WO2022211604A1 - Cellules souches modifiées avec un gène mutant de fe-fviii, cellules endothéliales différenciées à partir de celles-ci, et composition pharmaceutique les contenant pour la prévention ou le traitement de l'hémophilie - Google Patents

Cellules souches modifiées avec un gène mutant de fe-fviii, cellules endothéliales différenciées à partir de celles-ci, et composition pharmaceutique les contenant pour la prévention ou le traitement de l'hémophilie Download PDF

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WO2022211604A1
WO2022211604A1 PCT/KR2022/004816 KR2022004816W WO2022211604A1 WO 2022211604 A1 WO2022211604 A1 WO 2022211604A1 KR 2022004816 W KR2022004816 W KR 2022004816W WO 2022211604 A1 WO2022211604 A1 WO 2022211604A1
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fviii
cells
composition
pluripotent stem
coagulation factor
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김동욱
박철용
최상휘
김도훈
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연세대학교 산학협력단
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Definitions

  • the present invention relates to stem cells corrected with FE-FVIII mutant gene, endothelial cells differentiated therefrom, and a pharmaceutical composition for preventing or treating hemophilia comprising the same.
  • Hemophilia A is caused by various mutations in the blood coagulation factor FVIII (F8) (hereafter referred to as blood coagulation factor VIII, coagulation factor VIII, factor VIII, or factor 8) gene on the X chromosome. It is one of the most common genetic disorders.
  • FVIII is a cofactor of FIX. Active FVIII binds to active FIX and together form a tenase complex to activate FX.
  • FVIII is an essential protein in the coagulation pathway because the tenase complex initiates the positive feedback loop of coagulation. To date, there is no radical treatment for HA.
  • AAV vectors can only be applied to adults, and depending on the vector dose and the strength of the enhancer-promoter, carcinoma may develop.
  • iPSCs Patient-derived pluripotent stem cells
  • Pluripotent stem cells have pluripotency and self-renewal ability, so they are a good source of cell therapy to replace cells or tissues that do not function normally.
  • FVIII protein can be expressed normally when gene-edited iPSCs differentiate into endothelial cells (ECs) after correcting the reverse FVIII gene of patient-derived iPSCs using a programmable nuclease.
  • ECs endothelial cells
  • more than half of HA patients have other genetic variations, including large deletions, insertions, duplications, or point mutations. Therefore, there is a need for a universal strategy applicable to all types of genetic mutations occurring in HA patients.
  • DSB site-specific DNA double stranded-break
  • HDR homology-direct repair
  • NHEJ non-homologous end joining
  • the present inventors made intensive research efforts to develop a cell therapy agent capable of treating hemophilia by using gene editing in iPSCs.
  • the present inventors used CRISPR/Cas9 nickase-mediated knock-in (Knock-In, KI) to place the coagulation factor FVIII (F8) at the adeno-associated virus site 1 (AAVS1) locus of iPSCs derived from HA patients. ) (blood coagulation factor VIII, coagulation factor VIII, factor VIII, or factor 8) gene and a functionally enhanced FVIII gene were inserted to establish a universal gene editing strategy.
  • CRISPR/Cas9 nickase-mediated knock-in Knock-In, KI
  • AAVS1 adeno-associated virus site 1
  • the activity level of the F309S mutant FVIII gene (1.4-fold), the FVIII half-life of the E1984V mutant FVIII gene (1.35-fold), the activity level and half-life of FVIII in the F309S/E1984V- mutant FVIII gene (FE-FVIII) were confirmed. (1.56 fold and 1.55 fold, respectively).
  • FVIII and FE-FVIII expression cassettes were inserted into patient-derived iPSCs (WT-KI and FE-KI iPSCs), FVIII mRNA was expressed in iPSCs and differentiated endothelial cells (ECs), and FVIII protein was also secreted.
  • the FE-FVIII protein secreted from ECs derived from FE-KI iPSCs showed more improved activity level and stability of FVIII protein than FVIII protein analyzed by FVIII activity and decay assays of FVIII.
  • the present inventors confirmed that higher FVIII activity was shown in HA mice transplanted with ECs derived from FE-KI iPSCs than those derived from WT-KI iPSCs.
  • both WT-KI and FE-KI EC transplanted mice survived within 48 hours by tail clip challenge (17.6% and 22.2%, respectively), demonstrating the potential of cell-based hemophilia therapy. Therefore, the present invention was completed by identifying that FE-KI iPSCs can be usefully used as a cell therapy for HA treatment by differentiating them into ECs.
  • Another object of the present invention is to provide an induced pluripotent stem cell expressing a FVIII mutant gene comprising mutations at the F309 position and the E1984 position, an endothelial cell differentiated therefrom, and a method for producing the same.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating hemophilia A comprising the composition, induced pluripotent stem cells, or endothelial cells as an active ingredient.
  • the present invention comprises a polynucleotide encoding a mutant FVIII protein comprising mutations at positions F309 and E1984 of blood coagulation factor VIII (blood coagulation factor VIII).
  • a composition for factor FVIII knock-in is provided.
  • the blood coagulation factor FVIII may include full-length blood coagulation factor VIII (flFVIII), or a fragment thereof.
  • the fragment of the full-length blood coagulation factor FVIII may be B domain-deleted blood coagulation factor VIII (BDD-VIII) lacking the B domain, but is not limited thereto. .
  • the F309 position mutation may be a F309S mutation.
  • the E1984 position mutation may be an E1984V mutation.
  • the FVIII mutant protein may be F309S and E1984V mutant proteins.
  • the FVIII protein without the F309 position mutation and the E1984 position mutation may include the amino acid sequence of SEQ ID NO: 46.
  • the FVIII mutant protein having the F309 position mutation and the E1984 position mutation may include the amino acid sequence of SEQ ID NO: 48.
  • the composition further comprises a guide RNA targeting the AAVS1 locus or a polynucleotide encoding the same.
  • the guide RNA targets intron 1 of the PPP1R12C gene, but is not limited thereto.
  • the guide RNA has a length of about 15 nt to 30 nt, 15 nt to 25 nt, 15 nt to 24 nt, 15 nt to 23 nt, 15 nt to 22 nt, 15 nt to 21 nt, 15 nt to 20 nt, 16 nt to 30 nt, 16 nt to 25 nt, 16 nt to 24 nt, 16 nt to 23 nt, 16 nt to 22 nt, 16 nt to 21 nt, 16 nt to 20 nt, 17 nt to 30 nt, 17 nt to 25 nt, 17 nt to 24 nt, 17 nt to 23 nt, 17 nt to 22 nt, 17 nt to 21 nt, 17 nt to 20 nt, 18 nt to 30 nt, 18 nt to 25 nt, 18 nt to 24 nt to 24 nt
  • the guide RNA is to include a protospacer adjacent motif (Protospacer adjacent motif, PAM) sequence.
  • PAM protospacer adjacent motif
  • the polynucleotide encoding the guide RNA may be RNA, DNA, or PNA, and may be chemically modified.
  • the guide RNA may be a single chain guide RNA (sgRNA).
  • sgRNA single chain guide RNA
  • the chemical modification may include non-natural nucleotide linkages.
  • the chemical modification may include a phosphate in which a nonnatural inter-nucleotide bridging phosphate residue is modified.
  • the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates or phosphoroamidates. ) may be selected from
  • the composition further comprises an RNA-guided nuclease, or a polynucleotide encoding the same.
  • the RNA-guided nuclease may be a Cas polypeptide.
  • the Cas polypeptide is one of the protein components of the CRISPR/Cas system, and may be an activated endonuclease or a nick forming enzyme.
  • the Cas polypeptide may form a complex with crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) to exhibit its activity.
  • the Cas polypeptide may be, for example, from the genus Streptococcus (eg, Streptococcus pyogens), from the genus Neisseria (eg, Neisseria meningitidis), from the genus Pasteurella (eg, Pasteurella multocida), from the genus Francisella (eg, Francisella novicida), or It may be a polypeptide derived from a bacterium of the genus Campylobacter (eg, Campylobacter jejuni).
  • the Cas polypeptide may be a Cas9 polypeptide or a Cas12a (Cpf1) polypeptide.
  • the PAM sequence may be 5'-NGG-3', but is not limited thereto.
  • the N means any base.
  • the polynucleotide encoding the FVIII mutant protein and/or the polynucleotide encoding an RNA-guided nuclease may be included in the vector.
  • the vector may be a plasmid.
  • the composition is for correction in vitro, ex vivo, or in vivo.
  • the present invention provides an induced pluripotent stem cell derived from isolated somatic cells of a patient with Hemophilia A (HA) transformed to express the blood coagulation factor FVIII mutant protein. cell, iPSC).
  • HA Hemophilia A
  • the blood coagulation factor FVIII mutant protein contains mutations at the F309 position and the E1984 position of the blood coagulation factor FVIII (BDD-VIII).
  • the blood coagulation factor FVIII may include full-length blood coagulation factor VIII (flFVIII), or a fragment thereof.
  • the fragment of the full-length blood coagulation factor FVIII may be B domain-deleted blood coagulation factor VIII (BDD-VIII) lacking the B domain, but is not limited thereto. .
  • the F309 position mutation may be a F309S mutation.
  • the E1984 position mutation may be an E1984V mutation.
  • the FVIII mutant protein may be F309S and E1984V mutant proteins.
  • the isolated somatic cells of the hemophilia A patient refer to cells selected from the group consisting of muscle cells, nerve cells, epithelial cells, blood cells, mast cells, bone cells, and stem cells.
  • the somatic cells may be, for example, adipocytes, fibroblasts, epithelial cells, blood cells, or hematopoietic stem cells.
  • the fibroblasts may be skin-derived fibroblasts.
  • the epithelial cell may be a urinary tract-derived urinary epithelial cell.
  • the stem cell may be an adipose tissue-derived mesenchymal stem cell (MSC), but is not limited thereto.
  • induced pluripotent stem cell refers to a cell induced to have pluripotency through artificial dedifferentiation from differentiated cells.
  • the induced pluripotent stem cells are similar to embryonic stem cells in cell appearance, gene, or protein expression pattern, have pluripotency in vitro and in vivo, form teratoma, and germline transmission of genes is possible
  • the induced pluripotent stem cells may include iPS cells derived from all mammals such as human, cow, horse, pig, dog, sheep, goat or cat.
  • the induced pluripotent stem cells may be human-derived induced pluripotent stem cells.
  • the induced pluripotent stem cells may be induced pluripotent stem cells derived from a patient with hemophilia A.
  • the transformation can be performed using any known gene delivery system used for transformation of cells.
  • the gene delivery system of the present invention can be constructed in various forms, including (i) a naked recombinant DNA molecule, (ii) a plasmid, (iii) a viral vector, and (iv) the naked recombinant DNA molecule or It can be prepared in the form of liposomes containing plasmids, polymer nanoparticles, lipid nanoparticles, and the like.
  • the contacting step is carried out according to a viral infection method known in the art. Infection of host cells with viral vectors is described in the references cited above.
  • the gene delivery agent when the gene delivery agent is a naked recombinant DNA molecule or plasmid, the microinjection method (Capecchi, M.R., Cell, 22:479 (1980); and Harland and Weintraub, J. Cell Biol. 101:1094- 1099 (1985)), calcium phosphate precipitation (Graham, F.L. et al., Virology, 52:456 (1973); and Chen and Okayama, Mol. Cell. Biol. 7:2745-2752 (1987)), electroporation method (Neumann, E. et al., EMBO J., 1:841 (1982); and Tur-Kaspa et al., Mol.
  • the microinjection method Capecchi, M.R., Cell, 22:479 (1980); and Harland and Weintraub, J. Cell Biol. 101:1094- 1099 (1985)
  • calcium phosphate precipitation Graham, F.L. et al., Virology
  • the present invention provides an endothelial cell, which is differentiated from the induced pluripotent stem cell, and expresses the blood coagulation factor FVIII mutant protein.
  • the present invention provides a composition for the above-described blood coagulation factor FVIII knock-in, an induced pluripotent stem cell transformed to express a blood coagulation factor FVIII mutant protein, or the above It provides a pharmaceutical composition for preventing or treating hemophilia A, comprising endothelial cells expressing a blood coagulation factor FVIII mutant protein as an active ingredient.
  • the pharmaceutical composition may include a pharmaceutically acceptable carrier.
  • the carrier is used in the sense of including excipients, diluents or adjuvants.
  • the carrier may be, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pi It may be selected from the group consisting of rolidone, water, physiological saline, buffers such as PBS, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil.
  • the composition may include a filler, an anti-agglomeration agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, a preservative, or a combination thereof.
  • the pharmaceutical composition may be prepared in any formulation according to a conventional method.
  • the composition may be formulated, for example, as an oral dosage form (eg, a powder, tablet, capsule, syrup, pill, or granule), or a parenteral dosage form (eg, an injection).
  • an oral dosage form eg, a powder, tablet, capsule, syrup, pill, or granule
  • a parenteral dosage form eg, an injection.
  • composition may be prepared as a systemic formulation or a topical formulation.
  • the pharmaceutical composition may be administered orally, intravenously, intramuscularly, orally, transdermally, mucosally, intranasal, intratracheal, subcutaneously, or a combination thereof.
  • the composition may be administered by a subcutaneous route, and more specifically, it may be administered to subcutaneous fat.
  • the present invention provides a method for producing induced pluripotent stem cells expressing FVIII mutant protein in induced pluripotent stem cells derived from isolated somatic cells of a patient with hemophilia A (HA). .
  • the blood coagulation factor FVIII mutant protein in the method for producing pluripotent stem cells expressing the FVIII mutant protein, is prepared by treating the above-described composition for a blood clotting factor FVIII knock-in. transforming to express it.
  • the present invention provides a method for producing an endothelial cell expressing a blood coagulation factor FVIII mutant protein comprising the steps of:
  • iPSC induced pluripotent stem cell
  • the blood coagulation factor FVIII may include full-length blood coagulation factor VIII (flFVIII), or a fragment thereof.
  • the fragment of the full-length blood coagulation factor FVIII may be B domain-deleted blood coagulation factor VIII (BDD-VIII) lacking the B domain, but is not limited thereto. .
  • step (a) will include (a1) culturing the transformed induced pluripotent stem cells in a medium containing Y-27632 for 1 to 2 days.
  • the Y-27632 is about 1-20 ⁇ M, 1-18 ⁇ M, 1-15 ⁇ M, 1-14 ⁇ M, 1-13 ⁇ M, 1-12 ⁇ M, 1- 11 ⁇ M, 1-10 ⁇ M, 5-20 ⁇ M, 5-18 ⁇ M, 5-15 ⁇ M, 5-14 ⁇ M, 5-13 ⁇ M, 5-12 ⁇ M, 5-11 ⁇ M, 5-10 ⁇ M, 8- 20 ⁇ M, 8-18 ⁇ M, 8-15 ⁇ M, 8-14 ⁇ M, 8-13 ⁇ M, 8-12 ⁇ M, 8-11 ⁇ M, 8-10 ⁇ M, 5 ⁇ M, 6 ⁇ M, 7 ⁇ M, 8 ⁇ M, 9 ⁇ M, 10 ⁇ M, 11 ⁇ M, 12 ⁇ M, 13 ⁇ M
  • step (a) will further include (a2) culturing the transformed induced pluripotent stem cells in a medium containing CHIR99021 for 1 to 3 days.
  • the CHIR99021 is about 1 to 15 ⁇ M, 1 to 12 ⁇ M, 1 to 10 ⁇ M, 1 to 9 ⁇ M, 1 to 8 ⁇ M, 1 to 7 ⁇ M, 1 to 6 ⁇ M in the medium.
  • step (a3) the transformed induced pluripotent stem cells are cultured in a medium containing BMP4, bFGF, VEGF-A, or a combination thereof for 1 to 3 days. It will additionally include the step of
  • the BMP4 is 10-50 ng/mL, 10-40 ng/mL, 10-35 ng/mL, 10-30 ng/mL, 10-25 ng/mL, 15 in the medium. to 50 ng/mL, 15 to 40 ng/mL, 15 to 35 ng/mL, 15 to 30 ng/mL, 15 to 25 ng/mL, 20 to 50 ng/mL, 20 to 40 ng/mL, 20 to 35 ng/mL, 20-30 ng/mL, 20-25 ng/mL, 15 ng/mL, 16 ng/mL, 17 ng/mL, 18 ng/mL, 19 ng/mL, 20 ng/mL, 21 ng/mL, 22 ng/mL, 23 ng/mL, 24 ng/mL, or 25 ng/mL.
  • the bFGF is 1 to 25 ng/mL, 1 to 20 ng/mL, 1 to 15 ng/mL, 1 to 10 ng/mL, 3 to 25 ng/mL, 3 in the medium.
  • the VEGF-A is 20 to 100 ng/mL, 20 to 80 ng/mL, 20 to 70 ng/mL, 20 to 60 ng/mL, 20 to 50 ng/mL in the medium.
  • mL 30-100 ng/mL, 30-80 ng/mL, 30-70 ng/mL, 30-60 ng/mL, 30-50 ng/mL, 35-100 ng/mL, 35-80 ng/mL , 35-70 ng/mL, 35-60 ng/mL, 35-50 ng/mL, 40-100 ng/mL, 40-80 ng/mL, 40-70 ng/mL, 40-60 ng/mL, 40-50 ng/mL, 45-100 ng/mL, 45-80 ng/mL, 45-70 ng/mL, 45-60 ng/mL, 45-50 ng/mL, 45 ng/mL, 46 ng/mL mL, 47 ng/mL, 48 ng/mL, 49 ng/mL, 50 ng/mL, 51 ng/mL, 52 ng/mL, 53 ng/mL, 54 ng/mL, or 55 ng/mL.
  • step (a) may include steps (a1) to (a3) sequentially.
  • the culture in step (a) may be made in a culture vessel coated with a gel containing an extracellular matrix.
  • the gel containing the extracellular matrix is Matrigel TM , but is not limited thereto.
  • the steps (a1) to (a3) are to induce differentiation of the mesodermal lineage.
  • step (b) comprises isolating cells expressing a cell surface protein of CD31, CD34, VE-Cad, or a combination thereof from the population of endothelial progenitors.
  • step (b) comprises isolating cells expressing a cell surface protein of CD31, VE-Cad, or a combination thereof from the population of endothelial progenitors. More specifically, the method comprises isolating cells expressing the cell surface protein of CD31 from among the population of endothelial progenitors.
  • the step (c) will further include culturing the transformed induced pluripotent stem cells in a medium containing VEGF-A for 2 to 8 days.
  • the VEGF-A is 20 to 100 ng/mL, 20 to 80 ng/mL, 20 to 70 ng/mL, 20 to 60 ng/mL, 20 to 50 ng/mL in the medium.
  • mL 30-100 ng/mL, 30-80 ng/mL, 30-70 ng/mL, 30-60 ng/mL, 30-50 ng/mL, 35-100 ng/mL, 35-80 ng/mL , 35-70 ng/mL, 35-60 ng/mL, 35-50 ng/mL, 40-100 ng/mL, 40-80 ng/mL, 40-70 ng/mL, 40-60 ng/mL, 40-50 ng/mL, 45-100 ng/mL, 45-80 ng/mL, 45-70 ng/mL, 45-60 ng/mL, 45-50 ng/mL, 45 ng/mL, 46 ng/mL mL, 47 ng/mL, 48 ng/mL, 49 ng/mL, 50 ng/mL, 51 ng/mL, 52 ng/mL, 53 ng/mL, 54 ng/mL, or 55 ng/mL.
  • the present invention provides a composition for blood clotting factor FVIII knock-in.
  • the present invention provides an induced pluripotent stem cell expressing a FVIII mutant protein containing mutations at the F309 position and the E1984 position, an endothelial cell differentiated therefrom, and a method for producing the same.
  • the present invention provides a pharmaceutical composition for preventing or treating hemophilia A comprising the composition, induced pluripotent stem cells, or endothelial cells as an active ingredient.
  • composition of the present invention induced pluripotent stem cells, and endothelial cells differentiated therefrom have excellent efficacy in restoring the function of FVIII, and thus can be usefully used as a therapeutic agent for hemophilia.
  • FVIII activity of FVIII variants Activity was determined after supernatant obtained from transfected HEK293T cells.
  • B Disintegration analysis of FVIII variants. Each activity was measured after incubating the FVIII protein harvested from the transfected HEK293T cells at 37° C. for 0, 8, 16, and 24 hours.
  • C Decay rate and relative half-life of FVIII variants. Data are the mean ⁇ SEM of three independent experiments. The SD for the ratio decay value is estimated based on least squares curve fitting and is within about 10% of the mean value. *, p ⁇ 0.05 compared to BDD-FVIII transfected cells.
  • WT WT BDD-FVIII; F, F309S mutant BDD-FVIII; E, E1984V mutant BDD-FVIII; FE, F309S/E1984V mutant BDD-FVIII
  • FIG. 2 is a diagram showing the results of the primary PCR screening of the corrected iPSC cell line.
  • A The sgRNA target sequence at the AAVS1 locus located in intron 1 of the PPP1R12C gene located on chromosome 19.
  • B PCR-based genotyping to confirm targeted insertion of donor DNA in knock-in iPSC cell lines. Each primer set represents a 5' knock-in junction (F1/R1), a 3' knock-in junction (F2/R2) and the AAVS1 locus (F1/R2). ⁇ -actin was used as an internal reference.
  • C The partial sequence of the knock-in junction in the integrated iPSC cell line containing the template donor DNA. Both cancer sequences are shown in green. The donor plasmid sequences starting from pEF1 ⁇ and ending with the puromycin resistance gene are shown in blue and cyan, respectively. The original genome sequence is shown in black.
  • FIG. 3 is a schematic diagram illustrating the target insertion of the FVIII gene into the human AAVS1 locus of iPSCs derived from HA patients.
  • A Schematic of PCR target sites for primary PCR screening after knock-in at the AAVS1 locus of HA-derived iPSCs. Primers used for PCR-based genotyping are indicated by arrows.
  • B PCR-based genotyping to identify removal of the puromycin resistance cassette in corrected iPSC cell lines. F1/R1 and F2/R2 primer sets were used to detect 5' and 3' junctions of corrected iPSCs, respectively. The F3/R2 primer set was used to detect excision of the puromycin resistance cassette.
  • C Shows the sequence around the loxP site of a corrected iPSC cell line after generation of DNA amplicons using the F3/R2 primer set and excision of the resistance cassette.
  • FIG. 4 is a diagram showing the expression of pluripotency markers in corrected iPSC cell lines.
  • A Quantitative real-time PCR analysis of pluripotency markers (OCT4, SOX2, NANOG and LIN28) in parental patients and corrected iPSC lines. GAPDH was used to normalize gene expression. Data are the mean ⁇ SEM of three independent experiments.
  • B Immunofluorescence staining shows the expression of pluripotency marker proteins (OCT4 and SSEA4) of the corrected iPSC cell line. Nuclei were marked with 4',6-diamidino-2-phenylindole (DAPI) (Scale bar, 100 ⁇ m).
  • DAPI 4',6-diamidino-2-phenylindole
  • C Immunofluorescence staining shows expression of marker proteins representing ectoderm (NESTIN), mesoderm ( ⁇ -smooth muscle actin, ⁇ -SMA) and endoderm (hepatocyte nuclear factor-3 ⁇ , HNF-3 ⁇ ) of the corrected iPSC cell line. Nuclei were labeled with DAPI (scale bar, 100 ⁇ m).
  • DAPI scale bar, 100 ⁇ m.
  • Karyotyping was performed on corrected iPSC cell lines.
  • FIG. 5 is a view showing off-target analysis in iPSC cell lines corrected through target-deep sequencing.
  • A Four potential off-target sites different from the target site by up to 4 nucleotides were investigated in the corrected clones by target-deep sequencing. Mismatched nucleotide and PAM sequences (5'-NGG-3') are shown in blue and red, respectively.
  • B Total number of leads.
  • FIG. 6 is a diagram showing the phenotypic rescue (rescue) of FVIII gene expression in the corrected iPSC cell line.
  • A To confirm that the mutated sequence was translated in the FE-KI iPSC cell line, Sanger sequencing was performed on the mRNA of the FVIII gene in the corrected iPSC cell line. 309 Phe and 1984 Glu were successfully mutated to 309 Ser and 1984 Val in FE-KI iPSCs.
  • B qPCR analysis results showing FVIII expression levels in patient and knock-in iPSC cell lines. GAPDH was used to normalize gene expression. Data are the mean ⁇ SEM of three independent experiments.
  • FIG. 7 is a diagram showing the functional recovery of FVIII deficiency in the corrected iPSC cell line and the differentiated EC.
  • A qPCR analysis results showing EC marker expression (CD31, VWF, VE-cadherin) and FVIII expression levels in patient and knock-in iPSC cell lines. GAPDH was used to normalize gene expression. Data are the mean ⁇ SEM of three independent experiments.
  • B Immunofluorescence staining shows the expression of endothelial marker proteins (CD31 and VWF) of a knock-in iPSC cell line. Nuclei were labeled with DAPI (scale bar, 100 ⁇ m).
  • FIG. 8 is a diagram showing the functional recovery of FVIII in transplanted HA mice.
  • A RT-PCR analysis to detect expression of HA and human FVIII in transplanted mice. Human ACTIN was used to identify transplanted ECs. Mouse Gapdh was used as a control.
  • B FVIII activity was measured in HA and plasma collected from transplanted mice. Data represent detected activity per 1 ⁇ 10 6 EC.
  • FIG. 9 is a diagram showing a schematic diagram for the differentiation of corrected iPSCs and cell separation using MACS.
  • FIG. 10 and 11 are diagrams showing yields of corrected iPSCs for each cell surface marker. (FIG. 10, WT-BDD vs. FE-BDD; FIG. 11, Total)
  • FIG. 12 is a diagram showing a schematic diagram of the corrected iPSC differentiation, cell separation, and gene expression analysis.
  • 13 to 15 show EC-specific genes (CD31, FVIII, VE-Cad, vWF) for each experimental group (WT-BDD, FE-BDD, Total) and cell surface markers (CD31, VEGFR2, CD34, VE-Cad) It is a diagram showing the expression level.
  • 16 to 18 show cells that were isolated or unsorted with each cell surface marker (CD31+, VEGFR2+, CD34+, VE-Cad+) on day 4 of differentiating iPSCs into ECs, and microscopically on days 8-10 of differentiation ( 16) and CD31, vWF, and VE-Cad immunostaining (FIGS. 17 and 18) is a diagram showing the observed results.
  • FIG. 19 shows the differentiation of EC progenitor cells separated by each cell surface marker (CD31+, VEGFR2+, CD34+, VE-Cad+) on the 4th day of differentiating iPSCs into ECs by experimental group (WT-BDD, FE-BDD). It is a diagram showing the activity of FVIII contained in the culture medium of EC cells on days 8 to 10.
  • % used to indicate the concentration of a specific substance is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and Liquid/liquid is (volume/volume) %.
  • the present inventors used pcDNA4/BDD-FVIII plasmid (Addgene, #41035) as a backbone to construct a donor plasmid.
  • 5'-homology arm (5'-homology arm, left arm, LA) and 3'-homology arm (3'-homology arm, right arm, RA) were designed at the MunI/MluI and PacI/MauBI sites, respectively, and inserted.
  • the human EF1 ⁇ promoter sequence was cloned between the left arm and the FVIII open reading frame using the MluI/RruI site.
  • Streptococcus pyogenes (SpCas9)(D10A) nickase and 5'-GX 19 sgRNA (5'-GGGCCACTAG GGACAGGAT-3', SEQ ID NO: 1) expression plasmids were purchased from ToolGen (Seoul, Korea).
  • Cas9 nickase (D10A) plasmids, BDD-FVIII and FE-FVIII knock-in donor DNAs were electroporated into iPSCs of hemophilia A patients. Specifically, iPSCs from hemophilia A patients were washed once with DPBS and dissociated into single cells using ReLeSRTM (STEMCELL Technologies, Vancouver, Canada). 5x10 5 iPSCs were electroporated with 1 ⁇ g Cas9 nickase, 2 ⁇ g sgRNA expression vector and 2 ⁇ g each donor plasmid using the Neon transfection system (Invitrogen, Carlsbad, CA, USA).
  • cells were cultured in STEMMACS medium containing 10 ⁇ M Y-27632 (Sigma-Aldrich, St. Louis, MO, USA) for 2 days. Four days after transfection, cells were selected using 0.5 ⁇ g/mL puromycin.
  • a web-based in silico tool (www.rgenome.net) was used to search for 4 potential off-target sites that differed from the target site by up to 3 nucleotides.
  • Genomic DNA was isolated from patient and corrected iPSC cell lines using DNeasy Blood & Tissue Kits (QIAGEN) for target-deep sequencing, and off-target using the MiSeq system (Illumina, San Diego, CA, USA) in ToolGen. Sites were amplified and analyzed.
  • iPSCs were dissociated with ReleSRTM and transferred to Matrigel-coated culture dishes in STEMMACS medium containing 10 ⁇ M Y-27632 for 1 day. Then, the culture medium was replaced with STEMdiffTM APELTM2 medium (STEMCELL Technologies) containing 6 ⁇ M CHIR99021 (Tocris Bioscience, Bristol, UK) for 2 days to induce mesodermal lineages. On day 2, cells contained 25 ng/mL bone morphogenetic protein 4 (BMP4; Prospec, East Brunswick, NJ, USA), 10 ng/mL bFGF and 50 ng/mL vascular endothelial growth factor (VEGF)-A (PeproTech). Vascular lineages were induced by culturing in STEMdiffTM APELTM2 medium for 2 days.
  • BMP4 bone morphogenetic protein 4
  • VEGF vascular endothelial growth factor
  • FVIII activity was measured using the Coamatic ® Factor VIII chromogenic assay kit (Instrumentation Laboratory, Bedford, MA, USA) according to the manufacturer's instructions.
  • FVIII activity measurements were performed in 96-well microplates and absorbance at 405 nm was determined by endpoint readout used by a microplate reader (Molecular devices, San Jose, CA, USA). The standard curve was measured with HemosIL ® Calibration Plasma (Instrumentation Laboratory).
  • hemophilia A mice For transplantation into hemophilia A mice, 3-month-old hemophilia A mice (Jackson Laboratory, strain: B6;129S4-F8 tm1Kaz /J) were used, and patient-derived or corrected iPSCs were injected into each mouse 1x10 6 via tail vein injection. EC was injected. Cyclosporine A (210 mg/L; in negative water) was administered 3 days prior to transplantation and replaced once every 3 days. After 3, 5, 7, 10 and 14 days, blood samples were taken from each of the transplanted mouse tail veins to measure the expression and activity of FVIII, respectively.
  • HA and implanted mice were anesthetized and tail-clip analysis was performed. Briefly, a 2 mm diameter tail end was cut and bleeding was allowed for 5 min. After the tail was pressed firmly for 1 min, survival time was monitored in each mouse until 2 days after clipping.
  • A is the residual FVIII activity
  • a 0 is the initial activity
  • k is the apparent rate constant
  • t is the incubation time (hours) at 37°C.
  • HEK 293T cells Human embryonic kidney (HEK) 293T cells (ATCC, Manassas, VA, USA) contained 10% (vol/vol) fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 1% (vol/vol) P/S (Gibco) was maintained in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Grand Island, NY, USA).
  • Intron 22 inverted patient-derived iPSCs, BDD-FVIII and F309S/E1984V mutant FVIII (FE-FVIII) gene-inserted iPSCs were cultured in Matrigel (Corning, Corning, NY, USA)-coated culture dishes. Feeder-free culture was performed using STEMMACSTM iPSC-brew XF (STEMMACSTM iPSC-brew XF) culture medium (Miltenyi Biotec, Bergisch Gladbach, Germany).
  • Genomic DNA was isolated from cells using the DNeasy Blood & Tissue kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. To confirm the knock-in of the donor DNA into the AAVS1 locus, the DNA fragments of each junction were amplified using EmeraldAmp ® GT PCR Master Mix. The nucleotide sequence of each DNA amplicon was verified through Sanger sequencing at Cosmogenetech.
  • iPSC colonies were dissected to generate embryonic bodies (EBs), followed by EB culture medium [4 ng/mL basic fibroblast growth factor (bFGF; PeproTech, Rocky Hill, JN, USA), 20% knockout).
  • EB culture medium [4 ng/mL basic fibroblast growth factor (bFGF; PeproTech, Rocky Hill, JN, USA), 20% knockout.
  • DMEM/F12 medium Gibco
  • serum substitute Invitrogen
  • 1% non-essential amino acids Invitrogen
  • 2-mercaptoethanol Sigma-Aldrich
  • mouse anti-SSEA4 (1:200, Millipore, Billerica, MA, USA), rabbit anti-OCT4 (1:200, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-NESTIN (1:1000, Millipore), goat anti-HNF3 ⁇ (1:200, Santa Cruz Biotechnology), mouse anti- ⁇ -SMA (1:400, Sigma-Aldrich), mouse anti-CD31 (1:200, BD Biosciences, San Jose, CA, USA), and rabbit anti-VWF (1:500, Millipore).
  • target site Target Seq. (5' to 3') Forward primer Sequence.
  • Reverse primer Seq. (5' to 3') Off-target t 1 (OT1) , Chr.19) GGGCCCTTATGGACAGGAT GGG (SEQ ID NO: 33) GTGCCCGTATCCCAGAGT GAT (SEQ ID NO: 34) AGGTGGATGACAAGGTC AGG (SEQ ID NO: 35) Off-target 2 (OT2, Chr.19) GGGGCACTGGGGACAGGC TTGG (SEQ ID NO: 36) AGGAGGTCAGTCTGGGA GGT (SEQ ID NO: 37) GAGAGGGGCACAAACA GAAG (SEQ ID NO: 38) Off-target 3 (OT3, Chr.15) GGACCACTGGGCACAGGGAT CGG (SEQ ID NO: 39) ATGTTGGAAGAGGACGT TGG (SEQ ID NO: 40) TCACATGTCCTCCACCT GT
  • Antibodies used in this study Antibody Company Cat # OCT4 Santa Cruz SC9081 SSEA-4 Millipore MAB4304 NESTIN Millipore MAB5326 ⁇ -SMA Sigma A5228 HNF-3 ⁇ Santa Cruz SC6554 CD31 BD Bioscience 555444 vWF Millipore AB7356
  • FVIII mutants were transfected into HEK 293T cells and incubated at 37° C. for 24 hours, after which the supernatant was harvested and analyzed.
  • FVIII activity levels were increased in FVIII variants containing the F309S mutated-FVIII gene (1.40 fold in F, 1.56 fold in FE) compared to BDD-FVIII.
  • the E1984V mutated-FVIII gene reduced the level of FVIII activity compared to BDD-FVIII.
  • the E mutant FVIII gene reduces FVIII activity compared to WT, the increased FVIII activity in the FE mutant FVIII gene than the F mutant FVIII gene represents a synergistic effect.
  • the stability of the FVIII mutant was estimated through decay analysis (Fig. 1, B). After incubating the supernatant secreted from each cell line at 37° C. for 0-24 hours, each sample was checked by activity assay to determine the rate of decay compared to the initial stage. In the decay analysis, the F mutant FVIII gene was not significantly different from that of BDD-FVIII, but the stability and half-life were increased in the E1984V mutant FVIII gene (E, FE), which are factors related to stability ( FIGS. 1 and 1C ). Based on the above results, it was determined that the FE mutant FVIII gene is involved in both secretion and stability.
  • Example 2 Targeted knock-in of BDD-FVIII and FE-FVIII gene into the AAVS1 locus of HA patient-derived iPSCs )
  • sgRNA targeting intron 1 of the PPP1R12C gene was used ( FIG. 2A ).
  • Each donor plasmid with sgRNA and Cas9 nickase (D10A) vector was electroporated into iPSCs derived from HA patients ( FIG. 3A ).
  • genomic DNA was extracted from the emerging drug-resistant iPS colonies, and then knock-in colonies were identified by PCR analysis as previously described. To screen the corrected colonies, the 5' junction and the 3' junction of the knock-in site were amplified using a specific primer set (F1/R1, F2/R2).
  • WT-K1 and FE-K1 iPSCs were selected to excise the puromycin resistance cassette from the knock-in iPSC cell line.
  • transient Cre recombinase expression by electroporation we screened each iPSC cell line based on the PCR product generated by the F3/R2 primers. 3 of 8 colonies (37.5%, WT-K1 excision) and 4 of 8 colonies (50%, FE-K1 excision) were screened from each iPSC lineage.
  • the present inventors also confirmed the expression of pluripotent marker proteins (SSEA4, OCT4) in the corrected iPSC cell line (Fig. 4, B).
  • SSEA4, OCT4 pluripotent marker proteins
  • Fig. 4, B In vitro tripoderm analysis confirmed successful differentiation of ectoderm (NESTIN), mesoderm (alpha smooth muscle actin, ⁇ -SMA) and endoderm (hepatocyte nuclear factor-3beta, HNF-3 ⁇ ) (Fig. 4.C).
  • all corrected iPSC cell lines showed a normal 46,XY karyotype by G-banding (Fig. 4, D).
  • the present inventors confirmed whether the off-target mutation was induced in the iPSC lineage corrected by Cas9 nickase.
  • a web-based in silico tool was used to obtain a list of off-target sites that differed from the target site by up to 3 nucleotides, and identified 4 potential off-target sites for target-deep sequencing in 4 corrected iPSC cell lines and parental iPSCs. selected. It was confirmed that no significant mutations were found in the off-target site of the corrected iPSC cell line (FIG. 5).
  • EC is known to be the main source of FVIII production.
  • EC markers appeared on day 8 of differentiation and were confirmed by qRT-PCR analysis and immunocytochemistry.
  • qRT-PCR analysis showed that EC markers (CD31, VWF, VE-cadherin) were expressed in differentiated ECs compared to patient iPSCs (Fig. 7, A).
  • EC markers CD31, VWF, VE-cadherin
  • the present inventors further conducted FVIII activity assay to investigate whether the FVIII protein secreted from ECs differentiated from the corrected cell line.
  • the level of FVIII activity of FE-KI ECs (41.50 ⁇ 3.59 in FE-K1e1, 38.79 ⁇ 4.09 in FE-K1e2) was significantly higher than that of WT-KI ECs (19.17 ⁇ 5.15 in WT-K1e1, 20.88 ⁇ 2.14 in WT-K1e1). increased (Fig. 7, C).
  • a FVIII decay assay was then performed to determine the stability of the FVIII protein in each corrected EC cell line.
  • the supernatant obtained from each EC cell line was also incubated for 0-24 hours at 37°C as tested in 293T cells, and then each sample was analyzed by FVIII activity assay to investigate the rate of decay (FIG. 7, D).
  • FVIII activity assay to investigate the rate of decay (FIG. 7, D).
  • FVIII activity assay also showed no significant differences between HA mice and patient cell transplanted mice.
  • mice transplanted with WT-K1e1 and FE-K1e1 cells showed restoration of FVIII activity in HA mice.
  • the FVIII activity of mice transplanted with FE-K1e1 cells was more than 2-fold higher than that of mice transplanted with WT-K1e1 cells for 7 days after transplantation (7.00 ⁇ 1.23 in WT-K1e1, and 7.00 ⁇ 1.23 in FE-K1e1). 14.84 ⁇ 0.89). (Fig. 8, B).
  • the FE-KI cell line can increase the level of FVIII activity and the stability of FVIII protein in differentiated EC than the WT-KI cell line, and that FVIII activity can be restored in ECs transplanted into HA mice after 7 days. did.
  • cell surface markers CD31, VEGFR2, CD34, or VE-Cad
  • FIG. 9 A schematic diagram explaining the differentiation and separation process is shown in FIG. 9, and the yields for each cell surface marker are shown in Tables 4 and 10, and Tables 5 and 11.
  • the yield obtained by separating each cell surface marker was about 2% higher in the FE-BDD gene insertion group than in the WT-BDD gene insertion group in the case of CD31+ cell line.
  • VEGFR2+, CD34+, and VE-Cad+ cell lines showed that the WT-BDD gene insertion group was slightly higher than the FE-BDD gene insertion group, but the overall yield was significantly higher between the WT-BDD and FE-BDD gene insertion groups. There was no difference.
  • Example 8 Comparison of EC marker gene expression characteristics of each cell line after EC cell isolation using corrected iPSC cell surface markers
  • Example 7 the present inventors separated by each cell surface marker on the 4th day of EC differentiation (CD31+, VEGFR2+, CD34+, VE-Cad+), or after differentiating unsorted cell lines, differentiation 8 to 10 On day 1, expression levels of EC marker genes were compared using qPCR. A schematic diagram of cell differentiation, separation, and experimentation is shown in FIG. 12, and gene expression levels for each experimental group and cell line are shown in FIGS. 13 to 15.
  • the expression level of CD31 and VE-Cad genes which are endothelial cell-specific markers, was higher in the population of CD31-positive cells among the cell surface markers CD31, VEGFR2, CD34 and VE-Cad than in the unsorted cell population. was found to be significantly higher.
  • FIGS. 13 and 14 in the case of the cell line isolated with CD31 in the FE-BDD insertion group, it can be seen that the relative expression level of FVIII is increased compared to the cell line isolated by CD31 in the WT-BDD insertion group. there was.
  • Example 8 the present inventors separated by each cell surface marker on the 4th day of EC differentiation (CD31+, VEGFR2+, CD34+, VE-Cad+), or after differentiating unsorted cell lines, differentiation 8 to 10 On the first day, microscopy and immunostaining were performed with CD31, vWF, and VE-Cad and observed. The results are shown in FIGS. 16 to 18 .
  • EC progenitor cells were isolated by MACS on the 4th day, and then immunostained with EC markers CD31, vWF, and VE-Cad on the 10th day of final differentiation to observe the results. It was confirmed that the CD31+, CD34+, and VE-Cad+ cells were the cells uniformly expressing CD31 and vWF as a whole. In contrast, in the case of Unsorted and VEGFR2+ cells, it was confirmed that a large number of CD31-/vWF- cells were mixed (scale bar 200 ⁇ m).
  • CD31, CD34 and/or VE-Cad markers are used to isolate EC progenitor cells that are positive for the corresponding EC marker, it was found that cells differentiated into vascular endothelial cells can be separated with higher uniformity.
  • Example 10 Comparison of FVIII activity after EC cell isolation using cell surface markers of corrected iPSCs
  • Example 8 the present inventors differentiated cell lines separated by each cell surface marker (CD31+, VEGFR2+, CD34+, VE-Cad+) into EC cells on the 4th day of EC differentiation, and then differentiated WT- The activity of FVIII contained in the culture medium of BDD and FE-BDD cell lines was compared. The results are shown in Table 6 and FIG. 19 .

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Abstract

La présente invention concerne des cellules souches modifiées avec un gène mutant de FE-FVIII, des cellules endothéliales différenciées à partir de celles-ci, et une composition pharmaceutique les contenant pour la prévention ou le traitement de l'hémophilie. La composition, les cellules souches pluripotentes, et les cellules endothéliales différenciées à partir de celles-ci selon la présente invention ont une excellente activité de récupération de la fonction de FVIII et peuvent ainsi être avantageusement utilisées en tant qu'agent thérapeutique pour l'hémophilie.
PCT/KR2022/004816 2021-04-02 2022-04-04 Cellules souches modifiées avec un gène mutant de fe-fviii, cellules endothéliales différenciées à partir de celles-ci, et composition pharmaceutique les contenant pour la prévention ou le traitement de l'hémophilie WO2022211604A1 (fr)

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