WO2012123591A1 - Procédé pour la vectorisation d'acides nucléiques au noyau - Google Patents

Procédé pour la vectorisation d'acides nucléiques au noyau Download PDF

Info

Publication number
WO2012123591A1
WO2012123591A1 PCT/EP2012/054828 EP2012054828W WO2012123591A1 WO 2012123591 A1 WO2012123591 A1 WO 2012123591A1 EP 2012054828 W EP2012054828 W EP 2012054828W WO 2012123591 A1 WO2012123591 A1 WO 2012123591A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
acid molecule
sequence
sperna
rna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2012/054828
Other languages
English (en)
Inventor
Minoo Rassoulzadegan
Mitsuoki KAWANO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut National de la Sante et de la Recherche Medicale INSERM
Original Assignee
Institut National de la Sante et de la Recherche Medicale INSERM
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institut National de la Sante et de la Recherche Medicale INSERM filed Critical Institut National de la Sante et de la Recherche Medicale INSERM
Publication of WO2012123591A1 publication Critical patent/WO2012123591A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present invention relates to nucleic acid sequences that target nucleic acid molecules to the nucleus.
  • RNA-mediated inheritance of epigenetic traits (paramutations) in the mouse led us to the conclusion that gametic RNAs may act as transgenerational epigenetic determinants (Rassoulzadegan et al, 2006; Wagner et al, 2008; Grandjean et al, 2009).
  • Microarray analysis revealed the presence of microRNAs in sperm (Ostermeier et al, 2005; Amanai et al, 2006) and we are now led to consider other small noncoding RNAs as candidates for regulatory functions.
  • nucleic acid sequences that are able to target nucleic acid molecules to the nuclear compartment.
  • the inventors report the identification of two novel small noncoding RNAs, exclusively present in the mature sperm and efficiently transferred to the embryo where they are maintained in a tight association with the nucleus during all the preimplantation period.
  • the invention relates to a method for targeting nucleic acid molecules to the nucleus.
  • the inventors have identified two small noncoding RNAs, comprising the consensus sequence UGGG(G)CGGG which is sufficient for nuclear targeting of unrelated small RNAs.
  • This consensus sequence is identical in humans and can therefore be used in any mammalian species, as a nuclear targeting sequence.
  • the invention therefore relates to an isolated nucleic acid molecule comprising sequence UGGGCGGG and/or sequence UGGGGCGGG.
  • the invention also relates to the use of a single-stranded ribonucleic acid moiety comprising sequence UGGGCGGG and/or sequence UGGGGCGGG for targeting a nucleic acid molecule to the nucleus of a host cell.
  • the invention also relates to a method for targeting a nucleic acid molecule to the nucleus of a host cell comprising the step of adding a single- stranded ribonucleic acid moiety comprising UGGGCGGG and/or sequence UGGGGCGGG to said nucleic acid molecule.
  • the invention also relates to a method for obtaining an epigenetic regulation comprising introducing a nucleic acid as defined above into a fertilized egg or into an unfertilized oocyte.
  • the invention in another aspect, relates to a method for obtaining DNA recombination and/or mutagenesis comprising the step of introducing into a cell a nucleic acid molecule which is capable of provoking changes in the DNA sequence, such as DNA or RNA molecules involved in recombination and/or mutagenesis, wherein said nucleic acid molecule contains a single- stranded ribonucleic acid moiety comprising sequence UGGGCGGG and/or sequence UGGGGCGGG.
  • the invention therefore relates to an isolated nucleic acid molecule comprising sequence UGGGCGGG and/or sequence UGGGGCGGG.
  • said nucleic acid molecule is a ribonucleic acid molecule.
  • said nucleic acid molecule is chimeric DNA/RNA molecule, wherein the sequence UGGGCGGG or UGGGGCGGG is a single-stranded ribonucleic acid.
  • said nucleic acid molecule has a length comprised between 8 and 500 nucleotides. In a preferred embodiment, said nucleic acid molecule has a length of more than 8, preferably more than 9, even more preferably more than 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides.
  • said nucleic acid has a length of less than 500 nucleotides, preferably less than 200, less than 100, 50, 45, 40 nucleotides.
  • the nucleic acid molecule according to the invention may comprise the sequence UGGGCGGG or UGGGGCGGG in different positions. According to one embodiment, the UGGGCGGG or UGGGGCGGG sequence is located at the 5' end of the nucleic acid molecule.
  • the invention believe that, when the targeting sequence is located at the 5' end of the nucleic acid molecule, the nucleic acid molecule is not only targeted to the nucleus, but is also stably localized in the nucleus of the cells after mitosis.
  • the UGGGCGGG or UGGGGCGGG sequence is located at the 3' end of the nucleic acid molecule.
  • the UGGGCGGG or UGGGGCGGG sequence is located within the nucleic acid molecule, with other nucleotides or ribonucleotides on either end.
  • the invention also relates to the use of a single-stranded ribonucleic acid moiety comprising sequence UGGGCGGG and/or sequence UGGGGCGGG for targeting a nucleic acid molecule to the nucleus of a host cell.
  • the invention also relates to a method for targeting a nucleic acid molecule to the nucleus of a host cell comprising the step of adding a single- stranded ribonucleic acid moiety comprising UGGGCGGG and/or sequence UGGGGCGGG to said nucleic acid molecule.
  • targeting a nucleic acid molecule to the nucleus refers to a nucleic acid molecule which is predominantly located in the nucleus of a given cell.
  • said nucleic acid molecule is microinjected into the cytoplasm of the cell, it translocates into the nucleus.
  • said nucleic acid molecule is microinjected/transferred/transfected/infected into the nucleus of the cell, remains in the nucleus.
  • Nuclear translocation or nuclear localization can be assessed by any method known in the art. Typically, when bound to a fluorescent tag or when a fluorescent nucleic acid is used, the nuclear localization can be assessed by fluorescence microscopy.
  • the method for targeting a nucleic acid molecule to the nucleus has many potential applications, as described below.
  • it can be used to stabilize an exogenous nucleic acid in the nucleus.
  • said exogenous nucleic acid may be a regulatory RNA, a therapeutic RNA.
  • nucleic acid molecules according to the invention comprising the consensus sequence UGGG(G)CGGG, i.e. comprising UGGGCGGG or UGGGGCGGG, are not only targeted to the nucleus, but are also maintained stably during cell division. In other terms, they are also found in the nucleus.
  • the invention also relates to a method for obtaining an epigenetic regulation comprising introducing a nucleic acid as defined above into a fertilized egg.
  • the method for obtaining an epigenetic regulation can comprise introducing a nucleic acid as defined above into an unfertilized egg, and subsequently fertilizing said oocyte.
  • the inventors have observed that the nucleic acid according to the invention translocates into the nucleus following fertilization.
  • epidermatitis refers to heritable changes in gene function that occur without a change in the DNA sequence.
  • the method of the invention can also be used in order to target to the nucleus a nucleic acid molecule which is capable of provoking changes in the DNA sequence, such as DNA or RNA molecules involved in recombination and/or mutagenesis
  • the nucleic acids and methods of the invention can be used in order to target a regulatory RNA and/or a therapeutic RNA to the nucleus.
  • regulatory RNA and therapeutic RNA are often unstable and their effects are limited in time.
  • Prior art methods used in order to stabilize said RNA molecules rely on chemical modifications. These chemical modifications can lead to undesirable side effects.
  • the nucleic acids and methods of the invention rely on natural endogenous signals and therefore advantageously limit these undesirable side effects.
  • Fig. 1 Expression analysis of thirteen sperm small RNAs.
  • RNAs isolated from adult testis and sperm were polyadenylated. Reverse transcription was carried out using an RTQ primer with or without reverse transcriptase.
  • the cDNAs were amplified by PCR using a primer specific to each small RNA and an RTQ-UNIr universal primer (Supplemental Table 3). The expected cDNA sizes for the RNAs are ⁇ 120-bp.
  • the PCR products were electrophoresed on 3% agarose gels and stained using ethidium bromide. let-7a was used as a positive control.
  • Fig. 2 Genomic location and expression profiling of two speRNAs and their proximal piRNAs.
  • Obtained small RNA sequences of speRNA- 12 and -13 are shown as a blue arrowhead, and their adjacent piRNAs are indicated as a red arrowhead above the box. The size of the arrowhead is not scaled. The red lines in the box indicate that piRNAs registered in the piRNABank (http://pirnabank.ibab.ac.in/).
  • C Oligo primer sequences used in the analysis are shown as black arrows. The sequenced cDNA read of speRNA-12 and -13 are blue. The piRNAs adjacent to speRNA- 12 and -13 are red. Predicted stem-loop structure region as precursor- speRNA is indicated as upper case. The positions of the genome sequences are shown above each of the sequences.
  • speRNA-12 A minus strand of the mouse genome is shown for speRNA-12, and a plus strand of the genome is shown for speRNA-13.
  • D Expression of speRNA-12 and -13 and their flanking piRNAs was investigated using mouse total RNAs shown above the pictures. The expected cDNA sizes for the RNAs are ⁇ 120-bp. The PCR products were electrophoresed on 3% agarose gels and stained using ethidium bromide. let-7a was used as a positive control. A DNA size marker was loaded on each side of the gel. NTC stands for a non-template control.
  • E Model of the speRNA-12 and -13 production. Single-stranded precursor transcripts of piRNAs are expressed specifically in testis.
  • Mature piRNAs are preferentially expressed in testis and remain in sperm. During mature sperm formation, the speRNAs (blue arrows) are generated from piRNA precursor transcripts. (F) Alignment of mature speRNA-12 and -13. Identical nucleotide sequences are indicated by dots.
  • Fig. 3 Mitotic stability of speRNA-12 and -13 in the preimplantation embryo.
  • FITC(or Cy3)-tagged oligoribonucleotides were microinjected in fertilized eggs, the embryos were cultured for the next 48 hours and examined by fluorescence microscopy (Zeiss ApoTome). Controls were performed with micro RNA miR-124, previously used for this type of experiment (Grandjean et al, 2009).
  • Fig. 5 Map positions of speRNA-12 and -13 and known piRNAs in the piRNABank (http://pirnabank.ibab.ac.in/ .
  • Fig. 6 Analysis of putative piRNA precursors by strand-specific RT-PCR from speRNA-12 and -13 loci.
  • Total RNA from adult mouse testes and epididymides were reverse-transcribed with a gene-specific antisense primer (used for the detection of sense transcript), with a gene- specific sense primer, with no primer.
  • PCR was carried out using a set of gene-specific primers (Supplemental Table 3).
  • the PCR product size is 301-bp for the speRNA-12 locus, and 351 -bp for the speRNA-13 locus, whose nucleotide sequences are shown in Supplemental Fig. 3.
  • LINE 1 and ⁇ -actin were used as a positive and negative control for the existence of antisense transcripts, respectively.
  • Fig. 8 Expression profiling analysis for small RNAs mapped to piRNA loci identified in this study.
  • the PCR products were electrophoresed on 3% agarose gels and stained using ethidium bromide. let-7a was used as a positive control. A DNA size marker was loaded on each side of the gel. NTC stands for a non-template control. Primers used in the analyses are shown in Supplemental Table 3.
  • Fig. 9 Nuclear localization of the speRNA-12 is an exclusive property of the fertilized egg. Fluorescence-tagged oligoribonucleotides were microinjected and examined either one hour or 24 hours later. Unfertilized oocytes retain the FITC-tagged speRNA-12 uniformly distributed in the whole cell volume, similar to Cy3-tagged miR-124 RNA and contrasting with its strict nuclear localization in the fertilized eggs and subsequent stages.
  • RNA sequences ranging from 13 to 248 nt. It consists of fragments of long RNAs (rRNA and mRNA) as well as small regulatory RNAs such as microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs) (Supplemental Table 1).
  • rRNA and mRNA fragments of long RNAs
  • miRNAs microRNAs
  • piRNAs Piwi-interacting RNAs
  • both speRNA-12 and -13 were determined to be encoded within a ⁇ 40-kb region in a piRNA cluster region on chromosome 17 and oriented divergently (Fig. 2B). Since piRNAs are generated by repetitive cleavage a long transcript and the sperm small RNAs are oriented in the same way as their proximal piRNAs (Fig. 2B; Fig. 5), one could assume that the speRNA-12 and -13 are left over of the piRNA pathway.
  • speRNA-12 and -13 are cleaved out from stem-loop structured RNA rather than a long dsRNA and suggest that they represent a novel class of RNA (Fig. 2E). Their sequences show significant similarities, in particular 1st to 9th sequences are identical (CAGGGUGGG) (Fig. 2F) and do not match any known miRNAs registered in miRBase (http://mirbase.org/).
  • speRNA-12 the RNAs were detected in the fertilized egg, in all likelihood of sperm origin, since they were not present at a detectable level in oocytes. The same low copy numbers were maintained in the dividing blastomeres and the RNAs were not detectable any more starting at the blastocyst stage.
  • the labelled material injected into the male pronucleus was within minutes distributed between the two pronuclei (Figs. 3A,B). Both nuclear and cytoplasmic microinjection at the one or the two cell stages led to the same exclusive nuclear localization (data not shown). In contrast, their microinjection to unfertilized oocytes led to a homogenous distribution in the whole cellular volume (Supplemental Fig. 6).When the injected eggs were subsequently fertilized in vitro, and the female pronucleus regenerated, the fluorescence was progressively concentrated in the nuclear compartment. Interestingly, accumulation was first detected in the extruded second polar body and the incoming paternal nucleus, both endowed with a nuclear membrane.
  • miR-29b was of specific interest.
  • the microRNA present in our mouse sperm preparations, is reported to be translocated to the nucleus in HeLa cells (Hwang et al, 2007).
  • a fluorescence-tagged miR-29b oligoribonucleotide showed in the early embryo the behaviour different from that of the sperm small RNAs.
  • the label diffused from the nucleus to the cytoplasm, then was uniformly distributed in both compartments from the one- cell to the later stages (see 4-cell stage in Fig. 3B).
  • the stable nuclear association of the labelled speRNAs was maintained during preimplantation development.
  • the fluorescent oligoribonucleotides were detected in the nuclei of the blastomeres until the embryos reached the blastocyst stages (Fig. 3B; and data not shown). In the blastocyst, contrary to the previous stages, only a few nuclei of the inner cell mass and the trophectoderm were still labelled. Maintenance of full-length free oligoribonucleotides was independently ascertained by sequence analysis of individual embryos (data not shown).
  • speRNA-12 and -13 were maintained in the progeny of the microinjected blastomere without being transferred to the other one (data not shown). Maintenance during development and a strict nuclear localization, suggestive of an active role in early development, therefore appear as a unique property of the speRNA-12 and -13 RNAs.
  • RNAs are small, noncoding RNAs exclusively present in mature sperm and transferred to the fertilized egg. They appear to represent a novel class of small RNAs. Their generation at the final stages of spermatogenesis significantly enlarges our view of the role of piRNA genes in reproduction and development. Their strict, sequence dependent nuclear localization at the preimplantation stages (Fig. 3) indicates regulated transport mechanisms that remain entirely to be analyzed. Most importantly, sexual transfer of the two RNAs and their mitotic stability in the embryo are suggestive of paternal determinations in development. Their tight association with nuclear components makes somewhat unlikely that they could be involved in the classical microRNA-mediated posttranscriptional regulations.
  • microRNAs and other small RNA molecules may act in the early embryo as epigenetic determinants of the level of gene expression. Our current way of thinking would then rather consider these paternal RNAs transferred to the embryo as actors in epigenetic developmental controls.
  • spermatozoa were collected from -70 mouse caudae epididymides of strain C57BL/6. Motile spermatozoa were washed two times in MEM buffer (ImM Na pyruvate, EDTA, Penicillin, Streptomycin, and BSA) by centrifugation.sperm pellets were resuspended in phosphate- buffered saline (PBS), followed by centrifugation. Then the pellets were washed two times in frozen storage buffer (50mM HEPES buffer at pH 7.5, 10 mM NaCl, 5 mM Mg acetate, and 25% glycerol), then stored at -80°C.
  • MEM buffer ImM Na pyruvate, EDTA, Penicillin, Streptomycin, and BSA
  • PBS phosphate- buffered saline
  • RNA samples were evaluated using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA) and a 2100-Bioanalyzer with the RNA 6000 Nano Chip (Applied Biosystems, Carlsbad, CA, USA), respectively.
  • RNA cDNA library was generated from -0.8 ⁇ g of total RNA as previously described (Kawaji et al, 2008) but without cloning the library into a plasmid. Massively parallel pyrosequencing was performed using a 454 genome sequencer (Roche, Basel, Switzerland).
  • RNA samples isolated from various adult mouse tissues (8-9 weeks) and testes at various ages (1-8 weeks and 12 months) and embryo (15 days), and 0.7 ⁇ g of total RNA for sperm (9 weeks).
  • RNA was polyadenylated using poly(A) Tailing Kit (Ambion) and used to synthesize small RNA cDNA with PrimeScript II RTase (Takara Bio, Ohtsu, Shiga, Japan) and 2.5 ⁇ of an RTQ primer (Ro et al, 2006).
  • Individual RNAs were detected by PCR using AccuPrime Taq DNA SuperMix I (Invitrogen) and an RTQ-UNIr primer with gene-specific primers (Supplemental Table 3).
  • Oligoribonucleotides purchased from Proligo-Sigma (Sigma-Aldrich, St. Louis, MO, USA) were microinjected into the male pronuclei of embryos recovered at day 0.5pc from B6D2 Fl females, using the established methods of DNA transgenesis (Hogan et al, 1994) except for the exclusive use of naturally ovulated oocytes. The embryos were then cultured in M16 medium (Sigma-Aldrich) at 37°C. The microinjected molecules were observed over four days from the one-cell to the blastocyst stages by fluorescence microscopy (Zeiss, Oberkochen, Germany). Experiments were reproduced at least three times with a total of 10 embryos microinjected each time. For a given oligoribonucleotide, the same pattern was observed in 100% of the microinjected embryos.
  • RNA sequences were prepared from the genome sequence with additional CCA at the 3'-end, which was based on RefSeq annotation of NCBI Build 37 (mouse).
  • the remaining small RNA sequences which did not match to the RNA sequences above, were aligned with the mouse genome sequence (NCBI Build 37 or mm9), and their genomic coordinates were compared with the repeat masker track in the UCSC genome browser database. The small RNA sequences were classified based on the above hits to the known RNA sequences or overlaps on the genomic coordinates.
  • RNAfold http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi
  • the secondary structures of Fig. 2A and Fig. 4 were created by the mfold (http://mfold.bioinfo ⁇ i.edu/) 2 , followed by manual edition.
  • RNA precursors, protein-coding gene ( ⁇ -actin), and retrotransposon (LINE 1) were amplified using PrimeSTAR GXL DNA polymerase (Takara) with gene-specific primers (Supplemental Table 3) according to the manufacturer's instructions.
  • Example 2 spR-12 and spR13 are nuclear transfer elements
  • the inventors added either 3' or 5' extensions from the common spR sequence to miR-1 and miR-124 Cy3 -tagged oligoribonucleotides and checked their localization during early development. Results were identical for the two miRNA sequences.
  • oligoribonucleotides with sequences complementary to spR-12 and -13, nor the double stranded structures, nor deoxyribonucleotides with the same sequences were maintained in the injected nuclei.
  • chimeric molecules of oligodeoxyribonucleotides (of 20 bases) with the 5' extension with RNA motif of rUGGGCGGG were efficiently addressed to, and maintained in the nucleus.
  • RNA class No. of reads (%) rRNA 136362 (37.9) tRNA 24320 (6.76) sno/sc/snRNA 3078 (0.86) mRNA 2121 (0.59)
  • Novel small RNAs from the mouse sperm identified by deep-sequencing
  • bpiRNA mapped in piRNA locus
  • cStem-loop potential to pre-miRNA like secondary structure
  • dRT-PCR validated by poly(A)-tailed RT-PCR
  • RTQ-UNIr CGAATTCTAGAGCTCGAGGCAGG polyA-RT-PCR speRNA-1 (19) AGGAAACTGCCTCTCGGGGC polyA-RT-PCR speRNA-2 (20) CTGACAGCAAGGCCTCTC polyA-RT-PCR speRNA-3 (21) ACTCCGGGCTGCTCGGGAG polyA-RT-PCR speRNA-4 (22) TGGGAAAGACTCTGGGTCTC polyA-RT-PCR speRNA-5 (23) TGGCCTGGGCCTGGCAGTGG polyA-RT-PCR speRNA-6 (24) TCTGCCCTTCCCTCTGGAGA polyA-RT-PCR speRNA-7 (25) GTGTGTGCGTGTGGGC polyA-RT-PCR speRNA-8 (26) TGCCAGTGTGTGAGTGTG polyA-RT-PCR speRNA-9 (27) GTGCCCAGTCATGTCCGGGGG polyA-RT-PCR speRNA-10 (28) CGTGGGACCTTAGCGTTATGC polyA-RT-PCR speRNA-11 (29) GAACAGCCGAGTCCTTGCTG poly
  • TTACTGAGCCTTATCAGGGT polyA-RT-PCR piRNA12-F (38) CCCTCAACAAATAGTCTGTCT strand-specific RT-PCR piRNA12-R (39) TGGAAGCTTCCTTCCCTGTC strand-specific RT-PCR piRNA13-F (40) TCTCCCTTCTGTTTTCCGTG strand-specific RT-PCR piRNA13-R (41) CCCTGAGCAGTCACACTTTG strand-specific RT-PCR

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne de nouvelles molécules d'acide nucléique et leur utilisation pour le vectorisation nucléaire.
PCT/EP2012/054828 2011-03-17 2012-03-19 Procédé pour la vectorisation d'acides nucléiques au noyau Ceased WO2012123591A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201161453614P 2011-03-17 2011-03-17
US61/453614 2011-03-17

Publications (1)

Publication Number Publication Date
WO2012123591A1 true WO2012123591A1 (fr) 2012-09-20

Family

ID=45841508

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2012/054828 Ceased WO2012123591A1 (fr) 2011-03-17 2012-03-19 Procédé pour la vectorisation d'acides nucléiques au noyau

Country Status (1)

Country Link
WO (1) WO2012123591A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021072076A1 (fr) * 2019-10-08 2021-04-15 Massachusetts Institute Of Technology Mammifères mâles sans filles pour la suppression de la population non humaine

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6649749B2 (en) * 1993-03-26 2003-11-18 Gen-Probe Incorporated Detection of human immunodeficiency virus type 1
US20050261212A1 (en) * 2000-02-11 2005-11-24 Mcswiggen James A RNA interference mediated inhibition of NOGO and NOGO receptor gene expression using short interfering RNA
WO2007149521A2 (fr) * 2006-06-20 2007-12-27 The Johns Hopkins University Motifs nucléotidiques produisant des éléments de localisation et procédés d'utilisation
KR20100084459A (ko) * 2009-01-16 2010-07-26 차의과학대학교 산학협력단 인간 중배엽중기세포 유래의 신규 ｍｉＲＮＡ
WO2011001086A2 (fr) * 2009-06-29 2011-01-06 Universite Pierre Et Marie Curie Utilisation d'arn interferents pour le traitement d'une infection par le vih
WO2011146937A1 (fr) * 2010-05-21 2011-11-24 The Translational Genomics Research Institute Procédés et kits utiles dans le diagnostic du nsclc (cancer du poumon à grandes cellules)

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6649749B2 (en) * 1993-03-26 2003-11-18 Gen-Probe Incorporated Detection of human immunodeficiency virus type 1
US20050261212A1 (en) * 2000-02-11 2005-11-24 Mcswiggen James A RNA interference mediated inhibition of NOGO and NOGO receptor gene expression using short interfering RNA
WO2007149521A2 (fr) * 2006-06-20 2007-12-27 The Johns Hopkins University Motifs nucléotidiques produisant des éléments de localisation et procédés d'utilisation
KR20100084459A (ko) * 2009-01-16 2010-07-26 차의과학대학교 산학협력단 인간 중배엽중기세포 유래의 신규 ｍｉＲＮＡ
WO2011001086A2 (fr) * 2009-06-29 2011-01-06 Universite Pierre Et Marie Curie Utilisation d'arn interferents pour le traitement d'une infection par le vih
WO2011146937A1 (fr) * 2010-05-21 2011-11-24 The Translational Genomics Research Institute Procédés et kits utiles dans le diagnostic du nsclc (cancer du poumon à grandes cellules)

Non-Patent Citations (21)

* Cited by examiner, † Cited by third party
Title
AMANAI M; BRAHMAJOSYULA M; PERRY AC: "A restricted role for sperm-borne microRNAs in mammalian fertilization", BIOL REPROD, vol. 75, 2006, pages 877 - 884
DATABASE EMBL [online] 13 June 2006 (2006-06-13), "Homo sapiens piRNA piR-46797, complete sequence.", XP002677955, retrieved from EBI accession no. EMBL:DQ578685 Database accession no. DQ578685 *
GIRARD ANGELIQUE ET AL: "A germline-specific class of small RNAs binds mammalian Piwi proteins", NATURE: INTERNATIONAL WEEKLY JOURNAL OF SCIENCE, NATURE PUBLISHING GROUP, UNITED KINGDOM, vol. 442, no. 7099, 1 July 2006 (2006-07-01), pages 199 - 202, XP002500301, ISSN: 0028-0836, DOI: 10.1038/NATURE04917 *
GRANDJEAN V; GOUNON P; WAGNER N; MARTIN L; WAGNER KD; BERNEX F; CUZIN F; RASSOULZADEGAN M: "The miR-124-Sox9 paramutation: RNA-mediated epigenetic control of embryonic and adult growth", DEVELOPMENT, vol. 136, 2009, pages 3647 - 3655, XP055030276, DOI: doi:10.1242/dev.041061
HOFACKER, I.L.: "Vienna RNA secondary structure server", NUCLEIC ACIDS RES, vol. 31, 2003, pages 3429 - 3431, XP002460707, DOI: doi:10.1093/nar/gkg599
HOGAN B; COSTANTINI F; LACY L: "Manipulating the mouse embryo - a laboratory manual", 1994, COLD SPRING HARBOR LABORATORY
HWANG HUN-WAY ET AL: "A hexanucleotide element directs microRNA nuclear import", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, WASHINGTON, DC; US, vol. 315, no. 5808, 5 January 2007 (2007-01-05), pages 97 - 100, XP002548637, ISSN: 0036-8075, DOI: 10.1126/SCIENCE.1136235 *
HWANG HW; WENTZEL EA; MENDELL JT: "A hexanucleotide element directs microRNA nuclear import", SCIENCE, vol. 315, 2007, pages 97 - 100, XP002548637, DOI: doi:10.1126/SCIENCE.1136235
KAWAJI H; NAKAMURA M; TAKAHASHI Y; SANDELIN A; KATAYAMA S; FUKUDA S; DAUB CO; KAI C; KAWAI J; YASUDA J: "Hidden layers of human small RNAs", BMC GENOMICS, vol. 9, 2008, pages 157, XP021032882
KRAMER JA; KRAWETZ SA: "RNA in spermatozoa: implications for the alternative haploid genome", MOL HUM REPROD, vol. 3, 1997, pages 473 - 478
KRAWETZ SA: "Paternal contribution: new insights and future challenges", NAT REV GENET, vol. 6, 2005, pages 633 - 642
MINOO RASSOULZADEGAN ET AL: "RNA-mediated non-mendelian inheritance of an epigenetic change in the mouse", NATURE, vol. 441, no. 7092, 25 May 2006 (2006-05-25), pages 469 - 474, XP055030216, ISSN: 0028-0836, DOI: 10.1038/nature04674 *
OSTERMEIER GC; GOODRICH RJ; MOLDENHAUER JS; DIAMOND MP; KRAWETZ SA: "A suite of novel human spermatozoal RNAs", J ANDROL, vol. 26, 2005, pages 70 - 74, XP008150838
PERRY AC; WAKAYAMA T; KISHIKAWA H; KASAI T; OKABE M; TOYODA Y; YANAGIMACHI R: "Mammalian transgenesis by intracytoplasmic sperm injection", SCIENCE, vol. 284, 1999, pages 1180 - 1183
RASSOULZADEGAN M; GRANDJEAN V; GOUNON P; VINCENT S; GILLOT I; CUZIN F: "RNA-mediated non-mendelian inheritance of an epigenetic change in the mouse", NATURE, vol. 441, 2006, pages 469 - 474, XP055030216, DOI: doi:10.1038/nature04674
RO S; PARK C; JIN J; SANDERS KM; YAN W: "A PCR-based method for detection and quantification of small RNA", BIOCHEM BIOPHYS RES COMMUN, vol. 351, 2006, pages 756 - 763, XP024925751, DOI: doi:10.1016/j.bbrc.2006.10.105
TAM OH; ARAVIN AA; STEIN P; GIRARD A; MURCHISON EP; CHELOUFI S; HODGES E; ANGER M; SACHIDANANDAM R; SCHULTZ RM: "Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes", NATURE, vol. 453, 2008, pages 534 - 538, XP055153023, DOI: doi:10.1038/nature06904
V. GRANDJEAN ET AL: "The miR-124-Sox9 paramutation: RNA-mediated epigenetic control of embryonic and adult growth", DEVELOPMENT, vol. 136, no. 21, 1 November 2009 (2009-11-01), pages 3647 - 3655, XP055030276, ISSN: 0950-1991, DOI: 10.1242/dev.041061 *
WAGNER KD; WAGNER N; GHANBARIAN H; GRANDJEAN V; GOUNON P; CUZIN F; RASSOULZADEGAN M: "RNA induction and inheritance of epigenetic cardiac hypertrophy in the mouse", DEV CELL, vol. 14, 2008, pages 962 - 969
WATANABE T; TOTOKI Y; TOYODA A; KANEDA M; KURAMOCHI-MIYAGAWA S; OBATA Y; CHIBA H; KOHARA Y; KONO T; NAKANO T: "Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes", NATURE, vol. 453, 2008, pages 539 - 543, XP055153025, DOI: doi:10.1038/nature06908
ZUKER, M.: "Mfold web server for nucleic acid folding and hybridization prediction", NUCLEIC ACIDS RES, vol. 31, 2003, pages 3406 - 3415, XP002460708, DOI: doi:10.1093/nar/gkg595

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021072076A1 (fr) * 2019-10-08 2021-04-15 Massachusetts Institute Of Technology Mammifères mâles sans filles pour la suppression de la population non humaine

Similar Documents

Publication Publication Date Title
Santiago et al. All you need to know about sperm RNAs
Meister Argonaute proteins: functional insights and emerging roles
Fuchs Wightman et al. Target RNAs strike back on microRNAs
Morgan et al. Post-transcriptional regulation in spermatogenesis: all RNA pathways lead to healthy sperm
Watanabe et al. Identification and characterization of two novel classes of small RNAs in the mouse germline: retrotransposon-derived siRNAs in oocytes and germline small RNAs in testes
US10760081B2 (en) Compositions and methods for enhancing CRISPR activity by POLQ inhibition
Kawano et al. Novel small noncoding RNAs in mouse spermatozoa, zygotes and early embryos
He et al. Small RNA molecules in the regulation of spermatogenesis
Cao et al. Noncoding RNAs in the mammalian central nervous system
EP2925866B1 (fr) Arn circulaire destiné à l'inhibition de micro-arn
Amar et al. MicroRNA expression profiling of hypothalamic arcuate and paraventricular nuclei from single rats using Illumina sequencing technology
US11155870B2 (en) Methods and compositions for monitoring and enhancing early embryo development
Towler et al. Regulation of cytoplasmic RNA stability: Lessons from Drosophila
CN106604633A (zh) 作为禾本科植物和其他单子叶植物中雄性不育之决定子的调节性非编码rna
Pandey RNA-based regulation in human health and disease
CN1867672B (zh) 改良的siRNA分子和应用该改良的siRNA分子的抑制基因表达的方法
Cerutti et al. Turnover of mature miRNAs and siRNAs in plants and algae
WO2012123591A1 (fr) Procédé pour la vectorisation d'acides nucléiques au noyau
Chalupnikova et al. Production and application of long dsRNA in mammalian cells
US20050079614A1 (en) RNAs able to modulate chromatin silencing
Tang et al. Upregulation of PHLDA2 in Dicer knockdown HEK293 cells
Godden et al. An efficient CRISPR-Cas9 method to knock out miRNA expression in Xenopus tropicalis
EP2502997A1 (fr) Molécule d'acide nucléique à inhibition micro ARN
Jeong et al. Biogenesis of circular RNAs in vitro and in vivo from the Drosophila Nk2. 1/scarecrow gene
Nguyen The Internal Loops Stimulate Single Cleavage of Human Microprocessor on Primary MicroRNAs

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12709127

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12709127

Country of ref document: EP

Kind code of ref document: A1