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VIRAL VECTORS EXPRESSING THERAPEUTIC PROTEINS SPECIFICALLY IN MYELOID CELLS AND MICROGLIA

20230227847 · 2023-07-20

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention provides novel viral vectors for use in human therapy, particularly for use in in the treatment of a disease or disorder which has its origin in the brain or is brain based, particularly a PGRN-associated neurodegenerative disease or disorder including frontotemporal degenerative disease or disorder such as Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease. The invention also provides viral vectors for use in the treatment of brain tumors, particularly brain tumors selected from the group consisting of glioblastoma, glioma, ganglioneuroblastoma, astrocytoma, oligodendroglioma, PNET (primitive neuroectodermal), medulloblastoma, CNS lymphoma, and neuroblastoma, or any other CNS tumor and further in the treatment of brain metastasis, originating from any forms of breast, lung, colon, testicular, renal carcinomas and melanoma, or any other solid tumor, and any hematologic tumor, comprising all forms of leukemia and lymphomas. Further, the viral vectors may be used in the treatment of autoimmune diseases, inflammatory diseases and/or allergic diseases.

    Claims

    1. A viral vector comprising a nucleic acid molecule encoding a therapeutic polypeptide or a combination of therapeutic polypeptides under control of a promoter or promoter fragment, wherein the promoter or promoter fragment drives expression of the therapeutic protein or the combination of therapeutic proteins in myeloid cells and microglia, and wherein the promoter or promoter fragment is inactive in progenitor and/or stem cells.

    2. The viral vector according to claim 1, wherein the promoter is a) a miR223 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 1, or a functional fragment thereof; or b) an ITGAM promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 6, or a functional fragment thereof; or c) an AIF1 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 5, or a functional fragment thereof; or d) a TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 3, SEQ ID NO:23 or SEQ ID NO:24, or a functional fragment thereof, or e) a P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 2, SEQ ID NO: 21 or SEQ ID NO: 22, or a functional fragment thereof, or f) an OLFML3 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 4 or SEQ ID NO:25, or a functional fragment thereof; or g) a fusion promoter comprising a miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 1, or a functional fragment thereof, operably linked to i) a TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 3, SEQ ID NO:23 or SEQ ID NO:24, or a functional fragment thereof; and/or ii) a P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 2, SEQ ID NO: 21 or SEQ ID NO:22, or a functional fragment thereof; and/or iii) an OLFML3 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 4 or SEQ ID NO:25, or a functional fragment thereof; and/or iv) an ITGAM promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO:6, or a functional fragment thereof; and/or v) an AIF1 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO:5, or a functional fragment thereof.

    3. The viral vector according to claim 1 or 2, wherein the viral vector comprises at least one transcriptional regulatory element, wherein said at least one transcriptional regulatory element is arranged such that it inhibits or activates a transcriptional activity of the promoter.

    4. The viral vector according to claim 3, wherein the at least one transcriptional regulatory element comprises a binding site for a transcriptional activator or repressor, in particular wherein the transcriptional activator or repressor comprises: i) an antibiotic-binding domain, in particular a tetracycline/doxycycline-binding domain, a macrolide-binding domain or a pristinamycin-binding domain; ii) a hormone-binding domain, in particular a RU486-binding domain or an abscisic acid-binding domain; iii) a steroid-binding domain, in particular an ecdysone-binding domain; or iv) a dimerizer system, in particular a rapamycin-based of rapalog-based dimerizer system.

    5. The viral vector according to any one of claims 1 to 4, wherein the viral vector encodes a riboswitch, wherein the riboswitch controls translation of an mRNA encoding the therapeutic protein or the combination of therapeutic proteins.

    6. The viral vector according to any one of claims 1 to 5, wherein the therapeutic polypeptide is i) a polypeptide that restores a cellular function and/or elicits a cellular response in a cell; or ii) a polypeptide that enables and/or increases target specificity of a cell.

    7. The viral vector according to claim 6, wherein the polypeptide that restores a cellular function and/or elicits a cellular response in a cell comprises at least a fragment of one or more polypeptides selected from the group consisting of: PGRN, Presenilin1, Presenilin 2, IL-2, IL-12, IL-15, IL-21, IFN-alpha, IFN-alpha Receptor, IFN gamma, IFN-gamma Receptor, FasL/Fas, CD11b, selectins, such as L-Selectin or P-Selectin, PSGL (P-Selectin Ligand), TRAIL, TRAIL-R, Lymphotoxin beta (LT-β), LT-βR, decoyreceptors 1-3, TNF-alpha, TNF-alphaR, MSH, G-CSF, GM-CSF, IL-1, IL-6, IL-7, IL-8, IL31, IL1R, IL31R, IL-10, IL-23, CXCR3 ligands such as CXCL9 and CXCL-10, PD-1, PD-1L, PD-2 (PDC2), PD-2L, Granzyme B, Granulysine, CD11b, TIGIT, CD 112, CD 155, nitric oxide synthase, DNA methyltransferase 3b (DNMT3b), Jumonji domain-containing protein 1A (JMJD1A), somatostatine, histone deacetylases (HDAC) such as HDAC3 or HDAC 9, CSF1 receptor (CSF1R), IL-34, TAM, all chemokines and chemokine receptors, all cytokines and cytokine receptors.

    8. The viral vector according to claim 6, wherein the polypeptide that enables and/or increases target specificity of a cell enables and/or increases specificity to a tumor antigen, in particular wherein the tumor antigen is VEGF, a VEGF-Receptor, an antagonists to a metalloproteinase (e.g. MMP-9), CD40/CD40L, EGFR, Annexin1, FGFR-1, Her2, St6galnac5, MMP1-28, TIMPS1-4, Melanotransferrin, alpha4-beta1 Integrin, VCAM-1, E-cadherin, Alpha-v-beta3 integrin, Alpha-v-beta5 integrin, Alpha-v-beta6 integrin, Alpha-v-beta8 integrin, CCND1, BRCA, CEA, cancer-related antigen 72-4 (CA 72-4), cancer-related antigen 19-9 (CA 19-9), WT1, CD 11b, L-Selectin, NY-ESO-1, or a fragment thereof.

    9. A viral vector comprising a transgene under control of one or more promoters, wherein the transgene encodes a) PGRN, or a functional fragment thereof; or b) a polypeptide having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, or a functional fragment thereof.

    10. A viral vector comprising a transgene under control of one or more promoters, wherein the transgene encodes a) IL-12, or a functional fragment thereof; or b) a polypeptide having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 11, or a functional fragment thereof; and/or a polypeptide having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 12, or a functional fragment thereof.

    11. A viral vector comprising a transgene under control of one or more promoters, wherein the transgene encodes a) IFN-gamma, or a functional fragment thereof; or b) a polypeptide having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 10, or a functional fragment thereof.

    12. A viral vector comprising a transgene under control of one or more promoters, wherein the transgene encodes a) GM-CSF, or a functional fragment thereof; or b) a polypeptide having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 13, or a functional fragment thereof.

    13. A viral vector comprising a transgene under control of one or more promoters, wherein the transgene encodes a) G-CSF, or a functional fragment thereof; or b) a polypeptide having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 14, or a functional fragment thereof.

    14. A viral vector comprising a transgene under control of one or more promoters, wherein the transgene encodes a) GM-CSF and IFN-gamma, or functional fragments thereof; or b) a polypeptide having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 15, or a functional fragment thereof.

    15. A viral vector comprising a transgene under control of one or more promoters, wherein the transgene encodes a) G-CSF and IFN-gamma, or functional fragments thereof; or b) a polypeptide having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 16, or a functional fragment thereof.

    16. A viral vector comprising a transgene under control of one or more promoters, wherein the transgene encodes a) IL-2, or a functional fragment thereof; or b) a polypeptide having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 17, or a functional fragment thereof.

    17. A viral vector comprising a transgene under control of one or more promoters, wherein the transgene encodes a) IL-15, or a functional fragment thereof; or b) a polypeptide having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 18, or a functional fragment thereof.

    18. A viral vector comprising a transgene under control of one or more promoters, wherein the transgene encodes a) IL-21, or a functional fragment thereof; or b) a polypeptide having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 19, or a functional fragment thereof.

    19. A viral vector comprising a transgene under control of one or more promoters, wherein the transgene encodes a) IFN-alpha, or a functional fragment thereof; or b) a polypeptide having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 20, or a functional fragment thereof.

    20. The viral vector according to any one of claims 9 to 19, wherein the one or more promoters comprise: a) a myelo-specific promoter, or a functional fragment thereof; and/or b) a microglia-specific promoter, or a functional fragment thereof; and/or c) a fusion promoter comprising or consisting of i) a first promoter, wherein said first promoter is a myelo-specific promoter or a microglia-specific promoter, or a functional fragment thereof; and ii) a second promoter.

    21. The viral vector according to claim 20, wherein the myelo-specific promoter is a) a miR233 promoter, or a functional fragment thereof; or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 1, or a functional fragment thereof; b) an AIF1 promoter, or a functional fragment thereof; or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO:5, or a functional fragment thereof; or c) an ITGAM promoter, or a functional fragment thereof; or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO:6, or a functional fragment thereof.

    22. The viral vector according to claim 20 or 21, wherein the microglia-specific promoter is a) a TMEM119 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO:3, SEQ ID NO:23 or SEQ ID NO:24, or a functional fragment thereof; or b) a P2RY12 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; c) an OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO:4 or SEQ ID NO:25, or a functional fragment thereof.

    23. The viral vector according to any one of claims 20 to 22, wherein the first promoter is a myelo-specific promoter and wherein the second promoter is a microglia-specific promoter, or vice versa.

    24. The viral vector according to any one of claims 20 to 23, wherein the first promoter is a miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 1, or a functional fragment thereof; and wherein the first promoter is operably linked to i) a TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 5, SEQ ID NO:6 or SEQ ID NO:7, or a functional fragment thereof; ii) a P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or a functional fragment thereof; iii) an OLFML3 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 8 or SEQ ID NO:9, or a functional fragment thereof iv) an ITGAM promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 11, or a functional fragment thereof; and/or v) an AIF1 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 10, or a functional fragment thereof.

    25. The viral vector according to any one of claims 9 to 24, wherein the viral vector comprises at least one transcriptional regulatory element, and wherein said at least one transcriptional regulatory element is arranged such that it inhibits or activates a transcriptional activity of the promoter.

    26. The viral vector according to claim 25, wherein the at least one transcriptional regulatory element comprises a binding site for a transcriptional activator or repressor, in particular wherein the transcriptional activator or repressor comprises: i) an antibiotic-binding domain, in particular a tetracycline/doxycycline-binding domain, a macrolide-binding domain or a pristinamycin-binding domain; ii) a hormone-binding domain, in particular a RU486-binding domain or an abscisic acid-binding domain; iii) a steroid-binding domain, in particular an ecdysone-binding domain; iv) a dimerizer system, in particular a rapamycin-based of rapalog-based dimerizer system.

    27. The viral vector according to any one of claims 9 to 26, wherein the viral vector encodes a riboswitch, and wherein the riboswitch controls translation of an mRNA encoding the therapeutic protein or the combination of therapeutic proteins.

    28. The viral vector according to any one of claims 1 to 27, wherein the viral vector is a) a retroviral vector, in particular a lentiviral vector, more particularly a lentiviral SIN vector; or b) a foamy viral vector; or c) a viral vector selected from the group consisting of: an adenoviral vector, an adeno-associated viral vector, a herpes viral vector, a parvoviral vector, a coronaviral vector, and an alpha-retroviral vector.

    29. A fusion promoter comprising a) a miR223 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 1, or a functional fragment thereof; and b) a microglia-specific promoter, or a functional fragment thereof; wherein the miR223 promoter or the promoter having at least at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 1, or the functional fragment thereof, is operably linked to the microglia-specific promoter, or the functional fragment thereof.

    30. The fusion promoter according to claim 29, wherein the microglia-specific promoter is a) a TMEM119 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO:3, SEQ ID NO:23 or SEQ ID NO:24, or a functional fragment thereof; b) a P2RY12 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 2, SEQ ID NO: 21 or SEQ ID NO: 22, or a functional fragment thereof; c) an OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO:4 or SEQ ID NO:25, or a functional fragment thereof, d) an AIF1 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO:5, or a functional fragment thereof; or e) an ITGAM promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO:6, or a functional fragment thereof.

    31. The fusion promoter according to claim 29 or 30, wherein the fusion promoter comprises at least one transcriptional regulatory element, wherein said at least one transcriptional regulatory element is arranged such that it inhibits or activates a transcriptional activity of the promoter.

    32. The fusion promoter according to claim 31, wherein the at least one transcriptional regulatory element comprises a binding site for a transcriptional activator or repressor, in particular wherein the transcriptional activator or repressor comprises: i) an antibiotic-binding domain, in particular a tetracycline/doxycycline-binding domain, a macrolide-binding domain or a pristinamycin-binding domain; ii) a hormone-binding domain, in particular a RU486-binding domain or an abscisic acid-binding domain; iii) a steroid-binding domain, in particular an ecdysone-binding domain; iv) a dimerizer system, in particular a rapamycin-based of rapalog-based dimerizer system.

    33. The fusion promoter according to any one of claims 29 to 32, wherein the fusion promoter a) comprises any one of the sequences set forth in SEQ ID NO:26-29: or b) comprises a sequence having 90%, 91%, 92%, 93%, 94% or 95% sequence identity with any one of the sequence set forth in SEQ ID NO: 26-29, wherein the promoter drives expression in microglia and/or myeloid cells.

    34. A host cell comprising the viral vector according to any one of claims 1 to 28.

    35. The host cell according to claim 34, wherein the host cell is a hematopoietic stem cell, preferably a hematopoietic stem cell of a CD34-positive enriched cell population, or wherein the host cell is a myeloid cell.

    36. A pharmaceutical composition comprising the viral vector according to any one of claims 1 to 28 and/or the host cell according to claim 34 or 35.

    37. The viral vector according to any one of claims 1 to 28, the host cell according to claim 32 or 33 or the pharmaceutical composition according to claim 36 for use in medicine.

    38. The viral vector according to any one of claims 1 to 28, the host cell according to claim 34 or 35 or the pharmaceutical composition according to claim 36 for use in the treatment of a disease or disorder which has its origin or a manifestation in the brain or is brain-based.

    39. The viral vector according to any one of claims 9 or 20 to 28, the host cell according to claim 34 or 35 or the pharmaceutical composition according to claim 36 for use in the prevention and/or treatment of a PGRN-associated disease or disorder, in particular wherein the viral vector encodes PGRN, or a functional fragment thereof.

    40. The viral vector, the host cell or the pharmaceutical composition for use according to claim 39, wherein the PGRN-associated disease or disorder is a neurodegenerative disease or disorder.

    41. The viral vector, the host cell or the pharmaceutical composition for use according to claim 40, wherein the neurodegenerative disease or disorder is a degenerative disease or disorder.

    42. The viral vector, the host cell or the pharmaceutical composition for use according to claim 41, wherein the degenerative disease or disorder is selected from the group consisting of: Alzheimer's disease, amyotrophic lateral sclerosis, neuronal ceroid lipofuscinosis and Parkinson's disease.

    43. The viral vector according to any one of claims 10 to 28, the host cell according to claim 34 or 35 or the pharmaceutical composition according to claim 36 for use in the treatment of cancer, lymphoma and/or sarcoma in particular wherein the viral vector encodes at least one of IL-12, IFN-gamma, G-CSF, GM-CSF, IL-2, IL-15, IL-21 and/or IFN-alpha; or functional fragments thereof.

    44. The viral vector, the host cell or the pharmaceutical composition for use according to claim 43, wherein the cancer, lymphoma and/or sarcoma is a brain tumor or a brain metastasis.

    45. The viral vector, the host cell or the pharmaceutical composition for use according to claim 44, wherein the brain tumor is selected from the group consisting of: glioblastoma, glioma, ganglioneuroblastoma, astrocytoma, oligodendroglioma, PNET (primitive neuroectodermal tumor), medulloblastoma, CNS lymphoma, meningioma, retinoblastoma and neuroblastoma.

    46. The viral vector, the host cell or the pharmaceutical composition for use according to claim 44, wherein the brain tumor is a metastatic tumor originating from any form of breast cancer, lung cancer, colon cancer, testicular cancer, renal carcinomas, melanoma, ovary carcinomas, prostate carcinoma, neuroendocrine tumors or any other solid tumor or any sarcoma, or any hematologic tumor, comprising all forms of leukemia and lymphomas.

    47. The viral vector, the host cell or the pharmaceutical composition for use according to any one of claims 37 to 46, wherein the viral vector, the host cell or the pharmaceutical composition is administered in conjunction with a therapy that reduces the integrity of the blood-brain-barrier, in particular wherein the therapy that reduces the integrity of the blood-brain-barrier is a bone marrow conditioning therapy, a CNS conditioning therapy, and/or a blood-brain-barrier conditioning therapy.

    48. The viral vector, the host cell or the pharmaceutical composition for use according to claim 47, wherein the bone marrow conditioning therapy comprises the use of cytotoxic agents, alkylating agents, Busulphan, Treosulfan, Etoposide, Lomustin, radiotherapy, targeted radiotherapy (e.g. Yttrium-90 labeled anti-CD45 antibody, or Yttrium-90 labeled anti-CD66 antibody), ACK2 (anti-c-kit antibody), CD117 antibody-drug-conjugates, CD45-SAP, colony-stimulating factor 1 (CSF1) specific agents, PLX3397, BLZ9445, PLX5622, RG7155, PLX647, Ki20227, GW2580, IL-34 and/or desatinib.

    49. The viral vector, the host cell or the pharmaceutical composition for use according to claim 47 or 48, wherein the CNS conditioning therapy comprises the use of Busulphan.

    50. The viral vector, the host cell or the pharmaceutical composition for use according to any one of claims 47 to 49, wherein the blood-brain-barrier conditioning therapy comprises radiotherapy or targeted radiotherapy.

    51. The viral vector, the host cell or the pharmaceutical composition for use according to any one of claims 47 to 50, wherein the viral vector, the host cell or the pharmaceutical composition is administered after the therapy that reduces the integrity of the blood-brain-barrier, in particular wherein the viral vector, the host cell or the pharmaceutical composition is administered not earlier than half a day after the therapy that reduces the integrity of the blood-brain-barrier.

    52. The viral vector according to any one of claims 1 to 28, the host cell according to claim 34 or 35 or the pharmaceutical composition according to claim 36 for use in the treatment of autoimmune diseases.

    53. The viral vector according to any one of claims 1 to 28, the host cell according to claim 34 or 35 or the pharmaceutical composition according to claim 36 for use in the treatment of autoinflammatory diseases.

    54. The viral vector according to any one of claims 1 to 28, the host cell according to claim 34 or 35 or the pharmaceutical composition according to claim 36 for use in the treatment of allergic diseases.

    55. The viral vector according to any one of claims 1 to 28, the host cell according to claim 34 or 35 or the pharmaceutical composition according to claim 36 for use in hematopoietic and solid organ transplantation.

    56. A method for treating a disease or disorder which has its origin or a manifestation in the brain or is brain based in a subject in need, the method comprising the steps of: a) genetically modifying a hematopoietic stem cell and/or a population of enriched CD34-positive bone marrow cells, the modification step comprising a step of contacting the hematopoietic stem cell and/or the population of enriched CD34-positive bone marrow cells with the viral vector according to any one of claims 1 to 28; or genetically modifying a myeloid cell and/or a population of enriched myeloid cells, the modification step comprising a step of contacting the myeloid cell and/or the population of enriched myeloid cells with the viral vector according to any one of claims 1 to 28; b) administering the genetically modified cells from step (a) intravenously to the subject in need; and c) treating a disease or disorder which has its origin or a manifestation in the brain or is brain based in the subject in need.

    57. The method according to claim 56, wherein the hematopoietic stem cell and/or the population of enriched CD34-positive bone marrow cells, or the myeloid cell and/or the population of enriched myeloid cells have been obtained from the subject in need or from a foreign donor.

    58. A method for treating a disease or disorder which has its origin or a manifestation in the brain or is brain based in a subject in need, the method comprising the steps of: a) mobilizing hematopoietic stem cells in the subject in need; b) administering the viral vector according to any one of claims 1 to 28 intravenously to the subject in need subsequent to the mobilization of hematopoietic stem cells in step (a); and c) treating a disease or disorder which has its origin or a manifestation in the brain or is brain based in the subject in need.

    59. The method according to claim 58, wherein the mobilization of hematopoietic stem cells in the subject in need comprises the administration of G-CSF and/or Plerixafor.

    60. The method according to any one of claims 56 to 59, wherein the disease or disorder which has its origin or a manifestation in the brain or is brain based is a PGRN-associated disease or disorder, in particular wherein the PGRN-associated disease or disorder is a neurodegenerative disease or disorder, in particular wherein the neurodegenerative disease or disorder is a degenerative disease or a neurodegenerative disorder, in particular wherein the degenerative disease or neurodegenerative disorder is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, neuronal ceroid lipofuscinosis, and Parkinson's disease, in particular wherein the viral vector encodes PGRN, or a functional fragment thereof.

    61. The method according to any one of claims 56 to 59, wherein the disease or disorder which has its origin, or a manifestation, in the brain or is brain based is a brain tumor, in particular wherein the brain tumor is selected from the group consisting of: glioma, glioblastoma, ganglioneuroblastoma, astrocytoma, oligodendroglioma, PNET (primitive neuroectodermal tumor), medulloblastoma, CNS lymphoma, and neuroblastoma; or wherein the brain tumor is a metastatic tumor originating from any form of breast cancer, lung cancer, colon cancer, testicular cancer, renal carcinomas, melanoma, prostate cancer, or any other solid tumor or any sarcoma, or any hematologic tumor, comprising all forms of leukemia and lymphomas, in particular wherein the viral vector encodes IL-12, IFN-gamma, GM-CSF, G-CSF, 11-2, IL-15, IL-21 and/or IFN-alpha, or functional fragments thereof.

    62. The method according to any one of claims 56 to 61, wherein the method comprises an additional step of reducing the integrity of the blood-brain-barrier, in particular wherein reducing the integrity of the blood-brain-barrier comprises a bone marrow conditioning therapy, a CNS conditioning therapy, and/or a blood-brain-barrier conditioning therapy.

    63. The method according to claim 62, wherein the therapy that reduces the integrity of the blood-brain-barrier is performed prior to the administration of the genetically modified cells to the subject in need, in particular wherein the time interval between the therapy that reduces the integrity of the blood-brain-barrier and the administration of the genetically modified cells is carried out after the therapy that reduces the integrity of the blood-brain-barrier.

    64. A method for treating cancer in a subject in need, the method comprising the steps of: a) mobilizing hematopoietic stem cells in the subject in need; b) administering the viral vector according to the invention intravenously to the subject in need subsequent to the mobilization of hematopoietic stem cells in step (a); and c) treating cancer in the subject in need.

    65. The method according to claim 64, wherein the mobilization of hematopoietic stem cells in the subject in need comprises the administration of G-CSF and/or Plerixafor.

    66. A method for expressing a transgene in the brain and/or CNS of a subject, the method comprising the steps of: a) genetically modifying a hematopoietic stem cell and/or a population of enriched CD34-positive bone marrow cells, the modification step comprising a step of contacting the hematopoietic stem cell and/or the population of enriched CD34-positive bone marrow cells with the viral vector according to any one of claims 1 to 26; or genetically modifying a myeloid cell and/or a population of enriched myeloid cells, the modification step comprising a step of contacting the myeloid cell and/or the population of enriched myeloid cells with the viral vector according to any one of claims 1 to 28; b) administering the genetically modified cells from step (a) intravenously to the subject in need; and c) expressing the transgene encoded by the viral vector in the brain and/or CNS of the subject.

    67. The method according to claim 66, wherein the hematopoietic stem cell and/or the population of enriched CD34-positive bone marrow cells; or wherein the myeloid cell and/or the population of enriched myeloid cells has been obtained from the subject or from a foreign donor.

    68. A method for expressing a transgene in the brain and/or CNS of a subject, the method comprising the steps of: a) mobilizing hematopoietic stem cells in the subject; b) administering the viral vector according to any one of claims 1 to 28 intravenously to the subject in need subsequent to the mobilization of hematopoietic stem cells in step (a); and c) expressing the transgene encoded in the viral vector in the brain and/or CNS of the subject.

    69. The method according to claim 68, wherein the mobilization of hematopoietic stem cells in the subject comprises the administration of G-CSF or Plerixafor.

    70. The method according to any one of claims 66 to 69, wherein the method comprises an additional step of reducing the integrity of the blood-brain-barrier, in particular wherein reducing the integrity of the blood-brain-barrier comprises a bone marrow conditioning therapy, a CNS conditioning therapy, and/or a blood-brain-barrier conditioning therapy.

    71. The method according to claim 70, wherein the therapy that reduces the integrity of the blood-brain-barrier is performed prior to the administration of the genetically modified cells to the subject in need, in particular wherein the time interval between the therapy that reduces the integrity of the blood-brain-barrier and the administration of the genetically modified cells is carried out after the therapy that reduces the integrity of the blood-brain-barrier.

    72. A method for treating a disease or disorder which has its origin or a manifestation in the brain or is brain based in a subject in need, the method comprising the steps of: a) administering the viral vector according to any one of claims 1 to 28 into the brain of the subject in need or intrathecally; and b) treating a disease or disorder which has its origin or a manifestation in the brain or is brain based in the subject in need.

    73. The viral vector according to claim 72, wherein the viral vector is an AAV-based viral vector.

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0817] FIG. 1: SIN-lentiviral construct vUS6 comprising the transgene GRN under control of the promoter miR223 (SEQ ID NO:1). 2A: self cleaving peptide; GFP: green fluorescent protein; nls: nuclear localization sequence; PRE4: modified Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element.

    [0818] FIG. 2: FACS analysis with HEK293T cells after transduction. Left: Untransduced cells. Right: Cells transduced with the lentiviral construct shown in FIG. 1 (vUS6) (Titer: 6.04×10.sup.6 TU/mL; MOI=2).

    [0819] FIG. 3: FACS analysis with THP1 cells after transduction. Left: Untransduced cells. Right: Cells transduced with the lentiviral construct shown in FIG. 1 (vUS6) (Titer: 6.04×10.sup.6 TU/mL; MOI=2).

    [0820] FIG. 4: FACS analysis with human microglia after transduction. Left: Untransduced cells. Right: Cells transduced with the lentiviral construct shown in FIG. 1 (vUS6) (Titer: 6.04×10.sup.6 TU/mL; MOI=2).

    [0821] FIG. 5: Progranulin release by human microglia (GRN−/−) measured by ELISA. Left bar: untransduced cells. Right bar: Cells transduced with the lentiviral construct shown in FIG. 1 (vUS6).

    [0822] FIG. 6: SIN-lentiviral construct comprising the transgene IL-12 under control of the promoter miR223 (SEQ ID NO:1). IRES: internal ribosome entry site; PRE4: modified Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element.

    [0823] FIG. 7: FACS analysis with THP1 cells after transduction. Left: Untransduced cells. Right: Cells transduced with the lentiviral construct shown in FIG. 6 (Titer: 3.24×10.sup.6 TU/mL; MOI=2).

    [0824] FIG. 8: FACS analysis with human microglia after transduction. Left: Untransduced cells. Right: Cells transduced with the lentiviral construct shown in FIG. 6 (Titer: 3.24×10.sup.6 TU/mL; MOI=2).

    [0825] FIGS. 9A-9D: SIN-lentiviral constructs comprising the transgene GRN under control of the promoters (FIG. 9A) miR223-TMEM119 (vUS7; SEQ ID NO:28); (FIG. 9B) ITGAM (vUS8; SEQ ID NO:6); (FIG. 9C) miR223_P2RY12 (vUS11; SEQ ID NO: 26); and (FIG. 9D) miR223_OLFML3 (vUS12; SEQ ID NO:29). 2A: self cleaving peptide; GFP: green fluorescent protein; nls: nuclear localization sequence; PRE4: modified Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element.

    [0826] FIGS. 10A-10F: FACS analysis with THP-1 cells transduced with different vectors upon differentiation to macrophages. (FIG. 10A) non-transduced control; (FIG. 10B) vUS6-LV_miR223_GRN (FIG. 1); (FIG. 10C) vUS7-LV_miR223-TMEM119 GRN (FIG. 9A); (FIG. 10D) vUS8-LV_ITGAM_GRN (FIG. 9B); (FIG. 10E) vUS11-LV_miR223-P2RY12 (FIG. 9C); (FIG. 10F) vUS12-LV_miR223-OLFML3 (FIG. 9D.

    [0827] FIGS. 11A-11B: (FIG. 11A) Percentage of GFP positive cells (transduction rate) in differentiated THP-1 cells. (FIG. 11B) Mean fluorescent intensity of differentiated THP-1 cells.

    [0828] FIGS. 12A-12F: FACS analysis of human microglia cell line (GRN −/−) transduced with different vectors. (FIG. 12A) non-transduced control; (FIG. 12B) vUS6-LV_miR223_GRN (FIG. 1); (FIG. 12C) vUS7-LV_miR223-TMEM119_GRN (FIG. 9A); (FIG. 12D) vUS8-LV_ITGAM_GRN (FIG. 9B); (FIG. 12E) vUS11-LV_miR223-P2RY12 (FIG. 9C); (FIG. 12F) vUS12-LV_miR223-OLFML3 (FIG. 9D.

    [0829] FIGS. 13A-13B: (FIG. 13A) Percentage of GFP positive cells (transduction rate) in human microglia cell line (GRN −/−). (FIG. 13B) Mean fluorescent intensity of human microglia cell line (GRN −/−).

    [0830] FIGS. 14A-14C: Restoration of GRN secretion in human microglia cell line (GRN −/−). (FIG. 14A) Amount of Granulin in supernatant. (FIG. 14B) Mean fluorescence intensity normalized by vector copy number. (FIG. 14C) Amount of Granulin normalized by (transduction rate×vector copy number)

    [0831] FIGS. 15A-15F: Human CD34+ bone marrow cells were lentivirally transduced with vectors encoding GRN-2A-GFP as transgene, followed by differentiation to monocytes and FACS analysis in monocytes (day 12). (FIG. 15A) non-transduced control; (FIG. 15B) vUS6-LV_miR223_GRN (FIG. 1); (FIG. 15C) vUS7-LV_miR223-TMEM119 GRN (FIG. 9A); (FIG. 15D) vUS8-LV_ITGAM_GRN (FIG. 9B); (FIG. 15E) vUS11-LV_miR223-P2RY12 (FIG. 9C); (FIG. 15F) vUS12-LV_miR223-OLFML3 (FIG. 9D.

    [0832] FIG. 16: Percentage of GFP positive monocytes.

    [0833] FIGS. 17A-17D: Analysis of the activity of candidate promoters in human CD34+ cells upon differentiation into monocytes (day 7). (FIG. 17A) non-transduced control; (FIG. 17B) vUS6-LV_miR223_GRN (FIG. 1): (left) transduction in the presence of Amphotericin B, (right) transduction in the presence of Lentiboost; (FIG. 17C) vUS7-LV_miR223-TMEM119_GRN (FIG. 9A): (left) transduction in the presence of Amphotericin B, (right) transduction in the presence of Lentiboost; (FIG. 17D) vUS8-LV_ITGAM_GRN (FIG. 9B): (left) transduction in the presence of Amphotericin B, (right) transduction in the presence of Lentiboost.

    [0834] FIG. 18: Vector copy number of human CD34+ cells upon differentiation into monocytes (day 12). (Top) Transduction in the presence of Amphotericin B, (bottom) transduction in the presence of Lentiboost.

    [0835] FIG. 19: Summary of FIG. 18.

    [0836] FIG. 20: Transduction efficiency (% GFP-positive cells divided by vector copy number (VCN)) in myeloid cells obtained from human CD34+ cells.

    [0837] FIG. 21: miR223 promoter activity in monocytes, macrophages and iPSC-derived homologs to haematopoietic stem cells.

    EXAMPLES

    Example 1: Production of Gene Modified Haematopoietic Stem Cells (HSC) Appropriate for the Treatment of Patients Suffering from Frontotemporal Dementia Due to GRN Gene Mutation

    [0838] HSC obtained by leukapheresis upon HSC mobilization, are transduced with a lentiviral self-inactivating (SIN) vector, comprising human PGRN (progranulin) encoding cDNA according to SEQ ID NO: 7 under control of the miR223 promoter according to SEQ ID NO: 1. Preferentially, the genetically modified autologous HSC population is cryopreserved after genetic manipulation.

    Example 2: Production of Gene Modified CD34+ Cells Appropriate for the Treatment of Patients Suffering from Frontotemporal Dementia Due to GRN Gene Mutation

    [0839] A CD34+ cell population, obtained by leukapheresis upon HSC mobilization followed by CD34+ cell isolation, is transduced with a lentiviral SIN vector encoding PGRN according to SEQ ID NO: 7 under control of a miR223 fusion promoter construct consisting of the TMEM119 promoter contained in SEQ ID NO: 3 fused to the miR223 promoter according to SEQ ID NO: 1.

    Example 3: Production of Gene Modified CD34+ Enriched Bone Marrow Cells Appropriate for the Treatment of Patients Suffering from Frontotemporal Dementia Due to GRN Gene Mutation

    [0840] CD34+ enriched bone marrow cells are transduced with a lentiviral vector encoding PGRN according to SEQ ID NO:8 under control of the ITGAM promotor according to SEQ ID NO: 6.

    Example 4: Production of Gene Modified CD34+ Enriched Bone Marrow Cells Appropriate for the Treatment of Patients Suffering from Frontotemporal Dementia Due to GRN Gene Mutation

    [0841] CD34+ bone marrow cells are transduced with a foamy viral vector encoding PGRN according to SEQ ID NO: 8 under control of the ITGAM promotor according to SEQ ID NO: 6.

    Example 5: Production of Gene Modified Blood Derived Monocytes Appropriate for the Treatment of Patients Suffering from Glioblastoma

    [0842] Blood derived monocytes of a patient suffering from glioblastoma are transduced with a lentiviral vector encoding interferon-gamma under control of the ITGAM promoter according to SEQ ID NO: 6.

    Example 6: Production of Gene Modified Blood Derived Monocytes Appropriate for the Treatment of Patients Suffering from Renal Carcinoma and Brain Metastases

    [0843] Blood derived monocytes of a patient suffering from renal carcinoma and optionally suffering from brain metastases, are transduced with a lentiviral vector encoding interferon-gamma under control of the miR223 promoter according to SEQ ID NO: 1.

    Example 7: Experiments Using Lentiviral-SIN Vectors for Phagocyte-Specific Transgene Expression

    Testing of Transduction of Cell Lines

    [0844] The developed lentiviral SIN-vectors enable phagocyte-specific transgene expression and may therefore be used for applications in human diseases in which phagocytes are the disease-causing cells, or in which phagocytes can provide therapeutic elements to cure or treat these diseases. This is the case for disorders which have their origin in the brain, such as neurodegenerative disease, or brain cancer or metastasis, as well as for disorders outside of the brain, such as immunodeficiency or cancer.

    Transduction with a Vector Comprising a GRN Transgene:

    [0845] Following cell lines were analyzed: [0846] 1. HEK293Tcells (Human embryonic kidney cells) as positive control; [0847] 2. THP1 cells (Human phagocyte cell line) as model for phagocyte correction; [0848] 3. Immortalised human microglia cell line (Im-hMicro), in which the GRN gene was knocked-out (GRN−/−), as disease model.

    [0849] For transduction of cell lines, a lentiviral self-inactivating (SIN) vector was used, comprising the miR223 promoter as internal promoter, and hGRN encoding cDNA as transgene, as well as an EGFP-reporter linked to GRN by the 2A sequence (FIG. 1). Transduction experiments were conducted at MOI 2 (based on a titer of 6.04*TU/mL) in DMEM medium, supplemented with 10% FBS without addition of cytokines or transduction enhancers. GFP expression was measured by FACS 48 h after transduction and correlates with the expression of the GRN transgene.

    [0850] In the vector construct, miR223 promoter activity led to the translation of one mRNA species encoding both granulin and GFP, which were co-translated into two separate proteins in a molecular ratio of 1:1, of which GFP was detected by flow cytometry analysis. In all three cell lines, transduction with the LV-miR223-GRN vector led to high expression levels of GFP: HEK293T cells, 77% GFP-positive cells (FIG. 2); THP1 cells, 88.6% GFP-positive cells (FIG. 3), immortalized human microglia GRN −/− cell line, 63.9% GFP-positive cells (FIG. 4). Accordingly, it has been successfully demonstrated that the promoter miR223 drives expression of transgenes in phagocytic cells as well as in microglia.

    [0851] Additionally, in the immortalized human microglia GRN −/− cell line, an ELISA assay measuring the release of the progranulin protein into the cell culture supernatant was performed 10 days after transduction, showing reconstitution of progranulin protein production and release from microglia cells (FIG. 5). For the ELISA, the culture medium containing DMEM supplemented with 10% FBS, was exchanged for 24 h before analyses for DMEM medium without FBS. Untransduced cells were compared to transduced cells.

    Transduction with IL-12 Transgene Vector:

    [0852] Following cell lines were analyzed: [0853] 1. THP1 cells (Human phagocyte cell line) as model for phagocyte correction; and [0854] 2. Immortalised human microglia cell line (Im-hMicro), in which the GRN gene was knocked-out (GRN−/−) as model for microglia correction.

    [0855] For transduction of cell lines, a lentiviral self-inactivating (SIN) vector was used, comprising the miR223 promoter as internal promoter, and human IL12-beta and IL12-alpha encoding cDNA subunits fused by a protein linker to one protein with IL-12 activity, as well as an mCherry-reporter linked to IL12 subunits preceded by an internal ribosomal entry site (IRES) (FIG. 6). Experiments were conducted at MOI 2 based on a titer of 3.24*10.sup.6 TU/mL, in DMEM medium, supplemented with 10% FBS without addition of cytokines or transduction enhancers. mCherry expression was measured by FACS 48 h after transduction and correlates with the expression of the IL12 transgene.

    [0856] In both cell lines, transduction with the LV-miR223-IL12 vector led to high expression levels of mCherry: THP1 cells, 98.7% mCherry-positive cells (FIG. 7), immortalized human microglia GRN −/− cell line, 99.7% GFP-positive cells (FIG. 8). Accordingly, it has been successfully demonstrated that the promoter miR223 drives expression of transgenes in phagocytic cells as well as in microglia.

    Example 8: Analysis of Additional Promoters in a Phagocytic Cell Line

    [0857] It has been demonstrated in Example 7 that the promoter miR223 can drive expression in human phagocytic cells as well as in human microglia. In this Example, the activity of additional promoters was tested in phagocytic cells.

    [0858] The following lentiviral constructs were tested: [0859] LV-miR223-hGRN-2A-EGFP-NLS-WPRE (FIG. 1) [0860] LV-miR223-TMEM119-hGRN-2A-EGFP-NLS-WPRE (FIG. 9A) [0861] LV-ITGAM-hGRN-2A-EGFP-NLS-WPRE (FIG. 9B) [0862] LV-miR223-P2RY12-hGRN-2A-EGFP-NLS-WPRE (FIG. 9C) [0863] LV-miR223-OLFML3-hGRN-2A-EGFP-NLS-WPRE (FIG. 9D)

    [0864] THP-1 cells were seeded in a 96-well plate in a density of 40.000 cells per well. Transduction was carried out right after seeding by adding the appropriate amount of virus (MOI 2) and resuspending the cells in the well. For differentiation of untransduced or transduced THP-1 cells, cells were cultured in differentiation medium (RPMI 10% FBS, 1× GlutaMAX, 1× PenStrep, 10 ng/mL PMA) and incubated for 72 hours. Adherent cells were detached with StemPro Accutase and cells were washed with PBS. For analysis, Fc receptor was blocked by FcR blocking reagent at a dilution of 1:20, cells were strained with LIVE/DEAD Fixable Violet dye (1:1000) and PE-Cy7-CD11b (1:200) followed by a analysis in a LSR II Fortessa flow cytometer. Only single and viable cells (negative for LIVE/DEAD Fixable Violet staining) were analyzed. Differentiation to macrophages was followed by quantification of PE-Cy7-CD11b staining increasing upon differentiation to macrophages. GRN/GFP co-expression was assayed by quantification of GFP fluorescent signal intensity. All experiments were performed in duplicates.

    [0865] All tested promoter variants resulted in high transgene expression in THP-1 cells. The results of this assay are summarized in FIGS. 10A-10F and FIGS. 11A-11B.

    Example 9: Analysis of Additional Promoters in Microglia

    [0866] Activity of the lentiviral constructs described in Example 8 was also tested in microglia

    [0867] For that, immortalized human microglia were seeded in a 96-well plate in a density of 40.000 cells per well. Transduction was carried out right after seeding by adding the appropriate amount of virus (MOI 2) and resuspending the cells in the well. Adherent cells were detached with TrypLE Express and cells were washed with PBS. For analysis, cells were strained with LIVE/DEAD Fixable Violet dye (1:1000) followed by a analysis in a LSR II Fortessa flow cytometer. Only single and viable cells (negative for LIVE/DEAD Fixable Violet staining) were analyzed. GRN/GFP co-expression was assayed by quantification of GFP fluorescent signal intensity.

    [0868] For quantification of granulin secretion upon gene therapy treatment of granulin-deficient human microglia, cells were seeded at a density of 150.000 cells per well in a 24-well plate in 500 μL of medium. 24 hours after seeding, the culture medium was removed and replaced by 500 μL of fresh, antibiotics-free medium. Conditioned medium was collected after 24 hours of culture, debris were removed by centrifugation at 17,000 g for 10 minutes and samples were stored at −20° C. until processing. For quantification of granulin protein concentration, supernatant samples were concentrated with Amicon Ultra-0.5 3K centrifugal filter devices and the concentration of PGRN was determined with Progranulin (human) ELISA Kit (Adipogen, cat. #AG-45A-0018YEK-KI01) following the manufacturer's protocol. Results in ng represent total ng of PGRN released by 150.000 cells into 500 μL of medium within 24 hours. All experiments were performed in duplicates.

    [0869] All tested promoter variants resulted in high transgene expression in microglia. The results of this assay are summarized in FIGS. 12A-12F and FIGS. 13A-13B. Restoration of granulin secretion in GRN−/− cells is shown in FIGS. 14A-14C.

    Example 10: Transduction of Human CD34+ Bone Marrow Cells Followed by Differentiation to Monocytes

    [0870] Commercially available human CD34+ bone marrow cells were thawed and taken into culture (day 0) in X-VIVO 20, HSA 1%, SCF 300 ng/mL, Flt3-L 300 ng/mL, TPO 100 ng/mL. On day 1 and day 2, cells were transduced with MOI3 in the presence of protamine sulfate 4 μg/mL and amphotericin B 1 μg/mL. Cells were re-plated in expansion medium (X-VIVO 20, HSA 1%, PenStrep 1×, SCF 100 ng/mL, Flt3-L 100 ng/mL, TPO 100 ng/mL) on day 3. On day 5, medium was changed to pro-myelocytes expansion medium (IMDM, HEPES 5 mM, GlutaMax 2 mM, FBS 10%, PenStrep 0.5×, SCF 100 ng/mL, IL-3 100 ng/mL). And on day 8 to pro-myelocytes differentiation medium (IMDM, HEPES 5 mM, GlutaMax 2 mM, FBS 10%, PenStrep 0.5×, SCF 50 ng/mL, IL-3 20 ng/mL, GM-CSF 20 ng/mL, M-CSF 100 ng/mL). Cells were analyzed on day 12 for lineage markers and for marker gene expression by flow cytometry analysis.

    [0871] Analysis of differentiated cells for CD11b and CD14 expression revealed more than 80% differentiated monocytes. Within the monocytic cell populations, GFP marker gene expression co-expressed with granulin was analyzed (see FIGS. 15A-15F and 16).

    Example 11: Analysis of Additional Promoters in CD34+ Cells

    [0872] Human CD34+ cells were thawed (day 0) and taken into culture (in X-VIVO 20, HSA 1%, SCF 300 ng/mL, Flt3-L 300 ng/mL, TPO 100 ng/mL). On day 1 and 2, cells were consecutively transduced three times with viral infectious particles (in presence of transduction enhancers (Amphotericin B or Lentiboost)). From end of day 2 to day 5, cells were cultured n pro-myelocytes expansion medium (IMDM, FBS 10%, PenStrep 0.5×, SCF 100 ng/mL, IL-3 100 ng/mL). On day 5, cells were re-plated in pro-myelocytes differentiation medium (IMDM, FBS 20%, PenStrep 0.5×, SCF 100 ng/mL, IL-3 100 ng/mL, G-CSF 20 ng/mL). GFP marker expression was quantified by FACS analysis on day 7 (FIGS. 17A-17D); VCNs were quantified on days 12 of culture (FIG. 18).

    Example 12: Vector Copy Number Determination in Transduced CD34+ Cells

    [0873] Cells at day 12 of the differentiation protocol were pelleted and the cell pellets were stored at −20° C. until processing. [0874] Genomic DNA was isolated from the cell pellets using the QIAamp® DNA Blood Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. [0875] VCN was determined by qPCR with the delta-delta Ct method, using FOXP2 as reference gene and WPRE for the integrated vector gene. [0876] Clone H10 carrying 2 γ integrations was used as a reference.

    [0877] The results are summarized in FIGS. 19 and 20.

    Example 13: Transgene Expression in HSPCs and Myeloid Cells

    [0878] p47phox-deficient iPSCs were transduced with lentiviral vectors encoding p47phox under control of the miR223 promoter or under control of the constitutively active SFFV promoter. Cells were differentiated to embryoid bodies and further to monocytes and macrophages. iPSCs (CD133+). CD34-positive cells as iPSC-derived homolog to haematopoietic stem cells in embryoid bodies, monocytes (CD14+) and macrophages (CD206) were analyzed for transgenic p47phox expression.

    [0879] FIG. 21 shows that the miR223 promoter is active in monocytes and macrophages, but inactive in iPSC-derived homologs to haematopoietic stem cells.