KR20160137863A - Methods for producing Rh negative erythrocyte - Google Patents

Abstract

The present invention relates to a method for producing Rh negative red blood cells using Rh-positive erythroid precursor cells and Rh-positive erythroid precursor cells for producing Rh negative red blood cells. Rh-positive red blood cells can be effectively converted into Rh negative red blood cells by using the method of the present invention and erythroid precursor cells.

Description

Methods for producing Rh negative erythrocyte

The present invention relates to a method for producing Rh negative red blood cells using Rh-positive erythroid precursor cells.

Rh antigens, commonly referred to as Rh factors, are proteins that are generally expressed on the cell membrane of erythrocytes (Hartel-Schenk, S. and P. Agre (1992). "Mammalian red cell membrane Rh polypeptides are selectively palmitoylated subunits of a macromolecular complex." J Biol Chem 267 (8): 5569-5574.], Red blood cells can be classified into blood groups based on the presence and absence of Rh antigens. The basic test for determining the presence of Rh antigens in human blood utilizes the Rh designation derived from the blood of rhesus monkeys. In 1940, Karl Landsteiner and A S. Weiner discovered the above blood group system, since then a number of different distinct Rh antigens have been established. However, the D antigen remains the first most common and is expressed as a RhD protein; It is also a fundamental determinant of the Rh trait and is sometimes known to be the most serious cause of the immune response leading to death. D antigen is a collection of morpho-dependent epitopes following the entire RhD protein. While there are RHD deletions in most D-negative cocaine populations, the D-negative phenotype is associated with the most common RHD in other populations (especially Japanese and African blacks), while the D antigen The reason for the lack of expression is unknown (except for Africans). The molecular basis of the major silent RHD allele (called RHDψ ) found in people of African ancestry in 2000 is clear (11). RHDψ has a 37 base pair insertion of DNA that is a duplication of the intron 3 / exon 4 boundary, and a missense mutation in exon 5 and a nonsense mutation and a missense mutation in exon 6 . D-antigen, when transfused with Rh-positive blood, pose a risk to people who are Rh-negative without antigen. No side effects will occur during the first transfusion of Rh-incompatible blood; However, the immune system responds to foreign Rh antibodies by production of anti-Rh antibodies. The antibodies result in an immune response that induces aggregation when the same patient is transfused with Rh-positive blood again. This results in destruction of red blood cells or hemolysis and serious illness resulting therefrom and sometimes death.

Recently, the Yukio Nakamura group of Riken, Japan, has developed a novel approach to successfully establish immortal erythroid progenitor cells called human iPS cell-derived erythroid progenitor (HiDEP) cells from human iPS cells (19 ). HiDEP cells may be differentiated to produce the RBC from in Vitro, they are of the cell line to production, upregulation of red blood cells following the introduction of the in vitro differentiation of functional hemoglobin was confirmed that it is possible to express the specific markers. Although the efficiency of producing enucleated RBCs should be further enhanced, most importantly, all of these cell lines form enucleation following the introduction of in vitro differentiation.

Genomic editing by engineered nuclease agents is a technique in which genes are inserted from a genome using artificially engineered nucleases (Lombardo, A., D. Cesana, P. Genovese, B. Di Stefano, E. Provasi , D. Gritti, V. Broccoli, C. Bonini and L. Naldini (eds), DF Colombo, M. Neri, Z. Magnani, A. Cantore, P. Lo Riso, M. Damo, OM Pello, MC Holmes, PD Gregory 2011). "Site-specific integration and tailoring of cassette design for sustainable gene transfer." Nat Methods 8 (10): 861-869.) And rearranged (Lee, HJ, J. Kweon, E. Kim, S. Kim and JS Kim (2012). "Targeted chromosomal duplications and inversions in the human genome using zinc finger nucleases." Genome Res 22 (3): 539-548., Brunet, E., D. Simsek, M. Tomishima, DeKelver, VM Choi, P. Gregory, F. Urnov, DM Weinstock and M. Jasin (2009), "Chromosomal translocations induced at specified loci in human stem cells." Proc Natl Acad Sci USA 106 (26): 10620-10625. ), Or destroyed (21, 22, 23) It is a type of engineering. Zinc Finger Nucleases (ZFNs) (Kim, YG, J. Cha and S. Chandrasegaran (1996). "Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain." Proc Natl Acad Sci USA 95 (3): 1156-1160., Bitinaite, J., DA Wah, AK Aggarwal and I. Schildkraut (1998). "FokI dimerization is required for DNA cleavage." Proc Natl Acad Sci USA 95 ): 10570-10575.), TANENs (Transcription Activator-like Effector Nucleases) (Miller, JC, S. Tan, G. Qiao, KA Barlow, J. Wang, DF Xia, X. Meng, DE Paschon, E. Leung , SJ Hinkley, GP Dulay, KL Hua, I. Ankoudinova, GJ Cost, FD Urnov, HS Zhang, MC Holmes, L. Zhang, PD Gregory and EJ Rebar (2011). Nat Biotechnol 29 (2): 143-148.) And the CRISPER / Cas system (32). Among them, TALENs consist of TAL effectors including the customizable DNA binding repeats and the catalytic domain of Fokl endonuclease.

There has been no attempt to convert RH blood type using TALEN, and the present invention provides an optimal method for this.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to develop a method for producing Rh-negative red blood cells using Rh-positive erythroid precursor cells. As a result, the present inventors have completed the present invention by confirming that a nucleic acid sequence encoding a RhD antigen of Rh-positive erythroid precursor cells can be partially modified by using nuclease to produce RhD negative red blood cells.

Accordingly, an object of the present invention is to provide a method of producing Rh negative red blood cells using Rh-positive erythroid precursor cells.

Another object of the present invention is to provide Rh-positive erythroid precursor cells for Rh negative red blood cell production.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method of producing Rh negative red blood cells using Rh-positive erythroid precursor cells comprising the steps of:

(a) transfecting Rh-positive erythroid precursor cells so as to express TALEN (transcription activator-like effector nuclease) for knocking out the RHD gene; And

(b) differentiating the transfected erythroid precursor cells to obtain Rh negative red blood cells.

The present inventors have made extensive efforts to develop a method for producing Rh-negative red blood cells using Rh-positive erythroid precursor cells. As a result, it was confirmed that RhD negative rDNA could be produced by partially modifying the nucleic acid sequence encoding the RhD antigen of Rh-positive erythroid precursor cells with nuclease and knocking it out.

The method for producing Rh negative red blood cells using the Rh-positive erythroid precursor cells of the present invention will be described step by step.

(a) Rh-positive red blood cell progenitor cells, RHD  Transfection to express TALEN (transcription activator-like effector nuclease) to knock out the gene

The term "erythroid precursor cells" in this specification refers to all kinds of cells appearing in the process of differentiation into erythrocytes starting from hemocytoblasts. More specifically, erythroid precursor cells are differentiated from hematopoietic stem cells, which are CD34-positive cells, and prolythroblast, erythroblast, basophilic erythroblast, polychromatic erythroblast, (orthochromatic erythroblast), followed by reticulocyte stage, and the erythroid precursor cells can be used. Preferably, cells that only have the ability to differentiate into mature red blood cells and lose resolution to other blood cells can be used. Also, "erythroid precursor cell line" means an immortalized erythroid precursor cell capable of repeating cell division indefinitely. The term "blood stem cell" in the present specification means a stem cell that does not have the ability to differentiate into cells other than the blood system, and has the ability to differentiate into all kinds of hematopoietic cells, and has all the meanings as "hematopoietic stem cells ". The term "blood stem cell" refers to a cell population isolated and recovered by flow cytometry using antibodies specifically binding to hematopoietic stem cell surface antigen (CD34, etc.) from tissues such as cord blood, peripheral blood, bone marrow, fetal liver, It is known to be abundantly included. It is also possible to produce by inducing differentiation from human pluripotent stem cells as used in one embodiment of the present invention. Human pluripotent stem cells have self-replicating ability and can induce differentiation into hematopoietic stem cells. For example, ES cells (embryonic stem cells), EC cells (pluripotent tumor cells), EG cells (embryonic germ cells) , mGS cells (pluripotent germ cells), mesenchymal stem cells, mesenchymal stem cells, MUSE cells (Kuroda Y. et al., Proc. Natl. Acad. Sci. USA, 2010, 107, 19, 8639-8643 (See page), and the like, and the cell also includes a cell that has been artificially produced to have pluripotency such as iPS cells.

In one embodiment of the present invention, the erythroid precursor cells of the present invention are selected from the group consisting of proerythroblast, erythroblast, basophilic erythroblast, polychromatic erythroblast, (orthochromatic erythroblast). As described above, the erythroid precursor cells appear on the differentiation stage from the hematopoietic stem cell to the erythrocyte differentiation, and can be used to differentiate into Rh negative erythrocytes.

In one embodiment of the present invention, the erythroid precursor cells of the present invention can be erythroid precursor cells derived from blood stem cells derived from iPS cells. There are two kinds of erythroid precursor cells derived from iPS cell-derived blood stem cells, named HiDEP-1 and HiDEP-2. The erythroid precursor cells derived from iPS cell-derived blood stem cells do not need to sacrifice the embryo, so there is no ethical problem, and when the finally obtained blood is used for transfusion, the problem of compatibility between the donor and recipient is resolved There are advantageous advantages.

The RHD gene of the present invention is a gene encoding RhD erythrocyte membrane protein, which is also called Rh polypeptide 1 (RhPI). The RHD gene contains 11 exons and 10 introns and contains the nucleic acid sequence shown in Sequence Listing < RTI ID = 0.0 > 1 < / RTI >

The term "frame shift" in this specification refers to the deletion or insertion of nucleotides 3n + 1, 3n + 2 (where n is a positive integer including 0) relative to the nucleic acid sequence, Or translation of a translation frame consisting of three nucleotides that causes protein translation to prematurely terminate.

The term "knock-out" in this specification refers to termination of translation and loss of function of the protein by the frame shift described above. Techniques for knocking out specific genes are known in the prior art.

The present invention utilizes TALEN (Transcription Activator-like Effector Nucleases) nuclease to knockout the RHD gene. TALEN consists of TAL effectors including the customizable DNA binding repeats and the catalytic domain of Fokl endonuclease. In TAL effectors produced by plant pathogenic bacteria of the genus Xanthomonas, the intrinsic function of these proteins is to directly regulate host gene expression. Their targeting specificity is determined by the central domain of tandem, which is 33-35 amino acid repeats followed by a single truncated repeat of 20 amino acids (Mak, AN, P. Bradley, RA Cernadas, AJ Bogdanove and Science, 335 (6069): 716-719., Deng, D., C. Yan, X. Pan, M. Mahfouz, J. "The crystal structure of TAL effector PthXo1 bound to its DNA target. . "Wang, JK Zhu, Y. Shi and N. Yan (2012)." Structural basis for sequence-specific recognition of DNA by TAL effectors. " Science 335 (6069): 720-723.). A repeat-variable di-residue (RVD), a polymorphic pair of contiguous residues at positions 12 and 13 in each repeat, has four most common RVDs, each of which is preferentially associated with one of the four bases, and one for one nucleotide (1999), "Breaking "," Breaking ",< RTI ID = 0.0 & Science 326 (5959): 1509-1512. Moscou, MJ and AJ Bogdanove (2009). "A simple cipher governs DNA recognition by TAL effectors." Science 326 ): 1501.). Proc Natl Acad Sci USA 95 (18): 10570-10575 (1999), because of the Fokl domain functions as dimers (Bitinaite, J., DA Wah, AK Aggarwal and I. Schildkraut ). The pair of TALENs is designed to screen target genes with appropriate spacing in which DNA double-strand break (DSB) is specifically introduced. DSBs are often restored by non-homologous end-joining (NHEJ), which results in insertion or indels (Bibikova, M., M. Golic, KG Golic and D. Carroll (2002). "Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases." Genetics 161 (3): 1169-1175.). As a result, much of the 2/3 restoration by frame shift results in loss of translation termination and functionality. The Rh-positive erythroid precursor cells of the present invention are transfected with an expression vector expressing TALEN (transcription activator-like effector nuclease) engineered to knockout the RHD gene. In the present invention, the expression vector may be a replicon, for example, a plasmid, phage, or a cosmid, in which another DNA fragment may be introduced to cause replication of the inserted fragment. In general, vectors contain appropriate control elements. Suitable vector backbones include those commonly used in the art such as, for example, plasmids, viruses, artificial chromosomes, BAC, YAC, or PAC. As used herein, the term "vector" includes cloning and expression vectors as well as viral vectors and integrating vectors. An "expression vector" is a vector comprising one or more expression control sequences, and an "expression control sequence" is a DNA sequence that controls and / or regulates transcription and / or translation of another DNA sequence. Suitable expression vectors include plasmids and viral vectors derived from, for example, bacteriophage, baculovirus, tobacco mosaic virus, herpes virus, cytomegalovirus, retrovirus, vaccinia virus, adenovirus and adeno-associated virus, It is not. The term "control element" as used herein refers to a nucleotide sequence that affects transcription or translation initiation and rate, and stability and / or mobility of a transcript or polypeptide product. The regulatory region may include a promoter sequence, an enhancer sequence, a response element, a protein recognition site, an inducible element, a promoter control element, a protein, a protein, or the like, which may remain in a coding sequence, such as a secretion signal, a nucleotide sequence (NLS) But are not limited to, a binding sequence, a 5 'and a 3' untranslated region (UTR), a charge starting site, a termination sequence, an adenylic acid polymerisation sequence, an intron, and other regulatory regions.

More specifically, a plasmid can be used to transfect TALEN as described above, and the plasmid can use any plasmid having a promoter capable of expressing the TALEN in eukaryotic cells. Specifically, for example, the plasmid used for expressing TALEN in the present invention uses a CMV promoter capable of strongly expressing in eukaryotic cells and also uses a poly A signal to give intracellular stability to the expressed TALEN mRNA Can be used. In addition, an ampicillin resistance gene may be introduced to purify the plasmid in a pure manner to increase the yield and purity of the plasmid. The plasmid expressing TALEN can be used to transfect Rh-positive erythropoietic progenitor cells according to a conventionally known method.

In one embodiment of the present invention, TALEN of the present invention induces a premature termination codon (PTC) in an exon nucleic acid sequence selected from the group consisting of exons 1 to 4 of the RHD gene. More specifically, DNA double strand breaks are induced to induce early termination codons in the corresponding exons. The human RHD gene has a total of eight variant types, with variant 3 having an initiation codon in exon 4. Therefore, in the case of TALEN targeting exon 1, RHD of variant 3 can not be knocked out. Therefore, in this study, TALEN was designed to cleave the exon 1 and / or exon 4 regions. In addition, when the exon 4 site is targeted, the molecular form of the silent RHD allele found in African populations of the RHD negative blood group can be generated in 2000, leading to more reliable RHD blood type conversion.

In one embodiment of the present invention, the TALEN of the present invention includes NI, NN, NG and HD which bind to adenine, guanine, thymine or cytosine as RVD (repeat variable diresides) for binding to a target nucleic acid sequence, respectively.

(b) differentiating the transfected erythroid precursor cells to obtain Rh negative red blood cells

Induction of in vitro differentiation of erythroid precursor cells can be carried out according to a conventionally known method. Specifically, differentiation into erythrocytes can be induced by culturing an erythroid precursor cell line in a known erythrocyte differentiation medium, but not always limited thereto, and erythrocyte differentiation can be induced through various known methods. (20 ng / ml; Sigma), linoleic acid (4 ng / ml; Sigma), cholesterol (200 ng / ml; Sigma), sodium selenate Sigma), human insulin (10 μg / ml; Sigma), D-mannitol (14.57 mg / ml; Sigma), mifepristone (glucocorticoid receptor (Gibco-BRL) medium containing 1 μM antagonist, 1 μM; Sigma) and EPO (5 IU / ml) were used to induce erythrocyte differentiation by culturing erythroid precursor cells.

According to another aspect of the present invention, there is provided Rh-positive erythroid progenitor cells for producing Rh negative red blood cells, which comprises an expression vector expressing TALEN that induces a premature termination codon in the RHD gene nucleic acid sequence.

Rh-positive erythropoietic progenitor cells for producing Rh-negative red blood cells according to the present invention relate to erythroid precursor cells used for Rh negative erythropoietic production using Rh-positive erythroid precursor cells described above, and the excessive complexity described in the present specification Omit it to avoid.

In one embodiment of the present invention, TALEN of the present invention induces an early termination codon in the exon nucleic acid sequence selected from the group consisting of exons 1 to 4 of the RHD gene. Preferably designed to induce an early termination codon at the exon 1 and / or exon 4 site.

In one embodiment of the present invention, the erythroid precursor cells of the present invention are selected from the group consisting of proerythroblast, erythroblast, basophilic erythroblast, polychromatic erythroblast, (orthochromatic erythroblast).

In one embodiment of the present invention, the erythroid precursor cells of the present invention are erythroid precursor cells derived from blood stem cells derived from iPS cells.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method for producing Rh negative red blood cells using Rh-positive erythroid precursor cells.

(b) The present invention provides Rh-positive erythroid precursor cells for producing Rh negative red blood cells.

(c) Using the present invention, it is possible to effectively produce Rh-negative red blood cells using Rh-positive erythroid precursor cells.

Figure 1 A shows a schematic of the TALEN targeting site in the RHD gene. Blue boxes represent exons. RHD_E1_TALENs and RHD_E4_TALENs represent TALEN pairs targeting the nucleic acid sequences in exon 1 and exon 4, respectively. The red, yellow, green, and purple rectangular boxes in TALENs represent a TALE repeat unit that separates guanine, thymine, cytosine, and adenine, respectively. B and C in FIG. 1 show the T7E1 assay results using 293T cells after being transfected with plasmids encoding TALENs targeting RHD exon 1 (B, RHD_E1_TALENs) or exon 4 (C, RHD_E4_TALENs), respectively. The arrows indicate the expected positions of the DNA bands cleaved by T7E1. The numbers at the bottom of the gel represent the percentage of mutations measured by the band strength.
Figure 2 shows the generation of RHD -mutant erythroid precursor cells. Figure 2 A depicts a theoretical process illustrating the process of RHD-mutated clone generation. Clonal culture of HiDEP-1 erythroid precursor cells was initiated 3 days after transfection with a plasmid encoding TALENs targeting RHD. The genomic DNA from each clone was analyzed 17 days after the start of the clonal culture. Figure 2B shows a T7E1-based clonal assay. Genomic DNA isolated from each clone was used for T7E1 analysis. The arrows indicate the expected positions of the DNA bands cleaved by T7E1. Clones containing mutations at the targeting sites were labeled with a clone number. Untransfected cells and a population of cells transfected with the TALEN plasmid were used as negative control (NC) and positive control (PC), respectively. M represents a marker.
Figures 3a and 3b show the DNA sequences of RHD-mutated clones. RHD gene DNA sequences from parental cells, clones with a double allele mutation in exon 1 (E1_B) or exon 4 (E4_B), and a single allelic mutation in exon 4 (E4_M) Lt; / RTI > The TALE binding sites are represented by red letters, and the spacer regions are represented by green boxes. The deleted bases were represented by dashes and the inserted bases were represented by blue letters. The number of clones is indicated in parentheses (for example, X7 and X5 represent the number of each sequence). Sequence and sequence chromatograms for each allele were shown. The locus of each mutation in the schematic of the RHD gene, the premature termination codon (PTC) caused by the mutation, and the distance between the PTC and the exon-intron junction. The mutated protein sequences generated by nuclease-induced frame-shift mutations were represented by red letters and the translation termination was represented by dashes . nt represents a nucleotide.
Figure 4 shows the RHD mRNA of mutated clones. RT-PCR was performed to detect RHD mRNA in each clone, and the amplicons were (a) electrophoresis and (b) sequencing. Figure 4a shows representative representations of electrophoresis. ACTB was used as a control. The sizes of the marker (M) bands are shown on the left (kbp, kilo base pairs). Figure 4b shows a schematic of the RHD mRNA sequence. The number of clones is shown to the right of each transcript. Blue and yellow circles represent normal and mutant exons, respectively. Sequence and sequence analysis chromatograms for some appplicons (the spacer region is shown in green boxes).
Figure 5 shows flow cytometry analysis of D antigen expression in mutated cells. The parental and RHD -mutated (allelic, E1_B, E4_B; single allelic, E4_M) HiDEP-1 cells were induced for 4 days of differentiation and flow cytometry. D antigen expression was measured in glycophorin A ⁺ cells. Histograms show that D antigen is expressed in parental and single allelic mutant cells, but not in allelic mutant cells. RhD positive and negative blood cells were used as positive and negative control, respectively.
Figure 6 shows the presence of D antigen-mediated aggregation in RHD knockout cell lines . The parental, RHD knockout (E1_B, E4_B) and RHD single allelic mutation (E4_M) HiDEP-1 cells were induced for 4 days of differentiation and harvested using anti-D blood grouping reagents on 96-well plates and glass slides The cells were photographed and photographed with a microscope, and RhD positive and negative blood cells were used as positive and negative controls, respectively.
Figure 7 shows the morphology of parental and knockout HiDEP-1 cells after induction with erythrocytes. After the indicated times in induction of red cell differentiation, each HiDEP-1 cell line was stained with Giemsa and observed with a microscope. Representation of red blood cells obtained from HiDEP-1 cells is shown. The scale bar is 50 μm.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Methods

Example 1: Cell culture

Rh-positive blood group (dominant homozygous (DD)) The origin of HiDEP-1, an erythroid precursor cell line, has been known in the past [19]. Were cultured in the presence of feeder cells in the presence of doxycycline (1 μg / ml; Sigma), erythropoietin (EPO; 3 IU / ml; CJ Phama), and dexamethasone (10 ^-6 M; Sigma) The cells were maintained in a free StemSpan SFEM ^® medium (StemCell Technologies). 293T (human embryonic kidney cell line) cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS, Gibco-BRL) (Gibco-BRL). Rh D-positive and Rh D-negative marginal blood cells were supplied by Severance Hospital Blood Bank (Seoul, Korea).

Example 2: RHD  Targeting TALENs (transcription activator-like effector nucleases)

Plasmids encoding TALENs targeting exon 1 and exon 4 of the RHD gene were designed as previously described [23] (http://www.talenlibrary.net/) and were obtained from ToolGen. The repeat variable sequences (RVD) used to bind adenine, guanine, thymine and cytosine in the target sequence were NI, NN, NG and HD, respectively. A magnetic reporter was prepared as previously described [15]. To measure the TALEN activity using a 3.0 μg of TALEN plasmid (fleece mid 1.5 μg each encoding plasmids and other TALEN encoding a single TALEN) FuGENE ^® HD Transfection Reagent (Roche Diagnostics) and infected into 293T cells . After 72 hours of infection, cells were harvested and assayed for T7E1.

Example 3: RHD - Generation of knockout clones

HiDEP-1 cells were transfected with 4.5 μg of TALEN plasmid and reporter (1.5 μg plasmid encoding one TALEN, 1.5 μg plasmid encoding the other TALEN, 1.5 μg of plasmid encoding the other TALEN) using a human CD34 cell nucleofector kit (Lonza) from the Amaxa system ≪ / RTI > magnetic reporter plasmid). The transformed HiDEP-1 cells were mixed with antibody against magnetic bead-conjugated H-2K ^K (MACSelet K ^K microbead; Miltenyi Biotech) after incubation at 37 ° C for 3 days and cultured at 4 ° C for 20 minutes Respectively. H-2K ^K cells were separated on a column (MACS LS column; Miltenyi Biotech) according to the manufacturer's instructions and inoculated at a rate of 0.25 cells per well in 96-well plates. Cells were cultured for 2 weeks and each resulting clone was transferred to a 24-well plate for further expansion. RHD knockout clones were identified and sequenced using T7E1 analysis.

Example 4: T7E1 assay

T7E1 assays were performed as described previously [17, 18, 20, 37]. Genomic DNA purification kit (Promega) was used to isolate genomic DNA according to the manufacturer's instructions. Due to the high homology between RHD and RHCE , we performed nested PCR using the primers shown below, which are specific for RHD and bind but do not bind to RHCE : RHD exon 1 (first forward primer (FP) 5 'ATG GGA GCA CAG GGG AAG TT 3'; first reverse primer (RP) -5 'AAAAT TAG CCA GGC ATG GT 3'; second FP-5 'TTA AGA GCT CAC TGG GTG CC 3' RP-5 'GGG GAA GCA GAG AAG CAG AG 3') and RHD exon 4 (first forward primer -5 'GGT CAA AAG CAT ATA AGA GCT ACT G 3'; first RP-5 'ACT CCC CGT TAA GCA CTT TAC AT 3 '; second FP-5' GCA GTG GCT CAT GCC TGT AAT 3 '; second RP-5' CCT GCT CTG TGA AGT GCT TAA TTC 3 '). PCR amplicons were denatured by heating and annealed to form heterologous double stranded DNA treated with 5 units of mismatch-sensitive T7 endonuclease 1 (New England Biolabs) for 20 min at 37 < 0 > C And analyzed by 2% agarose gel electrophoresis.

Example 5: Sequencing analysis

The PCR amplicon was purified using Fragment DNA Purification Kit (iNtRON biotechnology) and cloned into pGEM-T vector. The replicated plasmid was sequenced using the T7 primer (5 'TAA TAC GAC TCA CTA TAG GG 3').

Example 6: RHD  RT-PCR of mRNA

Total cellular RNA was extracted by TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The isolated total RNA was then reverse transcribed using Oligo-dT primer (Qiagen) and AccuPower RT PreMix (Bioneer) according to the manufacturer's instructions. For the RT-PCR detection of mutant RHD mRNA expression, primers set 5 'ACA CAG GAT GAG CTC TAA GT 3' (in RHD exon 1) and 5 'GTG GCA GAG AAA GGA TTC AAC TCC 3' (in RHD exon 10) Respectively. The PCR amplicons were analyzed by 1% agarose gel electrophoresis and mRNA sequencing was performed as described above.

Example 7: Introduction of differentiation into RBC (red blood cell)

(20 ng / ml; Sigma), linoleic acid (4 ng / ml; Sigma), cholesterol (200 ng / Sigma), human insulin (10 [mu] g / ml; Sigma), D-mannitol (14.57 mg / ml; Sigma), sodium selenite (2 ng / ml; Sigma) ), Mifepristone (an antagonist of the glucocorticoid receptor, 1 [mu] M; Sigma) and IMDM (GIBCO-BRL) containing EPO (5 IU / ml).

Example 8: Flow cytometry

The harvested cells were incubated with antibodies (anti-D blood group labeling reagents (human IgG / IgM monoclonal; Millipore)) in FBS (Sigma) at room temperature for 20 minutes in 100 μl of staining medium (PBS containing 2% -Conjugated anti-human-CD235a (BD Biocsciences) and PE-conjugated anti-human-IgG [Fc gamma-specific] (eBioscience). The stained cells were analyzed using FACS Cantos II (BD Biosciences) and FlowJo (version X.0.7).

Example 9: Morphological analysis < RTI ID = 0.0 >

For morphology analysis, differentiated cells (1 × 10 ⁵ cells / slide) were centrifuged on a slide using a cytocentrifuge (Cytospin 3, Shandon Scientific) (1000 rpm, 5 min) and analyzed by Wright-Giemsa dye And observed using a microscope (BX51; OLYMPUS).

Example 10: Agglutination assay

Differentiated HiDEP-1 cells and control RBCs were placed in 96-well plates at 1x10 ⁶ cells per well in 10 μl PBS. Then 10 μl of anti-D blood grouping reagent was added in each well and cells were incubated at 37 ° C for 15 minutes. After the cultured cells were cultured in each well using a pipette tip, the mixture was observed using a stereo zoom microscope (SZ61; OLYMPUS) and photographed using a camera (G10; Canon). For extended observation in the mixture, the mixture was transferred from a 96-well plate to a glass slide and observed using an inverted microscope (IX71; OLYMPUS).

Experiment result

1. Design and verification of TALEN

The Rh blood group was determined according to the entire RhD protein by the D antigen, which is a collection of structure-dependent epitopes. The human RHD gene has a total of eight variant types, with variant 3 having an initiation codon in exon 4. In addition, some Africans with the RhD negative blood group phenotype have RHD pseudo-jeans ( RHDΨ ) containing 37 base pair replications and nonsense mutations [11]. Therefore, TALEN was designed to cleave the region between exon 1 and / or exon 4 (see Figure 1). Plasmids encoding TALEN targeting exon 1 or exon 4 of the human RHD gene were infected into 293T cells. 72 hours after transfection, the genomic DNA of the obtained cells was analyzed by T7E1 assay to assess the mutation frequency. The data show that the mutation frequencies in exon 1 and exon 4 from the RHD gene are 12% and 6% (see B and C in Fig. 1). In the above results, it was confirmed that the designed TALEN had activity in human 293T cells.

2. RHD  Screening of knockout cells

To establish RHD knockout cell lines , a whole experiment was planned. A magnetic separation reporter was constructed for enrichment of TALEN-induced mutant cells [15]. Therefore, in order to isolate enriched cells with TALEN-induced mutations, TALENs targeting exon 1 and exon 4 of the RHD gene with their respective magnetic reporters were identified as Rh-positive blood group (dominant homozygous (DD )) HiDEP-1 cell line. After 72 hours of transfection, cells expressing H-2K ^k membrane protein from magnetic reporters derived from TALEN activity were isolated and inoculated into 96-well plates for monoclonal expansion. After a single colony expansion, nine of 152 magnetically isolated clones that showed genetic modification in exon 1 by T7E1 analysis showed a target genomic frequency of 5.9%. In contrast, seven of 179 self-isolated clones that showed genetic modification of exon 4 by T7E1 analysis showed a target genomic frequency of 3.9%. As a result, seven clones with genetic modification in exon 9 and nine in exon 1 were obtained (see Fig. 2).

3. Sequencing analysis in mutated clones to establish biallelic mutant cell lines with early termination codons

PCR products of clones of genomic locus targeting exon 1 and exon 4 were inserted into pGEM-T vector by TA cloning to determine the exact sequence of the mutation. In a genomic locus targeting exon 1, one clone (# E1_B) with a double allelic mutation in 9 clones was obtained (see Figure 3a). In the clone (# E1_B), 12 sequences were analyzed, of which 7 sequence results showed 1 nt (nucleotide) deletion and the remaining 5 sequences showed 2 nt deletion. We confirmed that all mutated alleles in clone # E1_B have early termination codon (PTC) in exon 1. Among the 7 clones obtained by targeting exon 4, one double allelic mutation (# E4_B) (see Figure 3a) and one single-trait mutation (# E4_M) (see Figure 3b) were obtained. In the clone (# E4_B), 19 sequences were analyzed, 2 out of 19 showed 31 nt deletions and 236 nt insertions at the same site, and the remaining 17 showed only one nucleotide deletion. However, in the clone # E4_M, six sequences were analyzed, five of which showed 24 nt deletions and 7 nt insertions, and the remaining one retained the wild-type sequence. Alleles with 31 nt deletions and 236 nt insertions in the clone # E4_B have PTC on the inserted nucleotides in exon 4 and alleles with 1 nt deletion have PTC on exon 5. Alleles with 24 nt deletions and 7 nt insertions in the clone # E4_M also have PTC in exon 4. From these results we can clearly show that all mutated clones express immature or RhD-free proteins as a result of the Nonsense mutation due to error prone repair of NHEJ. HiDEP-1 cell line and one single transcript mutation (# E4_M) of two double allelic mutants (# E1_B and # E4_B) were established (see Figures 3a and 3b).

4. Detection of D antigens in biallelic mutants (# E1_B, # E4_B) and single-trait mutation (# E4_M) cell lines using flow cytometry

RT-PCR was first used to determine the levels of RHD gene expression in #Wild type, #E1_B, #E4_B, and #E4_M HiDEP-1 cell lines. RHD transcripts from all HiDEP-1 cell lines were amplified at the global stage (see Figure 4a). The #Wild type HiDEP-1 cell line expressed two major band and minor band RHD transcripts. Although the #E1_B and #E4_M HiDEP-1 cell lines expressed RHD transcripts of the same size bands, the expression level was lower than the #Wild type. However, we have identified third week bands of different sizes from other cell lines. RT-PCR products of all HiDEP-1 cell lines were then inserted into the pGEM-T vector by TA cloning to confirm that the RHD transcript sequences were identical to the genomic DNA sequences. It was confirmed that #Wild type and #E1_B express the same RHD transcript as the genomic DNA sequences of the respective cell lines (see FIG. 4B). However, mutated sequences in the RHD transcript could not be identified in #E4_B because #E4_B expresses various variant types except exon 4 (see Figure 4b). Also, # E4_M did not express transcripts from alleles with mutated sequences and only transcripts from alleles with wild-type were expressed (see Figure 4b). In the mRNA sequencing results, all HiDEP-1 cell lines expressing various variant types of RHD genes were identified. Next, in order to confirm the knockout of the RHD gene, the level of D antigen from the RhD protein was measured using flow cytometry. Since the undifferentiated HiDEP-1 cells of #Wild type do not express D antigen, D antigen was measured after induction-differentiation in all HiDEP-1 cell lines for 4 days (see FIG. 5). The induced differentiation of #Wild type and #E4_M was detected in 76% and 70% cells expressing D antigen. 7.6% and 9.0% cells were detected in the induced differentiation of # E1_B and # E4_B (all cells expressing D antigen were measured in glycophorin A (GPA) fraction), whereas expression of D antigen Of 76% and 70% cells were detected in the induced differentiation of #Wild type and #E4_M. As a result, it was confirmed that the established HiDEP-1 cell line of the two double allelic mutants (# E1_B, # E4_B) was RHD knockout cell host.

5. #Wild type, single allelic mutation (# E4_M) and RHD  Coagulation test in induced-differentiated cells of knockout cell line (# E1_B, # E4_B)

(Chen, YP, YY Qiao, XH Zhao, HS Chen, Y. Wang and Z. Wang (2007). "Rapid detection of hepatitis B virus surface antigen by an agglutination assay mediated by a bispecific diabody against both human erythrocytes and hepatitis B virus surface antigen." Clin Vaccine Immunol 14 (6): 720-725.]. Therefore, agglutination tests were conducted in induced differentiated cells of #Wild type, single allelic mutation (#E4_M) and RHD knockout cell lines (#E1_B, #E4_B). Induced-differentiated cells from all cell lines mixed with anti-D blood grouping reagent were transferred into 96-well plates. After 15 minutes of incubation at 37 ° C, the cells were shaken in each well using a pipette tip. As with Rh-negative cells, cells from #E1_B and #E4_B did not aggregate, while agglutination between #Wild type and #E4_M induced-differentiated cells, as well as Rh-positive RBCs, were identified (see Figure 6 ). Glass slides are more commonly used than 96-well plates for clinical flocculation testing [Chen, YP, YY Qiao, XH Zhao, HS Chen, Y. Wang and Z. Wang (2007). "Rapid detection of hepatitis B virus surface antigen by an agglutination assay mediated by a bispecific diabody against both human erythrocytes and hepatitis B virus surface antigen." Clin Vaccine Immunol 14 (6): 720-725.], And therefore flocculation tests from 96-well plates were applied to glass slides. The present inventors also confirmed the same results as before. The results confirmed that the Rh blood group of RHD knockout cell strain transformed by the programmable nuclease was converted from Rh-positive to Rh-negative blood group.

6. Morphometric analysis during induced differentiation of #Wild type and RHD knockout cell line (# E1_B)

All HiDEP-1 cell lines produce nucleated RBCs after induction of differentiation in vitro . Therefore, morphological analysis was performed to confirm the differentiation potential of the genomic modified cell line. # Wild type and # E1_B were differentiated in red cell differentiation medium for 12 days (see FIG. 7). Cell size and nuclei of induced-differentiated cells from the cell line were smaller during induction-differentiation. Twelve days after induction-differentiation, cells from both cell lines became almost fully enucleated cells. From the above results, it was confirmed that the cell line containing the TALEN-mediated genome modification in the RHD locus to determine the Rh blood group had the same differentiation potential as the #Wild type HiDEP-1 cell line.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

references

1. Goldstein J, Siviglia G, Hurst R, Lenny L, Reich L. Group B erythrocytes enzymatically converted to group O Science 215, 168-170 (1982).

2. Vosnidou NC , et al. Seroconversion of type B to O erythrocytes using recombinant Glycine max alpha-D-galactosidase. Biochem Mol Biol Int 46, 175-186 (1998).

3. Bakunina IY , et al. Alpha-galactosidase of the marine bacterium Pseudoalteromonas sp. KMM 701. Biochemistry (Mosc) 63, 1209-1215 (1998).

4. Liu QP , et al. Bacterial glycosidases for the production of universal red blood cells. Nat Biotechnol 25,454-464 (2007).

5. Landsteiner K WA. An agglutinable factor in human blood recognized by immune sera for rhesus blood. Proc Soc Exp Biol Med 43, 223 (1940).

6. Daniels G. The molecular genetics of blood group polymorphism. Hum Genet 126, 729-742 (2009).

7. Le van Kim C , et al. Molecular cloning and primary structure of the human blood group RhD polypeptide. Proc Natl Acad Sci USA 89, 10925-10929 (1992).

8. Cherif-Zahar B, Mattei MG, Le Van Kim C, Bailly P, Carton JP, Colin Y. Localization of the human Rh blood group gene structure to chromosome region 1p34.3-1p36.1 by in situ hybridization. Hum Genet 86, 398-400 (1991).

9. Daniels G. Human blood groups: Geoff Daniels; foreword to first edition by Ruth Sanger , 3rd edn. John Wiley & Sons (2013).

10. Colin Y, Cherif-Zahar B, Le Van Kim C, Raynal V, Van Huffel V, Cartron JP. Genetic basis of the RhD-positive and RhD-negative blood group polymorphism as determined by Southern analysis. Blood 78, 2747-2752 (1991).

11. Singleton BK , et al. The presence of an RHD pseudogene containing a 37 base pair duplication and a nonsense mutation in africans with the Rh D-negative blood group phenotype. Blood 95, 12-18 (2000).

12. Daniels G. Variants of RhD - current testing and clinical consequences. Br J Haematol 161, 461-470 (2013).

13. Kim H, Kim JS. A guide to genome engineering with programmable nucleases. Nat Rev Genet 15,321-334 (2014).

14. Kim H, Um E, Cho SR, Jung C, Kim JS. Surrogate reporters for enrichment of cells with nuclease-induced mutations. Nat Methods 8, 941-943 (2011).

15. Kim H, Kim MS, Wee G, Lee CI, Kim H, Kim JS. Magnetic separation and antibiotics selection Enrichment of cells with ZFN / TALEN-induced mutations. PLoS One 8, e56476 (2013).

16. Ramakrishna S, Kim YH, Kim H. Stability of zinc finger nuclease protein is enhanced by the proteasome inhibitor MG132. PLoS One 8, e54282 (2013).

17. Ramakrishna S , et al. Surrogate reporter-based enrichment of cells containing RNAguided Cas9 nuclease-induced mutations. Nat Commun 5, 3378 (2014).

18. Ramakrishna S, Kwaku Dad AB, Beloor J, Gopalappa R, Lee SK, Kim H. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res , (2014).

19. Kurita R , et al. Establishment of immortalized human erythroid progenitor cell lines can produce enucleated red blood cells. PLoS One 8, e59890 (2013).

20. Kim YH, Ramakrishna S, Kim H, Kim JS. Enrichment of cells with TALEN-induced mutations using surrogate reporters. Methods , (2014).

21. Steentoft C , et al. Mining the O-glycoproteome using zinc-finger nucleaseglycoengineered SimpleCell lines. Nat Methods 8, 977-982 (2011).

22. Kernstock S , et al. Lysine methylation of VCP by a novel human protein methyltransferase family. Nat Commun 3, 1038 (2012).

23. Kim Y , et al. A library of TAL effector nucleases spanning the human genome. Nat Biotechnol 31, 251-258 (2013).

24. Gutschner T, Baas M, Diederichs S. Noncoding RNA gene silencing through genomic integration of RNA destabilizing elements using zinc finger nucleases. Genome Res 21, 1944-1954 (2011).

25. Hu R, Wallace J, Dahlem TJ, Grunwald DJ, O'Connell RM. Targeting human microRNA genes using engineered Tal-effector nucleases (TALENs). PLoS One 8, e63074 (2013).

26. Kim YK , et al. TALEN-based knockout library for human microRNAs. Nat Struct MoI Biol 20,1458-1464 (2013).

27. Perez EE , et al. Establishment of HIV-1 resistance in CD4 + T cells by zinc-finger nucleases. Nat Biotechnol 26,808-816 (2008).

28. Holt N , et al. Human hematopoietic stem / progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo. Nat Biotechnol 28, 839-847 (2010).

29. Maierde , et al. Efficient clinical scale gene modification via zinc finger nuclease-mutated disruption of the HIV co-receptor CCR5. Hum Gene Ther 24, 245-258 (2013).

30. Lee HC, Oh N, Cho H, Choe J, Kim YK. Nonsense-mediated translational repression involved exon junction complex downstream of the premature translation termination codon. FEBS Lett 584, 795-800 (2010).

31. You KT , et al. Selective translational repression of truncated proteins from frameshift mutation-derived mRNAs in tumors. PLoS Biol 5, e109 (2007).

32. Hwang J, Kim YK. When a ribosome encounters a premature termination codon. BMB reports 46, 9-16 (2013).

33. Behm-Ansmant I, Kashima I, Rehwinkel J, Sauliere J, Wittkopp N, Izaurralde E. mRNA quality control: an ancient machinery recognizes and degrades mRNAs with nonsense codons. FEBS Lett 581, 2845-2853 (2007).

34. Chang YF, Imam JS, Wilkinson MF. The nonsense-mediated decay RNA surveillance pathway. Annu Rev Biochem 76, 51-74 (2007).

35. Neu-Yili G, Kulozik AE. NMD: multitasking between mRNA surveillance and modulation of gene expression. Adv Genet 62, 185-243 (2008).

36. Maquat LE. Nonsense-mediated mRNA decay in mammals. J Cell Sci 118, 1773-1776 (2005).

37. Kim HJ, Lee HJ, Kim H, Cho SW, Kim JS. Targeted genome editing in human cells with zinc finger nucleases. Genome Res 19, 1279-1288 (2009).

<110> IUCF-HYU (Industry-University Cooperation Foundation Hanyang University) <120> Methods for producing Rh negative erythrocyte <130> PN140328 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1381 <212> DNA <213> Human RHD gene <400> 1 atgagctcta agtacccgcg gtctgtccgg cgctgcctgc ccctctgggc cctaacactg 60 gaagcagctc tcattctcct cttctatttt tttacccact atgacgcttc cttagaggat 120 caaaaggggc tcgtggcatc ctatcaagtt ggccaagatc tgaccgtgat ggcggccatt 180 ggcttgggct tcctcacctc gagtttccgg agacacagct ggagcagtgt ggccttcaac 240 ctcttcatgc tggcgcttgg tgtgcagtgg gcaatcctgc tggacggctt cctgagccag 300 ttcccttctg ggaaggtggt catcacaacc atgagtgctt tgtcggtgct gatctcagtg 360 gatgctgtct tggggaaggt caacttggcg cagttggtgg tgatggtgct ggtggaggtg 420 acagctttag gcaacctgag gatggtcatc agtaataatg aacatgatgc acatctacgt 480 gttcgcagcc tattttgggc tgtctgtggc ctggtgcctg ccaaagcctc tacccgaggg 540 aacggaggat aaagatcaga cagcaacgat acccagtttg tctgccatgc tgggcgccct 600 cttcttgtgg atgttctggc caagtttcaa ctctgctctg ctgagaagtc caatcgaaag 660 gaagaatgcc gtgttcaaca cctactatgc tgtagcagtc agcgtggtga cagccatctc 720 agggtcatcc ttggctcacc cccaagggaa gatcagcaag acttatgtgc acagtgcggt 780 gttggcagga ggcgtggctg tgggtacctc gtgtgcttgc catggtgctg ggtcttgtgg 840 ctgggctgat ctccgtcggg ggagccaagt acctgccggg gtgttgtaac cgagtgctgg 900 ggattcccca cagctccatc atgggctaca acttcagctt gctgggtctg cttggagaga 960 tcatctacat tgtgctgctg gtgcttgata ccgtcggagc cggcaatggc atgattggct 1020 tccaggtcct cctcagcatt ggggaactca gcttggccat cgtgatagct ctcatgtctg 1080 gtctcctgac aggtttgctc ctaaatctta aaatatggaa agcacctcat gaggctaaat 1140 attttgatga ccaagttttc tggaagtttc ctcatttggc tgttggattt taagcaaaag 1200 catccaagaa aaacaaggcc tgttcaaaaa caagacaact tcctctcact gttgcctgca 1260 tttgtacgtg agaaacgctc atgacagcaa agtctccaat gttcgcgcag gcactggagt 1320 cagagaaaat tctttgagga gaatctcacc atttattatg cactgtagaa tacaacaata 1380 a 1381

Claims (11)

Rh-negative red blood cell producing method using Rh-positive erythroid precursor cells comprising the following steps:
(a) transfecting Rh-positive erythroid precursor cells so as to express TALEN (transcription activator-like effector nuclease) for knocking out the RHD gene; And
(b) differentiating the transfected erythroid precursor cells to obtain Rh negative red blood cells.
The method of claim 1, wherein the erythroid precursor cells are selected from the group consisting of proerythroblast, erythroblast, basophilic erythroblast, polychromatic erythroblast, and orthochromatic erythroblast. &Lt; / RTI &gt;
3. The method according to claim 2, wherein the erythroid precursor cells are erythroid precursor cells derived from blood stem cells derived from iPS cells.
The method according to claim 1, wherein the TALEN induces a premature termination codon (PTC) in at least one exon nucleic acid sequence selected from the group consisting of exons 1 to 4 of the RHD gene.
5. The method according to claim 4, wherein the TALEN induces an early termination codon in the nucleic acid sequence of Exon 1, Exon 4, or Exon 1 and Exon 4 of the RHD gene.
2. The method of claim 1, wherein the TALEN induces a biallelic mutation of the RHD gene.
2. The method of claim 1, wherein the TALEN comprises NI, NN, NG, and HD that bind to adenine, guanine, thymine, or cytosine as RVD (repeat variable diresides) for binding to a target nucleic acid sequence, respectively.
Rh-positive red cell progenitor cells for Rh negative red blood cell production, comprising an expression vector expressing TALEN that induces a premature termination codon in the RHD gene nucleic acid sequence.
The Rh-positive red cell precursor cell according to claim 8, wherein the TALEN induces a premature termination codon in any one or more exon nucleic acid sequences selected from the group consisting of exons 1 to 4 of the RHD gene.
The method of claim 8, wherein the erythroid precursor cells are selected from the group consisting of proerythroblast, erythroblast, basophilic erythroblast, polychromatic erythroblast, and orthochromatic erythroblast. Wherein the cell is selected from the group consisting of Rh-positive red cell progenitor cells.
[10] The Rh-positive red cell precursor cell according to claim 10, wherein the erythroid precursor cell is an erythrocyte precursor cell derived from blood stem cells derived from iPS cells.

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