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WO2016034679A1 - Micro-arn pour la suppression des antigènes de groupes sanguins - Google Patents

Micro-arn pour la suppression des antigènes de groupes sanguins Download PDF

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WO2016034679A1
WO2016034679A1 PCT/EP2015/070170 EP2015070170W WO2016034679A1 WO 2016034679 A1 WO2016034679 A1 WO 2016034679A1 EP 2015070170 W EP2015070170 W EP 2015070170W WO 2016034679 A1 WO2016034679 A1 WO 2016034679A1
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mir
cell
blood group
blood
erythrocyte
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Torsten Tonn
Erhard Seifried
Romy KRONSTEIN-WIEDEMANN
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Drk Blutspendedienst Nord-Ost Ggmbh
Technische Universitaet Dresden
DRK Blutspendedienst Baden Wuerttermberg Hessen gGmbH
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Drk Blutspendedienst Nord-Ost Ggmbh
Technische Universitaet Dresden
DRK Blutspendedienst Baden Wuerttermberg Hessen gGmbH
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    • 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
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • 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 pertains to micro RNAs (miR) regulating the expression of blood group antigens such as A, B, Rhesus or Kell.
  • the miRs of the invention were found to directly target glycosyltransferases, enzymes which are responsible for the generation of the glycosylation patterns that are characteristic for a given blood type.
  • the invention provides methods which harness miR expression-constructs for the generation of erythrocytes with a reduced expression of blood group antigens. Further provided are: erythrocytes produced with the methods of the invention, and a blood preparation suitable in transfusion medicine comprising the erythrocytes of the invention.
  • the invention additionally relates to a method for diagnosing blood group antigen suppression in a subject, the method comprising the detection of the copy-number of the miR target sites in the genetic loci encoding glycosyltransferases and other blood group receptors.
  • transfusion medicine when blood, or a blood fraction, such as red blood cells (erythrocytes), platelets (thrombocytes) and white blood cells (leukocytes), derived from a donor are administered to another person, serious adverse reactions may occur when the donor blood or blood fraction does not match properly with the blood of the recipient.
  • erythrocytes red blood cells
  • thrombocytes platelets
  • white blood cells leukocytes
  • transfusion reactions agglutination
  • blood from a donor of blood group A is given to a person of blood group B (blood group antigens A and B belong to the ABO system).
  • RhD rhesus D
  • RhD antibodies will lead to rapid destruction of RhD-positive red cells and to transfusion reactions. Furthermore, when a woman with red cell or platelet antibodies becomes pregnant, those antibodies can cross the placenta and can destruct the red cells or the platelets of the unborn child. This can lead to severe hemolysis resulting in anaemia, jaundice (after birth) and if not treated it can be fatal or lead to cerebral damage.
  • the ABO system of blood groups is the most extensively studied blood type antigen system. The ABO gene locus expresses glucosyltransferases in three alleleic forms: A, B, and 0.
  • the A allele encodes a glycosyltransferase that bonds a-N-acetylgalactosamine to D-galactose end of H antigen, producing the A antigen.
  • the B allele encodes a glycosyltransferase that joins a- D-galactose bonded to D-galactose end of H antigen, creating the B antigen.
  • the O allele differs slightly from the A allele by deletion of a single nucleotide - Guanine at position 261. The deletion causes a frameshift and results in translation of an almost entirely different protein that lacks enzymatic activity. This results in H antigen remaining unchanged in case of O groups.
  • MicroRNAs are a class of small (e.g., 18-24 nucleotides) non-coding RNAs that exist in a variety of organisms, including mammals, and are conserved in evolution. miRNAs are processed from hairpin precursors of about 70 nucleotides which are derived from primary transcripts through sequential cleavage by the RNAse III enzymes drosha and dicer. Many microRNAs can be encoded in inter- genic regions, hosted within introns of pre-mRNAs or within non-coding RNA genes.
  • miRNAs also tend to be clustered and transcribed as polycistrons and often have similar spatial temporal expression patterns.
  • MiRs have been found to have roles in a variety of biological processes including developmental timing, differentiation, apoptosis, cell proliferation, organ development, and metabolism.
  • no miRNA have been identified to date that are involved in the regulation of blood group antigens.
  • the objective of the present invention is to provide blood preparations with minimal antigenic potential in order to reduce immune reactions in a patient receiving a transfusion.
  • the above problem is solved in a first aspect by a method for the production of an erythrocyte with a suppressed expression of a blood group antigen, comprising the steps of
  • micro RNA selected from the group consisting of miR-331-3p, miR-1908, miR-4711, miR-99a, miR- 3613-3p, miR-522-3p, miR-224-3p, miR-26b-5p, miR-26a-5p, miR-4465, miR- 1297 and miR-100, or combinations thereof, and
  • said biological cell is a precursor cell of an erythrocyte, differentiating said precursor cell into an erythrocyte.
  • a method for suppressing the expression of a blood group antigen in a biological cell comprising
  • a genetic construct into said biological cell, wherein the genetic construct comprises an expressible miR sequence selected from the group consisting of miR-331-3p, miR-1908, miR-4711, miR-99a, miR-3613-3p, miR- 522-3p, miR-224-3p, miR-26b-5p, miR-26a-5p, miR-4465, miR- 1297 and miR-100, or combinations thereof,
  • said biological cell is a precursor cell of an erythrocyte, differentiating said precursor cell into an erythrocyte.
  • miR-331-3p, miR-1908, miR-4711, miR-99a and miR-100 were identified to target the expression of glycosyltransferases, in particular glycosyltransfer- ases A, which are responsible for the expression of blood group antigens.
  • the miRs miR- 3613-3p, miR-522-3p, miR-224-3p, miR-26b-5p, miR-26a-5p, miR-4465 and miR-1297 where identified to target the rhesus D gene. Therefore, ectopic expression of these miR molecules, in particular of miR-331-3p and miR-1908, is surprisingly effective in order to reduce blood group antigen expression in a cell.
  • the above miR which are characterized by a mature miR sequences according to Table 1.
  • micro RNA refers to a particular class of "small RNA molecules" as defined above.
  • micro RNA refers to a non-coding RNA comprising from about 3 to about 200, from about 4 to about 180, from about 5 to about 150, from about 6 to about 120, from about 7 to about 90, from about 8 to about 80, from about 10 to about 60, from about 20 to about 40 or from about 20 to about 30 nucleotides in length, which hybridizes to and regulates the expression of a coding RNA.
  • the mi-RNAs as referred to may be single or double-stranded and may be obtained from a micro-RNA precursor, such as a hairpin RNA precursor, by natural processing routes (e.g. using intact cells or cell ly sates) or by synthetic routes (e.g. using isolated processing enzymes, such as the dicer enzyme or RNAase III). Alternatively, the mi-RNAs may be obtained directly by biological or chemical synthesis without the involvement of a precursor. The mi-RNAs can silence the activity of an oligonucleotide encoding for a protein by blocking its translation.
  • mi-RNA micro-RNA
  • miR mi-RNA sequence
  • the term "genetic construct” is meant to refer to a nucleic acid molecule that comprises a nucleic acid sequence that encodes a protein or miR operably linked to elements necessary for expression of either the protein or miR sequence.
  • the genetic construct is DNA, preferably a plasmid or genome of a viral vector.
  • the genetic construct is RNA, preferably a genome of a retroviral vector.
  • the expression of said miR in step b. involves the transduction or transfection of said biological cell with a genetic construct comprising an expressible sequence of said miR.
  • the miR molecules are ectopically expressed in biological systems.
  • ectopically or “ectopic” as used herein in reference to the expression of a nucleic acid molecule, preferably a miR sequence is distinct from the expression pattern in a wild type biological cell.
  • ectopic expression of a miR of the invention can refer to expression in a cell type other than a cell type in which the nucleic acid molecule normally is expressed, or at a time other than a time at which the nucleic acid molecule normally is expressed, or at a level other than the level at which the nucleic acid molecule normally is expressed.
  • biological cell refers to a cell selected from the group consisting of an erythrocyte or a precursor cell of an erythrocyte, such as a stem cell, an adult- or embryonic stem cell, a hematopoietic stem cell, an immortalized erythrocyte precursor cell, an induced pluripotent stem cell (IPS cell), a bone marrow derived stem cell, a hematopoietic stem cell derived from cord blood, or a hematopoietic stem cell derived from mobilized peripheral blood.
  • an embryonic stem cell is not exclusively obtainable by a method involving the destruction of human embryos.
  • micro RNA selected from the group consisting of miR-331-3p, miR- 1908, miR-4711, miR-99a, miR-3613-3p, miR-522-3p, miR-224-3p, miR-26b-5p, miR-26a-5p, miR-4465, miR- 1297 and miR- 100, or combinations thereof, d.
  • miR micro RNA
  • said biological cell is a precursor cell of an erythrocyte, differentiating said precursor cell into an erythrocyte.
  • Transcription factor Spl also known as specificity protein 1
  • SP1 Transcription factor 1
  • the protein encoded by this gene is a zinc finger transcription factor that binds to GC-rich motifs of many promoters.
  • the encoded protein is involved in many cellular processes, including cell differentiation, cell growth, apoptosis, immune responses, response to DNA damage, and chromatin remodelling and cancer (Beishline Kl, Azizkhan- Clifford J.FEBS J. 2015 Jan;282(2):224-58. doi: 10.111 l/febs.13148 "Spl and the 'hallmarks of cancer'").
  • step (c) is mandatory and not optionally.
  • step (d) is mandatory and not optionally.
  • steps (c) and (d) are both mandatory.
  • CpG island (- 650 to + 50). This region contains a lot of binding sites for several transcription factors, i.e. Spl, AP2, GATA.
  • PTGS post-transcriptional gene silencing
  • miRNAs are known to mediate post-transcriptional gene silencing (PTGS) in the cytoplasm, recent evidence indicates that at least some fraction of mammalian miRNA's may also have nuclear roles in regulating gene expression. They provide evidence that the endogenous miRNA pathway may direct transcriptional silencing in the nucleus, in addition to the cytoplasmic PTGS and translational activation pathways.
  • the inventors now identified binding sites for miR-331-3p and miR-1908-5p in the 5'UTR of the ABO gene, which also were located within the Spl transcription factor binding domains in the ABO gene regulatory area.
  • the Spl gene also contained target sites of miR-331-3p itself, and therefore is a target of the miR. Therefore, Spl is negatively regulated by the miR of the invention, and its further inhibition leads to the suppression of ABO gene expression.
  • Spl inhibitors are known in the art. However preferred Spl inhibitors of the invention are Withaferin A and Mithramycin A.
  • the inhibitor of Spl is contacted with the biological cell in a concentration of ⁇ , ⁇ - ⁇ .
  • the methods of the invention are preferably ex-vivo or in vitro methods.
  • the methods in accordance may further include an additional step of expanding and/or purifying said erythrocytes.
  • the expansion or purification can simply take place by culturing the erythrocytes of the invention.
  • the person of skill in the art may apply immuno- adsorption or centrifugation in order to expand and/or isolate the erythrocyte preparation.
  • the term "blood group antigen” means in context of the invention an antigen pattern of the ABO system, such as A, or B, or alternatively rhesus D and/or Kell.
  • the blood group antigen to be supressed by the method of the invention is A and/or B
  • said miR is preferably miR-331-3p and/or miR-1908.
  • said miR is selected from the group consisting of miR-3613-3p, miR-522-3p, miR-224-3p, miR-26b-5p, miR-26a-5p, miR-4465, and miR- 1297.
  • a recombinant erythrocyte cell comprising a genetic construct, wherein said genetic construct comprises at least one expressible miR- sequence selected from the group consisting of miR-331-3p, miR-1908, miR-4711, miR-99a, miR-3613-3p, miR-522-3p, miR-224-3p, miR-26b-5p, miR-26a-5p, miR-4465, miR-1297 and miR- 100, or combinations thereof.
  • Another aspect of the invention relates to an expression vector comprising an expressible sequence of a miR selected from the group consisting of miR-331-3p, miR-1908, miR4711, miR-99a, miR-3613-3p, miR-522-3p, miR-224-3p, miR-26b-5p, miR-26a-5p, miR-4465, miR-1297 and miR- 100, or combinations thereof, wherein the vector is suitable for the expression of said miR in a biological cell selected from the group consisting of an erythrocyte or an erythrocyte precursor cell, such as a stem cell, an adult- or embryonic stem cell, a hematopoietic stem cell, an immortalized erythrocyte precursor cell, an induced pluripotent stem cell (IPS cell), a bone marrow derived stem cell, a hematopoietic stem cell derived from cord blood, or a hematopoietic stem cell derived from mobilized peripheral blood.
  • Yet a further aspect pertains to a method for identifying a miR involved in the suppression of the expression of a blood group antigen, the method comprising,
  • a genetic construct comprising an expressible reporter gene sequence with a 3 ' untranslated region (UTR) of a glycosyltransferase gene or a rhesus D gene,
  • the object of the present invention is also solved by a method for diagnosing a low expression phenotype of a blood group antigen in a subject, comprising
  • the control shall in this aspect refer to a copy number of said miR-target sites which is characteristic for the majority of individuals within the population - therefore a copy-number that would qualify as "normal".
  • the population may in certain preferred aspects be the human population.
  • a copy number of 3 miR- 1908-target sites within the glycosyltransferases A gene is considered as a "normal" copy- number.
  • Individuals with an increased number of target sites have the low expression phenotype of blood group antigen A.
  • step (b) said blood group antigen is A and said glycosyltransferase gene is glycosyltransferase isoform A, or wherein said blood group antigen is B and said glycosyltransferase gene is glycosyltransferase isoform B; or wherein in step (c) said blood group antigen is rhesus D, and said rhesus blood group system gene is the rhesus D (RhD) gene.
  • miR-target site refers to a sequence stretch within a genetic locus of a given gene that when expressed into mRNA mediates the binding of a mature miR molecule to said complementary sequence-stretch on the mRNA. Via the binding of the mature miR to the miR- target site on the mRNA, the translation of the mRNA is inhibited which results into a decreased protein expression.
  • the copy number of miR-target sites therefore refers to the number of sequences stretches within a genetic locus which allow mature miR to bind to the transcribed mRNA molecules. The more binding sites are present within a genetic locus, the stronger is the repression of protein expression by the miR.
  • copy number preferably, relates to the average copy number of a miR target site (binding site) per gene locus in the genome of a subject to be diagnosed.
  • the average copy number is the arithmetical average of miR binding sites in a gene locus of a subject.
  • a copy-number of 4 or higher in preferred embodiments of the invention is usually indicative for a low expression phenotype of blood group antigen A and/or B.
  • said miR target site is a target site ofmiR-331-3p, miR-4711 or miR-1908.
  • the terms “subject” and “patient” are used interchangeably.
  • the terms “subject” and “subjects” refer to an animal, preferably a mammal including a non- primate (e.g., a cow, pig, horse, cat, dog, rat, and mouse) and a non-primate (e.g., a monkey such as a cynomolgous monkey and a human), and more preferably a human.
  • a non- primate e.g., a cow, pig, horse, cat, dog, rat, and mouse
  • a non-primate e.g., a monkey such as a cynomolgous monkey and a human
  • the copy-number of miR target-sites in the 3'UTR is in a preferred embodiment determined by DNA-sequencing or allele-specific PCR.
  • the person of skill is aware of methods suitable for the detection of the number of sequence motifs in a genetic sequence.
  • a method for increasing the expression of a blood group antigen in a biological cell, comprising a.
  • a miR selected from the group consisting of miR-331-3p, miR-1908, miR-4711, miR-99a, miR-3613-3p, miR- 522-3p, miR-224-3p, miR-26b-5p, miR-26a-5p, miR-4465, miR-1297 and miR-100
  • a method for increasing the expression of a blood group antigen in a biological cell, comprising a.
  • the biological cell is contacted with an SPl protein by ectopically expressing SPl in the biological cell, for example by introducing an SPl expression construct into the biological cell.
  • a further aspect relates to the use of a miR selected from the group of miR-331-3p, miR-1908, miR-4711, miR-99a, miR-3613-3p, miR-522-3p, miR-224-3p, miR-26b-5p, miR- 26a-5p, miR-4465, miR-1297 and miR-100, for the suppression of the expression of a blood group antigen.
  • the use is an ex vivo or in vitro use, and/or said blood group antigen is selected from A, B rhesus D and/or Kell.
  • An alternative aspect of the invention is a use of an antisense molecule against a miR selected from the group of miR-331-3p, miR-1908, miR-4711, miR-99a, miR-3613-3p, miR-522-3p, miR-224-3p, miR-26b-5p, miR-26a-5p, miR-4465, miR-1297 and miR-100, for increasing the expression of a blood group antigen.
  • the use is an ex vivo or in vitro use, and/or said blood group antigen is selected from A, B rhesus D and/or Kell.
  • kit for performing a method as described herein before wherein the kit is adapted to any of said methods.
  • Another aspect relates to an erythrocyte produced according to a method of the invention, for use in transfusion medicine.
  • a blood preparation suitable for blood transfusion comprising an erythrocyte produced according to a method of the invention, or comprising an erythrocyte according of the invention as described herein before.
  • FIG. 1 Luciferase assay, a. Renilla normalized activity of luciferase in K562 cell transduced with a 3' UTR - reporter of the normal expression phenotype of the glycosyltransferase A gene. The cells were treated with mock, unrelated miR and miR-331, miR- 1908 and miR-4711. b. The presence of 5 versus 3 mi- croRNA-1908 binding sites in the 3 ' untranslated region of individuals with weak A blood group phenotypes leads to further reduced blood group A expression. Luciferase assay with 3 'UTR of glycosyltransferase A and altered 3 'UTR of glycosyltransferase A weak variants. The cells were treated mock, unrelated miR or miR- 1908 and renilla luciferase control vector in K562 cells. Error bars depict s.e.m. (*) indicate significance.
  • Figure 2 Blood group antigen A expression in hematopoietic stem cells transduced with miR. Erythrocytes derived from hematopoietic stem cells transduced with miR of the invention were tested for blood group antigen A expression 11 days post-transduction in the flow cytometer. Glycophorin A was used as a marker for erythrocytes.
  • Figure 4 Downregulation of endogenous miR-1908-5p, but not miR-331-3p enhance blood group A antigen expression on red blood cell surface, a, Real-time PCR expression analysis of miR-331 -3p and -1908-5p in erythroid cells derived from CD34+ hematopoietic stem cells (day 8 of differentiation) transduced with locker-miR-331-3p or -1908-5p compared to mock control, b, Amount of erythroid cells (day 8 of differentiation) after lentiviral transduction of locker-miR-331, -1908 or control (mock) within the GFP positive cell population measured by flowcytometry using anti-glycophorin A antibody, c, Relative expression of blood group A antigen per erythoid cell after lentiviral transduction of locker-miR-331, -1908 within the GFP positive and glycophorin A positive cell population compared to mock control, d, Real-time PCR expression analysis of glycosyltransfera
  • FIG. 5 3'UTR of glycosyltransferase A in hybrid variants of A-0 v reveals the presence of more miRNA binding sites for miR-1908-5p.
  • a Nucleotide sequence of the 3' flanking region in the ABO gene of normal glycosyltransferase A (upper sequence) and the hybrid variant A-0 lv (lower sequence). Binding sites for miR-1908-5p are marked with gray. The additional two binding sites in the hybrid variant are indicated with dark gray
  • b Known hybrid alleles modified from Chester and Olsson (Chester and Olsson 2001). Only changes from the consensus sequence are shown. Mutations causing amino acid changes are shown in bold type. The dark vertical bars indicate the position of the in- trons.
  • Thick-sided boxes indicate regions where a crossing-over event is likely to have occurred.
  • a thick vertical line indicates the end of the reading frame.
  • the shadowed area indicates a reading frame shift.
  • ABO gene from samples 1, 3, 5, 7, 8 and 12 were sequenced in this study and compared with known alleles.
  • SEQ ID NO: 6 to 17 (cloning primer sequences)
  • Recombinant human SCF, Flt3-ligand, IL-3 and TPO are from Peprotech (Hamburg, Germany).
  • Recombinant human erythropoietin (EPO) was from Cellscience (Canton, MA, USA).
  • Insulin solution, ho lo -transferrin, heparin, hydrocortisone and Polybrene were from Sigma/ Aldrich (Schnelldorf, Germany).
  • Cells were derived from cord blood, mobilized peripheral blood, bone marrow, induced pluripotent stem cells or embryonic stem cells or immortalized erythrocyte precursor cells.
  • CD34+ cells from different sources with blood group A were isolated by centrifugation over Biocoll (Biochrom) and subsequently magnetic microbead selection of MNCs by the use of LS-MACS columns (Miltenyi Biotec; 94% ⁇ 3% purity).
  • the cells were cultured in IMDM (Biochrom) supplemented with 1% L-glutamin (Biochrom), lOOunits/ml penicillin and 10( ⁇ g/ml streptomycin (Biochrom), 330 ⁇ g/mL holo-human transferrin, 10 ⁇ g/mL recombinant human insulin, 2 IU/mL heparin Choay, and 5% AB serum (pooled from three different donors, DRK blood donor center North-East, Dresden, Germany).
  • the expansion procedure is performed in three steps.
  • first step day 2 to day 7 post- transduction
  • 2 x 10 4 /mL CD34+ cells were cultured in the presence of 10 6 M hydrocortisone, 100 ng/niL SCF, 5 ng/niL IL-3 and 3 IU/mL Epo.
  • second step day 7 to day 11
  • the cells were resuspended at 10 5 /mL of SCF and Epo.
  • the third step day 11 to day 18
  • the cells were cultured with Epo alone. Cell counts were adjusted to 1 x 10 6 and 1 x 10 7 cells/mL on days 11 and 15, respectively.
  • Human embryonic kidney 293T cells were cultured in a humidified atmosphere of 5% C0 2 /air in Dulbecco's modified Eagle's medium with stable glutamine and high glucose (Life technologies, Darmstadt, Germany) supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin.
  • K562 cells were cultured in a humidified atmosphere of 5% C0 2 /air in RPMI 1640 medium (Biochrom, Berlin, Germany) supplemented with 10% fetal bovine serum (Biochrom), lOOunits/ml penicillin and 100 ⁇ / ⁇ 1 streptomycin (Biochrom), 1% L-glutamine (Life Technologies, Darmstadt, Germany), 1% MEM non-essential amino acids (Life Technologies) and 1% sodium pyruvate (Life Technologies).
  • Viral supernatants were produced by transient transfection of 293T cells using 23 ⁇ g of pLV-[hsa-miRNA] or pLV- [miR A- locker] (Biosettia, San Diego, USA), 9 ⁇ g of the vesicular stomatitis virus envelope glycoprotein envelope plasmid (pMD2.G) and 17 ⁇ g of the packaging plasmid pR8.94.
  • CD34+ stem cells were isolated and plated at a density of 1 x 10 5 /well in a 24 well plate.
  • lentiviral supernatants at an MOI of 10 were added to pre-coated wells with retronectin (Takara, Otsu, Japan) for 1 hour at 37°C.
  • CD34+ stem cells were spin-infected, and then cultured for 48 hours.
  • the medium for transduction was STEMSpan (STEMCELL technologies, Grenoble, France) supplemented with 50ng/ml SCF, 5ng/ml IL-3 and lOng/ml TPO.
  • the 3'UTR of the glycosyltransferase A was synthesized from GeneArt (life technologies) and cloned into the Hindlll and Spel site downstream of firefly luciferase gene in the pMIR- REORT vector (Applied biosystems, Darmstadt, Germany). To assess the effect of different miRNAs on glycosyltransferase A 3'UTR activity, expression constructs encoding miRNA- 26, -99a, -100, -331, -1908 and -4711 were inserted into a CMV-based pcDNA3.1 (-) (life technologies).
  • the following primers were used to amplify the expression constructs from the genomic DNA of human whole blood and cloned into the BamHIII and EcoRI site of pcDNA3.1 : miRNA-26a (forward: GGATTGAATTCTATCACAAGGTCCCAGGGCTGG (SEQ ID NO: 6); reverse: CAATAGGATCCGGCTGCAACAGCTGCAGACTC (SEQ ID NO: 7)),
  • miRNA-99a forward: CCAGAG AATTCTGAAGGCCTTTAATGGAGAA (SEQ ID NO: 8); reverse: CAATAGGATCCAGTATGCACTGA AGTTTCTG (SEQ ID NO: 9)
  • miRNA-100 forward: ATATAGGATCCGCTGCCTCACCTGTAAGCTC (SEQ ID NO: 10); reverse: CGATAGAATTCAAAGTGGAAACCAAGGGAAGC (SEQ ID NO: 11)),
  • miRNA-331 forward: CCATAGAATTCGACAACGTACAGAAGGCTCC (SEQ ID NO: 12); reverse: TAATAGGATCCGGCTG GGAAGACTTTGTTACC (SEQ ID NO: 13)),
  • miRNA-1908 forward: GCATGGAATTCGCAGGCGTGTC CCGGCGCATG (SEQ ID NO: 14); reverse: ATATAGGATCCGGTTGTGCAAGGTAAGGTCC (SEQ ID NO: 15)) and
  • miRNA-4711 forward: GCATGGAATTCGACAATCAATACAGAGCCTGAG (SEQ ID NO: 16); reverse: ATATAGGATCCCTGTCCACGTCCTACACACTC (SEQ ID NO: 17)).
  • K562 cells were cotransfected with 0.25 ⁇ of reporter construct, 2.5ng renilla luciferase construct pGL4.75 (Promega, Mannheim, Germany) and O ⁇ g of indicated miRNA expression constructs, all combined with Lipofectamine 2000 (Life technologies).
  • the luciferase activities (both firefly and renilla luciferase) in the same sample were determined after 48 hours with the Dual-Glo Luciferase Assay (Promega) and measured by Luminometer (Perkin Elmer, MA, USA). The relative reporter activities were obtained by normalization of firefly to the renilla luciferase activities.
  • FITC labeled anti- human blood group A antibody BD bioscience
  • 100000 cells were labeled with FITC-conjugated anti-Blood group A, V450-conjucated anti- CD45 and APC-conjugated anti-CD235a (BD Bioscience, Heidelberg, Germany) for 25 minutes in darkness at room temperature. Cells were washed with PBS containing 1%FBS, 5mM EDTA and 0,09% Na-azid by centrifugation at 300 x g for 5 minutes.
  • Transduced cells were identified by red fluorescent puromycin-N-acetyl-transferase (mCherry), which is inserted into the pLV-[hsa-miR] -vectors and green fluorescent protein, which is inserted into the pLV-[miR- locker] -vectors. Analyses were performed on a FACSCanto II flow cytometer (BD Bioscience) with Flow Jo Software.
  • RNA isolation and Reverse transcription polymerase chain reaction RT-PCR
  • the primers and probes for blood group A were 5' FAM-CCC CAG CCA AAG GTG CTG AC A CC-TAMRA 3', sense 5' CTC GTT GCC AAG GAT GGT CTA 3' and antisense 5' CCA CGA GGA CAT CCT TCC TAC A 3'.
  • reverse transcription was performed with TaqMan MicroRNA Reverse Transcription Kit using Megaplex primer pool v3.0 and subsequently specific TaqMan MicroRNA Assays for miRNA-26a, - 99a, -100, 331-3p, -1908-5p and -4711-3p (Applied Biosystems).
  • Realtime PCR was performed with TaqMAN Universal PCR Master Mix according to manufacturer's instruction.
  • mRNAs For mRNAs, the data were normalized using the endogenous GAPDH control (5' FAM-TGT TGC CAT CAA TGA CCC CTT CAT TG-TAMRA 3', sense 5' AGG GCT GCT TTT AAC TCT GGT AA 3', antisense 5' CAT GGG TGG AAT CAT ATT GGA AC 3').
  • endogenous GAPDH control 5' FAM-TGT TGC CAT CAA TGA CCC CTT CAT TG-TAMRA 3', sense 5' AGG GCT GCT TTT AAC TCT GGT AA 3', antisense 5' CAT GGG TGG AAT CAT ATT GGA AC 3'.
  • U6 snRNA was used as the endogenous control. Gene expression was analyzed using DCt method and normalized to untransduced control.
  • Example 1 MiRs Reduce Activity of a Glycosyltransferase A 3'UTR Reporter
  • the 3'UTR of glycosyltransferases A genes of the normal- and the weak- expression allele were cloned into a firefly luciferase reporter vector. Either of the firefly- reporter constructs was then co-transduced into K562 cell together with a constitutive active Renilla-reporter for normalization.
  • MiR-vectors as described above were co-transduced and both firefly and renilla activity was measured for each experiment. The results are shown in figure 1.
  • MiR-331, miR-1908 as well as miR-4711 significantly reduce firefly-activity for the 3'UTR of glycosyltransferase A gene.
  • MiR-26 was used as unrelated control.
  • the weak blood group A allelic variant of the reporter construct with more binding sites for miR-1908 was also significantly more sensitive to miR- 1908 treatment than the normal expression variant.
  • miR-331, miR-1908 and miR-4711 target the 3'UTR of the glycosyltransferase A gene.
  • miR of the invention were transduced into hematopoietic stem cells via a lentiviral expression system. After transduction the stem cells were differentiated into mature erythrocytes. Blood group antigen A expression was checked via fluorescent activated cell cytometry as described before. Only cells that were transduced and positive for the erythrocyte marker glycophorin A were gated for the analysis of blood group antigen A expression. Blood group antigen A expression was determined 11 days post-transduction with miR. The results are shown in figure 2. Both, miR-331 as well as miR- 1908 was found to reduce expression of blood group antigen A.
  • FIG. 5a An altered 3'UTR in 6 of the donors was found (Fig. 5a).
  • the 3'UTR of the consensus sequence from blood group Al revealed three binding sites for miR-1908-5p and one for miR- 33 l-3p.
  • the 3'UTR of the 6 A weak variants included five binding sites for miR- 1908-5p.
  • the whole gene of these samples was sequenced and it was identified that all variants with an altered 3'UTR were hybrid variants of allele A and 0 lv (A-0 lv ) (Fig. 5b).
  • These A weak variants express almost no blood group A antigen on the surface of RBCs, as compared to normal blood group A expressing cells.
  • CpG island - 650 to + 50. This region contains a lot of binding sites for several transcription factors, i.e. Spl, AP2, GATA.
  • miRNAs are known to mediate post-transcriptional gene silencing (PTGS) in the cytoplasm, recent evidence indicates that at least some fraction of mammalian miRNA's may also have nuclear roles in regulating gene expression. They provide evidence that the endogenous miRNA pathway may direct transcriptional silencing in the nucleus, in addition to the cytoplasmic PTGS and translational activation pathways.
  • the inventors analyzed the promoter sequence in the 5'UTR of the ABO gene for potential miRNA binding sites. Interestingly, there are one for miR-331-3p and three binding sites for miR-1908-5p, of which two are in the binding site for transcription factor Spl . In addition, using microRNA target prediction tools the inventors further identified Spl as a potential target gene for miR- 33 l-3p with two binding sites for this miRNA. Therefore increased expression of miR-1908- 5p and miR-331-3p leads to downregulation of transcription factor Spl and to a competition of miR-1908-5p with Spl which in turn results in blood group gene repression.

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Abstract

Cette invention concerne des micro-ARN (miR) régulant l'expression des antigènes de groupes sanguins, tels que les antigènes A, B, les antigènes du système Rhésus ou du système Kell. Les miR selon l'invention se sont avérés cibler directement les glycosyltransférases, les enzymes qui sont responsables de la génération des motifs de glycosylation caractéristiques d'un type de sang donné. L'invention concerne des procédés qui exploitent des constructions d'expression de miR pour générer des érythrocytes caractérisés par une expression réduite d'antigènes de groupes sanguins. Des érythrocytes produits par les procédés selon l'invention, et une préparation de sang se prêtant à la médecine transfusionnelle comprenant les érythrocytes selon l'invention sont en outre décrits. L'invention concerne en plus une méthode permettant de diagnostiquer la suppression des antigènes de groupes sanguins chez un sujet, la méthode comprenant la détection du nombre de copies des sites cibles miR dans les loci génétiques codant pour des glycosyltransférases et autres récepteurs de groupes sanguins.
PCT/EP2015/070170 2014-09-03 2015-09-03 Micro-arn pour la suppression des antigènes de groupes sanguins Ceased WO2016034679A1 (fr)

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CN110042165A (zh) * 2019-03-21 2019-07-23 苏州西山生物技术有限公司 猕猴abo血型基因分型方法
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3027739A4 (fr) * 2013-09-06 2016-06-08 Shanghai Sidansai Biotechnology Co Ltd Cellules modifiées pour la production de cellules sanguines
CN110042165A (zh) * 2019-03-21 2019-07-23 苏州西山生物技术有限公司 猕猴abo血型基因分型方法
CN110042165B (zh) * 2019-03-21 2022-08-12 苏州西山生物技术有限公司 猕猴abo血型基因分型方法
US11162079B2 (en) 2019-05-10 2021-11-02 The Regents Of The University Of California Blood type O Rh-hypo-immunogenic pluripotent cells
US12221622B2 (en) 2019-05-10 2025-02-11 The Regents Of The University Of California Modified pluripotent cells
US12492382B2 (en) 2019-05-10 2025-12-09 The Regents Of The University Of California Blood type O Rh—hypo-immunogenic cells

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