EP2329024A1 - Arn endogène court en épingle à cheveux et son utilisation - Google Patents

Arn endogène court en épingle à cheveux et son utilisation

Info

Publication number
EP2329024A1
EP2329024A1 EP08784123A EP08784123A EP2329024A1 EP 2329024 A1 EP2329024 A1 EP 2329024A1 EP 08784123 A EP08784123 A EP 08784123A EP 08784123 A EP08784123 A EP 08784123A EP 2329024 A1 EP2329024 A1 EP 2329024A1
Authority
EP
European Patent Office
Prior art keywords
seq
cells
sequence
shr
bmscs
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.)
Withdrawn
Application number
EP08784123A
Other languages
German (de)
English (en)
Other versions
EP2329024A4 (fr
Inventor
Robert C. Zhao
James Q. Yin
Chunjing Bian
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.)
Institute of Basic Medical Sciences of AMMS
Institute of Basic Medical Sciences of CAMS
Original Assignee
Institute of Basic Medical Sciences of AMMS
Institute of Basic Medical Sciences of CAMS
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 Institute of Basic Medical Sciences of AMMS, Institute of Basic Medical Sciences of CAMS filed Critical Institute of Basic Medical Sciences of AMMS
Publication of EP2329024A1 publication Critical patent/EP2329024A1/fr
Publication of EP2329024A4 publication Critical patent/EP2329024A4/fr
Withdrawn legal-status Critical Current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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • the present invention relates to a RNA molecule, particularly to an endogenous short hairpin RNA as well as the application of the invented RNA to induce the formation of hematopoietic cells by repressing ElA-like inhibitor of differentiation- 1 (referred to hereinafter as EIDl).
  • EIDl ElA-like inhibitor of differentiation- 1
  • BMSCs bone marrow mesenchymal stem cells
  • HSCs hematopoietic stem cells
  • BMSCs expanded in culture can be distinguished from HSCs by the lack of expression of CD45 and CD34 (G. Chamberlain, J. Fox, B. Ashton, J. Middleton, Stem Cells. 25, 2739-49 (2007).). Since it is a great challenge to obtain a large number of HSCs for clinical application (B.P. Sorrentino, Nat Rev Immunol.
  • BMSCs would serve as an alternate source for HSCs if BMSCs can be induced to differentiate into hematopoietic cells.
  • mesenchymal stem cells BMSCs
  • BMSCs mesenchymal stem cells
  • HSCs hematopoietic stem cells
  • miRNAs are differentially expressed in different hematopoietic cell types and play important regulatory roles.
  • miR-181 is associated with B lymphoid development (CZ. Chen et al., Science 303, 83-86 (2004)), miRNA-142 and -223 with T lymphopoiesis (S. H. Ramkissoon et al., Leukemia Research, 30, 643-647 (2005).), miRNA-221 and -222 with erythropoiesis (N. Felli et al., Proc Natl Acad Sci U S A. 102, 18081-18086 (2005).), miRNA-223 with granulocytic differentiation (F.
  • miRNAs have been shown to prevent the differentiation of HSCs (III, R. W. Georgantas et al., Proc Natl Acad Sci U S A.104, 2750-2755 (2007).).
  • miRNAs such as miR-130a and miR-10a have been found to target the transcription factor genes HOXAl and MAFB, which are important for cellular differentiation (R.
  • the first object of the invention is to provide an endogenous short hairpin RNA or a complement thereof having a sequence of SEQ. ID. NO.: 1 that induces the formation of hematopoietic cells particularly by repressing EIDl.
  • the said SEQ. ID. NO.: 1 shows:
  • the said sequence of SEQ. ID. NO.: 1 essentially includes the nucleic acid of 5'-CAA AUA CUC A-3'.
  • the second object of the invention is to provide an expression construct including the sequence of SEQ. ID. NO.: 1.
  • the third object of the invention is to provide a vector comprising an expression construct having an expression construct including the sequence of SEQ. ID. NO.: 1.
  • the vector preferably is a retrovirus plasmid (retrovirus vector pMSCV ) that expresses the sequence of SEQ. ID. NO.: 1.
  • the fourth object of the invention is to provide an application of the invented RNA or a complement thereof having a sequence of SEQ. ID. NO.: 1 to induce the formation of hematopoietic cells or a method of treatment for a subject suffering from dysfunction of formation of blood cells or a method of treatment for hematopoiesis in mammalian including human.
  • the invention also provides a use of an endogenous short hairpin RNA having SEQ. ID. NO.: 1 or SEQ. ID. NO.: 2 in manufacture of medicament for treatment of a subject suffering from dysfunction of hematopoiesis in mammalian including human.
  • the said SEQ. ID. NO.: 2 shows 5'-UGU AAC ACA AUG GUG AGU
  • the query sequence is at most 23 nucleotides in length.
  • the sequence is at least 10 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at most 23 nucleotides. And the GAP analysis aligns the two sequences over a region of at least 10 nucleotides.
  • a polynucleotide of the invention comprises a sequence which is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99
  • Polynucleotides of the present invention are natural ones, and may possess one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site- directed mutagenesis on the nucleic acid).
  • Oligonucleotides and/or polynucleotides of the invention hybridize to a sill- gene of the present invention, or a region flanking said gene, under stringent conditions.
  • stringent hybridization conditions refers to parameters with which the art is familiar, including the variation of the hybridization temperature with length of an oligonucleotide. Nucleic acid hybridization parameters may be found in references which compile such methods, Sambrook, et al. (supra), and Ausubel, et al. (supra).
  • stringent hybridization conditions can refer to hybridization at 65°C in hybridization buffer (3.5xSSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin (BSA), 2.5 mM NaH2PO4 (pH7), 0.5% SDS, 2 niM EDTA), followed by one or more washes in 0.2.xSSC, 0.01% BSA at 500C.
  • hybridization buffer 3.5xSSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin (BSA), 2.5 mM NaH2PO4 (pH7), 0.5% SDS, 2 niM EDTA
  • nucleic acid and/or oligonucleotides hybridize to the region of the an insect genome of interest, such as the genome of a honeybee, under conditions used in nucleic acid amplification techniques such as PCR.
  • BMSCs transduced with a newly identified short hairpin RNA (shRNA) of the present invention can differentiate into hematopoietic stem cells (HSCs) and their descendant multipotent progenitor cells that have the capacity of further differentiating into blood cells in vitro, according to the invention, on transplantation into sublethally irradiated NOD/SCID mice, transduced human BMSCs engrafted and differentiated into all hematopoietic lineages including lymphocytes and myelocytes. Furthermore this new shRNA alters BMSC fate by repressing the translation of EIDl.
  • shRNA short hairpin RNA
  • shRNAs Like artificial shRNAs, the two strands of their stems are perfectly complementary with a length of at least 21 nucleotides.
  • shRNAs important in regulating the self-renewal and directional differentiation of stem cells we employed a custom microarray for a high-throughput screen (see. table 1).
  • sRNA BMSC
  • HSC HSC/BMSC
  • *ZK-249 is referred as shR-CH in this paper.
  • shR-CH The expression of shR-CH was found to be at least 10 times higher in HSCs than in MSCs. To determine the length of mature shR-CH, this endogenous shRNA has been cloned. As shown in Figure 2B, mature shR-CH was about 21nt in length and its precursor formed a perfectly complementary hairpin structure. The said shR-CH resides within the first intron of SH3PXD2B gene that is located on chromosome 5 and is not phylogenetically conserved as most endogenous shRNAs (17 Accompanying manuscript).
  • EIDl (ElA-like inhibitor of differentiation- I)(NM O 14335.2) was selected for validation by qRT-PCR and Western blot assay because it contains five naturally-occurring putative shR-CH binding sites at its 3'UTR region (see Fig 1C). More importantly, EIDl has been shown to have a close relationship with two essential hematopoiesis-related transcriptional coactivators (S. Miyake et al., MoI Cell Biol. 20, 8889-902 (2000) ; W. Xu et al., Blood. 107, 4407-16 (2006).).
  • CBP Cyclic adenosine monophosphate response element binding protein
  • p300 interacts with over 312 proteins, at least 65 of which are encoded by genes that are essential for hematopoiesis
  • CBP/p300 was thought to provide both an assembly platform as well as protein acetyltransferase functions with many transcription factors and histones that regulate gene expression (V. V.
  • shR-CH mesenchymal stem cells from human bone marrow are infected with those vectors.
  • FIG. 2B the levels of shR-CH were significantly elevated by transduction of shR-CH vectors, compared with those in control or mock infected cells. Elevated levels of shR-CH can be sustained for a long time in BMSCs infected with shR-CH (data not shown).
  • Levels of EIDl mRNA were analyzed via application of RT-PCR at day 2 following transduction of shR-CH vectors.
  • BMSCs and HSCs it was noted that a reciprocal relationship between the expression of EIDl and the expression of the CD45 marker that is characteristic for hematopoietic cells. Moreover, CD45 was upregulated after shR-CH induced repression of EIDl, raising the possibility that EIDl is involved in suppressing the hematopoietic program in BMSCs. According to the present invention a single BMSC was cloned by the single cell cloning method to reach this end and rule out the possibility of HSC contamination.
  • results from a fluorescence activated cell sorter illustrated that the cloned BMSCs were characterized with the presence of specific CD29, CD 105 and CD44 surface antigens and FIk-I nuclear antigen, and without the CD34, CD45 and CD133 specific for HSCs (Suppl. Fig. 1). Then these cloned cells were infected with vectors expressing shR-CH or mock shRNA and seeded onto the 24-well plate coated with f ⁇ bronectin and collagen, and supplemented with a cocktail of hematopoietic cytokines and growth factors.
  • BMSCs originating from transduced BMSCs were identified on the basis of the green fluorescent protein (GFP) marker carried by the vector, and differentiation of BMSCs to hematopoietic cells was characterized by expression of specific CD45 surface antigen (Fig. 3A).
  • GFP green fluorescent protein
  • Fig. 3A Ex vivo expansion of infected BMSCs was determined by using a modified CFU assay. 5000 seed cells from each group were selected and cultured for an additional 14 days.
  • shR-CH overexpression rendered a substantial growth advantage during the 14-day culture as determined by in vitro colony-forming capacity (CFU-GM). This was also reflected by the predominance of GFP- and CD45- positive cells in the shR-CH culture (Fig. 3B).
  • si-EIDl is highly specific for its target mRNA since mock siRNAs showed no effects on the differentiation of BMSCs. Therefore, like the ectopic expression of shR-CH, the knockdown of EIDl expression in BMSCs can result in the differentiation of these stem cells toward hematopoietic cells. Together, the findings described above provide the direct evidence that shR-CH dictates the differentiation of MSCs via the repression of EIDl.
  • the cells were analyzed by flow cytometry by using three monoclonal antibodies against human leukocyte and stem cell markers. Consistent with the observation on the bone marrow sections, FACS profiles of bone marrow cells from recipients also showed robust engraftment of the BMSC-derived cells in medullary canal (Fig. 4B). After 8 weeks, the lineage composition of medullary canal cells descending from infected human BMSC (GFP + cells) was examined. The shR-CH expression in human BMSCs led to a significant increase in lymphoid (CD3) cells in medullary canal (2.8% vs 0.5% in the control).
  • CD3 lymphoid
  • Fig. 4B there was also a substantial elevation in CD33-positive myeloid lineage cells.
  • shR-CH The preferential expression of shR-CH in HSCs implicates an important role of shR-CH in hematopoiesis.
  • the findings in the invention show that ectopic expression of shR-CH is able to initiate and direct the human BMSCs to differentiate into hematopoietic cell lines in vitro and vivo (Fig. 3 and 4) substantiate that notion.
  • EIDl EIDl
  • shR-CH probably up-regulates the activities of hematopoiesis-related genes such as CBP and p300 (Fig 4C).
  • CBP and p300 knockout or point-mutant mice were reported to have dramatically reduced numbers of definitive erythroid, myeloid, and B-lymphocytic progenitors in bone marrow (L. H. Kasper, Nature. 419, 738-43 (2002); Y. Chen, P. Haviernik, K.D. Bunting, Y.C. Yang, Blood. 110, 2889-98 (2007); A.L. Kung et al, Genes Dev. 14, 272-7 (2000)).
  • Other studies further showed that CBP and p300 were fate decision factors for HSCs, responsible for HSC self-renewal and hematopoietic differentiation, respectively (V.I. Rebel et al., Proc Natl Acad Sci U S A.
  • Figure 1 shows an identification and characterization of a novel shR-CH in human stem cells, wherein Figure IA indicates the differential expression of newly identified shR-CH. Data are averages of at least three independent determinations. Error bars indicate standard deviations. *P ⁇ 0.01 and **P ⁇ 0.001, expression of shR-CH detected by microarrays or qRT-PCR assay in BMSCs compared with HSCs, respectively.
  • Figure IB indicates the cloning and sequencing of mature shR-CH and the secondary structure of the same. Solid red line stands for the sequence of mature shR-CH.
  • Figure 1C indicates the alignment of the sequences of mature shR-CH and its three putative binding sites at the 3'UTR region of EIDl mRNA.
  • FIG. 2 shows that the ectopic expression of shR-CH can effectively inhibit the expression of endogenous EIDl gene at the translational level.
  • Fig 2A indicates the construction of the mock-sRNA /GFP and shR-CH/GFP retroviral vectors used in this study.
  • Fig 2B indicates the differential expression of newly identified shR-CH in different cases. Data are averages of at least three independent determinations. Error bars indicate standard deviations. *p ⁇ 0.001.
  • Fig 2C indicates Real Time-PCR analysis on the expression levels of EIDl mRNA under various conditions were carried out and normalized to that of GAPDH, and the resultant expression levels in different cases are normalized to their levels in the control. Data are averages of at least three independent determinations. Error bars indicate standard deviations.
  • Fig 2D indicates the levels of EIDl protein from different cases were analyzed by Western blotting. The Western blot was stripped and re-probed with actin antibody to check for equal loading of total protein.
  • Figure 3 shows that the enforced expression of shR-CH can induce the proliferation and differentiation of human BMSCs in vitro.
  • Cells from a single clone were used.
  • Fig 3A indicates the morphological alterations of normal BMSC, mock-, shR-CH- and siEIDl -infected BMSCs illustrated by different staining methods.
  • Fig 3B indicates CFC assay of CD45 + hematopoietic cells. Cells were cultured in growth medium for 24 hours after transduction and then transferred into differentiation medium for 12 hours before immunostaining for CD45 specific for hematopoietic cells.
  • Fig 3C indicates quantitative analysis of colonies from three different cases. At Day 14, the colony number from the BMSCs infected by shR-CH, siEIDl or mock vectors gave the result of n>6/case; *p ⁇ 0.001. Value inside bars represents fold increase.
  • Figure 4 shows the effects of shR-CH forced expression of on hematopoietic lineage differentiation in vivo, wherein Fig 4A indicates the immunofluorescent and Hochest33342 staining of mouse bone marrow sections at 60 days post-trans. Enumeration of cells were stained with the human- specific antibodies against CD45, CD3 or CD33 in control, mock-shRNA and shR-CH sections of bone marrow obtained at 60 days post-transplantation.
  • Fig 4B indicates hematopoietic reconstitution from human BMSCs grafted in NOD-SCID mice. 10 5 GFP + CD45 + BMSCs were transplanted in sublethally irradiated NOD-SCID mice.
  • Fig 4C and Fig 4D indicate that Real time -PCR analysis on the expression levels of RUNXl mRNA under various conditions were carried out and normalized to that of GAPDH, and the resultant expression levels in different cases are normalized to their levels in the control. Data are averages of at least three independent determinations. Error bars indicate standard deviations.
  • BM Human bone marrow
  • mononuclear cells were separated by a Ficoll-Paque gradient centrifugation (specific gravity 1.077 g/mL; Nycomed Pharma AS, Oslo, Norway) and cultured in DF12 medium containing 5% FCS, 20 ng/niL EGF, 100 U/mL penicillin and 100 g/mL streptomycin (Gibco Life Technologies) at 37°C and a 5%CO 2 humidified atmosphere. The floating cells were discarded at 18 hours. Culture media were changed every 2 days.
  • cells were harvested and further depleted of hematopoietic cells with magnetic-activated cell separation (MACS) CD45, GIyA, and CD34 micromagnetic beads (Miltenyi Biotec, Auburn, CA). Cells were then replated and passaged. At passages 4 cells were harvested by trypsinization, transfected with retrovirus vectors and used in the transplantation assay. Cells derived from single clone were used in other assays. To ensure single-cell originality of each cell colony, sorted cells were plated in wells coated with fibronectin (Sigma, St Louis, MO) and collagen (Sigma) for each patient.
  • fibronectin Sigma, St Louis, MO
  • collagen Sigma
  • Culture medium was Dulbecco modified Eagle medium and Ham F 12 medium (DF 12) containing 40% MCDB-201 medium complete with trace elements (MCDB) (Sigma), 2% fetal calf serum (FCS; Gibco Life Technologies, Paisley, United Kingdom), 1: insulin-transferrin-selenium (Gibco Life Technologies), 10 - " 9 M dexamethasone (Sigma), 10 ⁇ 4 M ascorbic acid 2-phosphate (Sigma), 20 ng/mL interleukin-6 (Sigma), 10 ng/mL epidermal growth factor (Sigma), 10 ng/mL platelet-derived growth factor BB (Sigma), 50 ng/mL fetal liver tyrosine kinase 3 (Flt-3) ligand (Sigma), 30 ng/mL bone morphogenetic protein-4 (Sigma), and 100 LVmL penicillin and 100 g/mL streptomycin.
  • MCDB 2% fetal calf serum
  • a retrovirus vector pMSCV encoding shRNAs expressed from the U6 promoter was generated by the method in the art (Michael T Hemann 2003)., the short hairpin DNA sequence (SEQ. ID. NO.: 3 and SEQ. ID. NO.: 4, Table 4) coincident with the pre-shR-CH was chemically synthesized as a construct.
  • the retroviruses containing the shR-CH-expressing cassette were packaged by H293T cells.
  • the retroviral titers of the mock-GFP and shR-CH-GFP producer cells were 3 x 105/mL and 4 x 105/mL respectively, as assessed by transfer of GFP expression to H293T cells. (Fig.2 A)
  • Short hairpin RNAs were polyadenylated at 37 ° C for 30 min in a 50-ul reaction volume including 1.5 ug RNA and 5 U poly(A) polymerase (Takara). Poly(A)-tailed small RNA was recovered by phenol/chloroform extraction and ethanol precipitation. Reverse transcription was performed by applying 1.5 ug RNA and 1 ug of RT primer (SEQ. ID. NO.: 5, Table 4) with 200 U of Superscript III reverse-transcriptase (Invitrogen). The cDNA amplification was carried out for 40 cycles at a final annealing temperature of 60 ° C by using primers SEQ. ID. NO.: 6 (Table 4) targeting specifically the novel shRNA shR-CH and SEQ.
  • a 5' adapter (SEQ. ID. NO.: 8, Table 4) was ligated to small RNA by using T4 RNA ligase (TAKARA) and the ligation products were recovered by phenol/chloroform extraction followed by ethanol precipitation. Reverse transcription was performed by means of 1.5 ug RNA and 1 ug specific RT primer, SEQ. ID. NO.: 9 (Table 4). The cDNA amplification was carried out for 40 cycles at a final annealing temperature of 60 ° C via primers, SEQ. ID. NO.: 10 and SEQ. ID. NO.: 11 (Table 4). The PCR products were separated on 2% agarose with Goldview staining.
  • RNA and related genes were determined by real-time RT-PCR.
  • SYBR green assays with SYBR Premix Ex TaqTM (TAKARA) were run on the 7500HT real-time PCR instrument (Applied Biosystems).
  • Reverse transcription of small RNA was performed by using 1.5 ug RNA and 1 ug shR-CH specific RT primer .
  • beta-actin was used as loading control.
  • the antibodies used included beta-actin (Santa Cruz Biotechnology), and EIDl (UPSTATE).
  • the membranes then were incubated for one hour with HRP-conjugated rabbit anti-goat secondary antibody. Immunocomplexes were visualized with a commercial ECL kit.(Fig.2D) Example 7 Bioinformatic Analysis
  • miRanda 3.0 and targetscan 3.1 ( Miranda KC et al., Cell. 126, 1203-1217 (2006); Grimson A, Farh KK et al., MoI CeI. 27, 91-105 (2007)), were used in the invention for target prediction for shRNA.
  • HSCs-overexpressed human-shR-CH was selected for target verification.
  • one potential targets for human shR-CH, EIDl was chosen for further experimental validation. The result was shown in Fig.1 C .
  • CFU-GM Granulocyte-macrophage colony-forming unit
  • Cells from a single clone were fixed with paraformaldehyde 4% for 30 min at room temperature and then washed three times with PBS. The cells were permeabilized with Triton 0.2% in PBS for 5 min. After four washes in PBS, the cells were blocked with a blocking buffer (Dako) for 30 min and then incubated in PBS/BSA 0.1% with an anti-albumin (Dako) or anti-CD45, CD34, CD19, CD71, CD3 and CD33 antibody (PE; Becton Dickinson), or with rabbit IgG (Jackson ImmunoResearch) as a negative control for one hour at room temperature.
  • Dako anti-albumin
  • PE Becton Dickinson
  • rabbit IgG Jackson ImmunoResearch
  • the cells were washed three times in PBS and incubated with an antirabbit whole IgG-Cy3 (Jackson ImmunoResearch) or anti-rabbit whole IgG-Cy2 (Jackson ImmunoResearch) in PBS/BSA 0.1% for one hour at room temperature.
  • the cells were washed again (four times in PBS) and the nuclei were stained with Hochest 33342 diluted in PBS for 5 min at room temperature.
  • the stained cells were visualized by using a fluorescence microscope (Leica DMIRB) and images captured using Magnaf ⁇ re software. The results refer to Fig.3A, Fig.4A.
  • mice Six to eight week-old male nonobese diabetic (NOD)/ LtSz-scid/scid (SCID ) mice were bred and maintained under defined flora conditions in individually ventilated (high-efficiency particle-arresting filtered air) sterile micro-isolator cages (Techniplast, Milan, Italy). All animal handle and experiment procedures were approved by the Animal Care and Use Committee of the Chinese Academy of Medical Sciences. Mice were sublethally irradiated (300 cGy) with a cesium source (MDS Nordion; Gammacell, Ottawa, QC, Canada) prior to transplantation.
  • NOD nonobese diabetic
  • SCID LtSz-scid/scid mice
  • BMSCs 2.5 ⁇ l O 5 cells/mice transduced without or with shR-CH vectors or mock vectors in 0.4 ml of physiological saline (PS) were respectively injected via tail vain into the irradiated mice.
  • PS physiological saline
  • the peripheral white blood cell count was done once a week. Mice were killed 2 months later by cervical dislocation.
  • the BM from both femora and tibiae were collected. They were air dried and then stained with Jenner-Giemsa (BDH Ltd, Poole, United Kingdom). Conventional 4-um histologic sections of decalcified tibia were cut from formalin-fixed, paraffin-embedded material and stained.
  • mice that received a transplant was assessed for presence of human cells by FACS.
  • Mononuclear cells were harvested as described in the previous paragraph and red blood cells were lysed by adding 8.3% ammonium chloride. Single-cell suspensions were then prepared. After blocking of Fc receptors with human serum, cells were determined by labeling with anti-CD45, -CD33, or -CD3 phycoerythrin (PE; Becton Dickinson). Cells labeled with anti-immunoglobulin G (anti-IgG) monoclonal antibody (mAb) were used as control. In addition, cells were gated to include both lymphoid and myeloid fractions. (Fig.4B)

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Diabetes (AREA)
  • Biochemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L’invention concerne un ARN endogène court en épingle à cheveux ayant une séquence de SEQ ID NO: 1 ou un de ses compléments qui induit la formation de cellules hématopoïétiques, notamment en réprimant EID1, et son utilisation. L’invention concerne également une construction d’expression, un vecteur et un polynucléotide comprenant la séquence de SEQ ID NO: 1. La SEQ ID NO: 1 est représentée par 5’-CAA AUA CUC ACC GUG UUA CA -3’.
EP08784123A 2008-08-25 2008-08-25 Arn endogène court en épingle à cheveux et son utilisation Withdrawn EP2329024A4 (fr)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2008/072133 WO2010022553A1 (fr) 2008-08-25 2008-08-25 Arn endogène court en épingle à cheveux et son utilisation

Publications (2)

Publication Number Publication Date
EP2329024A1 true EP2329024A1 (fr) 2011-06-08
EP2329024A4 EP2329024A4 (fr) 2012-02-22

Family

ID=41720773

Family Applications (1)

Application Number Title Priority Date Filing Date
EP08784123A Withdrawn EP2329024A4 (fr) 2008-08-25 2008-08-25 Arn endogène court en épingle à cheveux et son utilisation

Country Status (4)

Country Link
EP (1) EP2329024A4 (fr)
CN (1) CN102131929A (fr)
AU (1) AU2008361210A1 (fr)
WO (1) WO2010022553A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103088065B (zh) * 2013-01-25 2014-12-10 北京银杏德济生物技术有限公司 一种能够大规模并快速高纯度地诱导间充质干细胞转决定成造血干细胞的方法
EP2805705B1 (fr) * 2013-05-23 2016-11-09 IP Gesellschaft für Management mbH Emballage comprenant des unités d'administration des sels de sodium de l'acide (R)-3-[6-amino-pyridin-3-yl]-2-(1-cyclohexyl-1 H-imidazol-4-yl)-propionique
CN116904469B (zh) * 2023-09-12 2024-01-23 首都儿科研究所 一种p300蛋白表达的抑制剂和制备方法及其用途

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040265849A1 (en) * 2002-11-22 2004-12-30 Applera Corporation Genetic polymorphisms associated with Alzheimer's disease, methods of detection and uses thereof
WO2009017670A2 (fr) * 2007-07-26 2009-02-05 University Of Massachusetts Effecteurs d'extinction épigénétique à médiation par ras et leurs utilisations

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7612195B2 (en) * 2003-10-22 2009-11-03 Aventis Pharmaceuticals Inc. Retroviral vectors for delivery of interfering RNA
CN101024830A (zh) * 2007-02-01 2007-08-29 王鸿艳 一种ShRNA的克隆方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040265849A1 (en) * 2002-11-22 2004-12-30 Applera Corporation Genetic polymorphisms associated with Alzheimer's disease, methods of detection and uses thereof
WO2009017670A2 (fr) * 2007-07-26 2009-02-05 University Of Massachusetts Effecteurs d'extinction épigénétique à médiation par ras et leurs utilisations

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
A. BAVNER: "A transcriptional inhibitor targeted by the atypical orphan nuclear receptor SHP", EMBO REPORTS, vol. 3, no. 5, 15 May 2002 (2002-05-15), pages 478-484, XP55010059, ISSN: 1469-221X, DOI: 10.1093/embo-reports/kvf087 *
BIAN CHUNJING ET AL: "An endogenous short hairpin RNA shr337 induces the formation of hematopoietic stem cells from messenchymal stem cells in human bone marrow", EXPERIMENTAL HEMATOLOGY; 37TH ANNUAL SCIENTIFIC MEETING OF THE INTERNATIONAL-SOCIETY-FOR-EXPERIMENTAL-HEM ATOLOGY/6TH INTERNAT; BOSTON, MA, USA; JULY 09 -12, 2008, ELSEVIER INC, US, vol. 36, no. 7 supplement 1, 1 July 2008 (2008-07-01), page S6, XP008144301, ISSN: 0301-472X, DOI: 10.1016/J.EXPHEM.2008.05.007 *
BIAN CHUNJING ET AL: "An endogenous short hairpin RNA shr337 induces the formation of hematopoietic stem cells from messenchymal stem cells in human bone marrow", EXPERIMENTAL HEMATOLOGY; 37TH ANNUAL SCIENTIFIC MEETING OF THE INTERNATIONAL-SOCIETY-FOR-EXPERIMENTAL-HEM ATOLOGY/6TH INTERNAT; BOSTON, MA, USA; JULY 09 -12, 2008, ELSEVIER INC, US, vol. 36, no. 7 supplement 1, 1 July 2008 (2008-07-01), pages S47-S48, XP008144300, ISSN: 0301-472X, DOI: 10.1016/J.EXPHEM.2008.05.007 [retrieved on 2008-06-20] *
Gu, Tongjun et al.: "A novel class of endogenous shRNAs in human cells", Nature Precedings, 22 January 2008 (2008-01-22), XP002661697, Retrieved from the Internet: URL:http://precedings.nature.com/documents/1531/version/1/files/npre20081531-1.pdf [retrieved on 2011-10-20] *
See also references of WO2010022553A1 *
WANG ET AL: "Bcl2 enhances induced hematopoietic differentiation of murine embryonic stem cells", EXPERIMENTAL HEMATOLOGY, ELSEVIER INC, US, vol. 36, no. 2, 26 November 2007 (2007-11-26), pages 128-139, XP022425429, ISSN: 0301-472X, DOI: 10.1016/J.EXPHEM.2007.09.004 *
ZOU GANG-MING ET AL: "Reduction of Shp-2 expression by small interfering RNA reduces murine embryonic stem cell-derived in vitro hematopoietic differentiation", STEM CELLS, ALPHAMED PRESS, DAYTON, OH, US, vol. 24, no. 3, 1 March 2006 (2006-03-01), pages 587-594, XP002489842, ISSN: 1066-5099, DOI: 10.1634/STEMCELLS.2005-0272 *

Also Published As

Publication number Publication date
AU2008361210A1 (en) 2010-03-04
WO2010022553A1 (fr) 2010-03-04
EP2329024A4 (fr) 2012-02-22
CN102131929A (zh) 2011-07-20

Similar Documents

Publication Publication Date Title
Pei et al. AMPK/FIS1-mediated mitophagy is required for self-renewal of human AML stem cells
Herrera-Merchan et al. miR-33-mediated downregulation of p53 controls hematopoietic stem cell self-renewal
Kong et al. MIR-23A microRNA cluster inhibits B-cell development
Deng et al. HoxBlinc RNA recruits Set1/MLL complexes to activate Hox gene expression patterns and mesoderm lineage development
Antonchuk et al. HOXB4 overexpression mediates very rapid stem cell regeneration and competitive hematopoietic repopulation
Wojtowicz et al. Ectopic miR-125a expression induces long-term repopulating stem cell capacity in mouse and human hematopoietic progenitors
Hu et al. Bmi1 suppresses adipogenesis in the hematopoietic stem cell niche
Sommerkamp et al. Differential alternative polyadenylation landscapes mediate hematopoietic stem cell activation and regulate glutamine metabolism
JP2016513974A (ja) 造血幹細胞系列を初期化するための組成物および方法
WO2010017551A2 (fr) Procédé pour mir-125a dans la promotion de renouvellement automatique et d'extension de cellules souches hématopoïétiques
Zou et al. Duplexes of 21‐nucleotide RNAs mediate RNA interference in differentiated mouse ES cells
US20090220465A1 (en) Methods and compositions for modulation of stem cell aging
US9163234B2 (en) Culture method
US8828965B2 (en) MiR-150 for the treatment of blood disorders
Wang et al. E3-ligase Skp2 regulates β-catenin expression and maintains hematopoietic stem cell homing
WO2010022553A1 (fr) Arn endogène court en épingle à cheveux et son utilisation
Suzuki et al. Homeostasis of hematopoietic stem cells regulated by the myeloproliferative disease associated-gene product Lnk/Sh2b3 via Bcl-xL
US20080305085A1 (en) Compositions And Methods For Stem Cell Expansion
Zaidan et al. Endothelial-specific Gata3 expression is required for hematopoietic stem cell generation
CN101368180A (zh) 一类能将间充质干细胞分化为造血细胞的小rna分子及其作用靶点
US20150299712A1 (en) Modulation of Hematopoietic Stem Cell Differentiation
Li et al. Zfp521 is essential for the quiescence and maintenance of adult hematopoietic stem cells under stress
Ihme Characterization of the Leukemia Initiating Cell in Human Acute Myeloid Leukemia
US20150283164A1 (en) Treatment of Myelodysplastic Syndrome by Inhibition of NR2F2
Sun MicroRNA-18a prevents senescence of mesenchymal stem 2 cells by targeting CTDSPL

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20110325

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL BA MK RS

DAX Request for extension of the european patent (deleted)
A4 Supplementary search report drawn up and despatched

Effective date: 20120125

RIC1 Information provided on ipc code assigned before grant

Ipc: A61P 7/00 20060101ALI20120117BHEP

Ipc: A61K 31/7105 20060101ALI20120117BHEP

Ipc: C12N 15/113 20100101AFI20120117BHEP

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20120824