to a patient, according to techniques known in the art.
  • Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free.
  • pharmaceutical formulations include formulations for human and veterinary use. Methods for preparing pharmaceutical compositions of the invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference.
  • the present pharmaceutical formulations comprise compound (e.g., 0.1 to 90% by weight), or a physiologically acceptable salt thereof, mixed with a pharmaceutically- acceptable carrier.
  • the pharmaceutical formulations of the invention can also comprise compound which are encapsulated by liposomes and a pharmaceutically-acceptable carrier.
  • Preferred pharmaceutically-acceptable carriers are water, buffered water, normal saline, 0.4% saline, 0.3%> glycine, hyaluronic acid and the like.
  • compositions of the invention can also comprise conventional pharmaceutical excipients and/or additives.
  • Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents.
  • Suitable additives include, e.g., physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (such as, for example, calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
  • Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.
  • conventional nontoxic solid pharmaceutically acceptable carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a solid pharmaceutical composition for oral administration can comprise any of the carriers and excipients listed above and 10-95%), preferably 25%>-75%>, of the compound.
  • a pharmaceutical composition for aerosol (inhalational) administration can comprise 0.01-20%) by weight, preferably 1%>-10%> by weight, of the compound encapsulated in a liposome as described above, and a propellant.
  • a carrier can also be included as desired; e.g., lecithin for intranasal delivery.
  • Pharmaceutical compositions of the invention may include any further agent which is used in the prevention or treatment of acute leukemia.
  • the anti- acute leukemia may include an anticancer agent.
  • said anticancer agents include but are not limited to fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, ir
  • additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photo sensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, anthracyclines, MDR inhibitors and Ca2+ ATPase inhibitors.
  • Additional anticancer agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi-specific antibodies, monobodies, polybodies.
  • Additional anticancer agent may be selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, and growth factor mimetics thereof.
  • the further therapeutic active agent can be an antiemetic agent.
  • Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoemanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dunenhydrinate, diphenidol, dolasetron, meclizme, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiefhylperazine, thioproperazine and tropisetron.
  • the further therapeutic active agent can be an hematopoietic colony stimulating factor.
  • Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alpha.
  • the other therapeutic active agent can be an opioid or non-opioid analgesic agent.
  • opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, nomioiphine, etoipbine, buprenorphine, mepeddine, lopermide, anileddine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazodne, pemazocine, cyclazocine, methadone, isomethadone and propoxyphene.
  • Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam and sulindac.
  • the further therapeutic active agent can be an anxiolytic agent.
  • Suitable anxiolytic agents include, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.
  • said additional active agents may be contained in the same composition or administrated separately.
  • the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of acute leukemia.
  • FIGURES Figure 1: The DLK1-DI03 locus is deregulated in APL samples. Inside the DLK1- DI03 locus (a), three classes of ncRNA (long ncRNA, microRNA and clustered snoRNA) are represented. Due to imprinting, the genes are either paternally (Pat) or maternally (Mat) expressed under the control of an intergenic differentially-methylated region (IG-DMR). The microRNA and snoRNA loci are represented by diagonally and vertically hatched boxes, (b) The signal intensities of DLK1-DI03 snoRNAs in AML samples assayed on GeneChip microRNA arrays demonstrated that only a small set of snoRNA was expressed.
  • ncRNA long ncRNA, microRNA and clustered snoRNA
  • IG-DMR intergenic differentially-methylated region
  • FIG. 2 DLK1-DI03 snoRNA expression was lost by ATRA treatment and increased by PML-RARalpha.
  • 14q(II-l) [SNORD 114-1] was cloned and transfected into a K562 cell line. Its over- expression was verified by qPCR 48h after transfection.
  • sequences containing the intron (in which the snoRNA sequence was present) and its flanking exon were cloned. We ensured that only the mature snoRNA sequence was over- expressed by RT-PCR for the snoRNA and intronic lariat sequences. "Blank” corresponds to RT-PCR on the mature snoRNA sequence using cDNA template from DLK1-DI03 negative cells.
  • Figure 5 14q(II-l) snoRNA effect on cell growth and Rb/pl6 pathways is confirmed in APL blasts, (a) Blasts from APL patients were transfected with siRNA- 14q(II-l) or siRNA negative control (siRNA-Neg). Effective targeting of the 14q(II-l) snoRNA was evaluated by qPCR and means ⁇ SEM of expression fold change were represented on the graph, (b) Cell growth of APL blasts after 48h of siRNA treatment was estimated using Mallassez cell counting. Paired t-tests were used as the statistical test (p ⁇ 0.05 : *; p ⁇ 0.01 : ** and p ⁇ 0.001 :
  • AML acute myeloblasts leukemia
  • ALL acute lymphoblastic leukemia
  • RNA samples Four hundred nanograms of total RNA were hybridized on GeneChip microRNA array (Affymetrix). Unlogged data were treated with dChip software with a specific cut-off (filter) to generate dendrograms. The cut-off value of 0.5 applied to microRNAs could not be used for snoRNAs because their expression was lower, thus, we used a cut-off value of 0.25. Rows (corresponding to RNAs of interest) were standardized by respective RNA means of expression and distant metric system applied is correlation. Sample clustering was carried out by the centroid method. Significance analysis of microarrays (SAM) was used to analyze statistical significance of the results. High-throughput quantitative PCR
  • the 25 ⁇ reaction contained lxSYBR Green PCR Master Mix (Sigma Aldrich), with each primer at 0.3 ⁇ , and 5 ⁇ 1 of cDNA (see above) diluted at 1 :5.
  • the primer efficiency >85% was checked prior to the experiments.
  • 2-ACT threshold cycle values were used for the analysis (5S rRNA was selected as the housekeeping gene).
  • APL Acute Promyelocytic Leukemia
  • APL blasts were cultured in the presence of ⁇ ATRA (Sigma) diluted in ethanol, or in and 5 days, the percentage of differentiated cells was assessed by cell morphology with May-Grunwald-Giemsa (MGG) stained cyto centrifuge slides and by expression of CD l ib as determined by flow cytometry.
  • the monoclonal antibodies used for staining were: CDl lb- PE-Cy7 (BD Biosciences) and CD33-PE-Cy5 (BD Biosciences) and isotype-matched control conjugates.
  • Flow cytometry was performed using a BD-LSRII flow cytometer (BD Biosciences).
  • cDNA encoding PML-RARalpha was cloned into an expression vector pCDNA3 (Invitrogen).
  • KG-1 cells ATCC CCL-246 were transfected with pCDNA3 or pCDNA3-P/R using nucleofection Amaxa Technology (Lonza) and Ingenio universal solution (Minis Bio LLC).
  • SnoRNA expression was monitored by RT-qPCR after 48 and 72h of transfection.
  • PML-RARalpha expression was monitored by RT-PCR using primers.
  • Cells were washed with PBS and fixed in cold 70% ethanol for 20min. Cells were then washed twice with PBS-0.1% BSA and once with PBS then labeled using propidium iodide staining for 30min (Invitrogen). Cell cycle distribution was evaluated by fluorescence analysis on a FACScan cytometer. Cell doublets were excluded and 20,000 events per condition were analyzed.
  • Non-supervised clustering indicated that the snoRNA expression profiles of the AML samples were different from those of CD33+ myeloid cells (data not shown), CD34+ progenitors or total bone marrow (data not shown). It is worth mentioning, however, that Flt3-ITD-mutated AML co-segregated with controls, suggesting that this group of leukemia is distinct with regard to snoRNA profiles (data not shown).
  • Flt3-ITD-mutated AML co-segregated with controls, suggesting that this group of leukemia is distinct with regard to snoRNA profiles (data not shown).
  • snoRNAs appeared to be globally down-regulated in leukemia cells, few snoRNAs located into clusters are over-expressed in some AML cases. Their expression was associated or not to specific chromosomal translocations (data not shown). Thus, through microarray analysis we noticed ectopic expression of a set of snoRNAs from the DLK1-DI03 locus in APL samples carrying identical PML-RARalpha bcrl translocations (data not shown).
  • DLK1-DI03 snoRNAs are located within the introns of the maternally expressed gene 8 (Meg8), which carries 1 , 9 and 31 highly related copies of SNORD 1 12 [14q(0)], SNORD113 [14q(I>] and SNORD114 [14q(II)] snoRNA genes respectively ( Figure la). As expected (due to their restricted expression pattern), they were not detected in controls (healthy CD33+ myeloid cells) or in PML/RARa-negative AML samples ( Figures lb and lc). Using microarrays, we observed that only eight sequence variants of SNORD114, five variants of SNORD113 and one SNORD112 were expressed in APL patients ( Figure lb). Of note, the level of expression of these particular snoRNAs varied significantly within the same cluster.
  • micro RNA genes are located around the SNOR112-114 gene cluster in the DLK1-DI03 locus and seem to have the same expression pattern as their neighboring snoRNAs.
  • To validate snoRNA results from microarrays in APL we examined the expression of the micro RNAs from the DLK1-DI03 locus in APL and PML-RARalpha-negative cells using GeneChip microRNA arrays. When compared to AML samples, we confirmed the significant over-expression of hsa-miR-127, miR-370 and miR-154, as already reported in APL, as well as eleven other micro RNAs (Figure Id) (Dixon-Mclver A. et al, 2008).
  • PML-RARalpha impacts on DLK1-DI03 snoRNA expression.
  • DLK1-DI03 snoRNAs played a role in APL pathogenesis we selected the SNORD114-1 [14q(II-l)] variant to analyze its impact on cell growth. This variant was chosen because among the snoRNAs it displayed the highest expression of the locus and because expression of this variant was sustained the longest following treatment with ATRA. The 14q(II-l) variant was cloned and transiently trans fected into the K562 cell line which represents the best model described for studying the DLK1-DI03 locus (i.e., this locus is transcriptionally active in these cells).
  • the Rb/ l6 pathways are involved in snoRNA-mediated cell growth.

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Abstract

Cette invention concerne une méthode permettant d'identifier un patient présentant ou à risque de présenter ou de développer une leucémie aiguë, ladite méthode comprenant la mesure, dans un échantillon prélevé chez ledit patient, d'au moins un petit ARN nucléolaire du locus DLK1-DI03. L'invention concerne également un composé qui inhibe l'expression d'un petit ARN nucléolaire du locus DLK1-DI03 à utiliser dans le traitement de la leucémie aiguë chez un patient le nécessitant.
PCT/EP2013/057530 2012-04-11 2013-04-11 Méthodes pour le traitement et le diagnostic de la leucémie aiguë WO2013153139A1 (fr)

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CN111808946A (zh) * 2020-07-06 2020-10-23 浙江大学 一种骨髓增生异常综合征标记物及其试剂盒
WO2021175966A1 (fr) * 2020-03-04 2021-09-10 Ninovax Produits pour la suppression ou la réduction de l'expression ou de l'activité d'un snoarn et leurs utilisations dans le traitement du cancer

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