Viral vectors that specifically express therapeutic proteins in myeloid cells and microglia

Abstract

The present invention provides novel viral vectors for use in human therapy, particularly for use in the treatment of diseases or disorders that originate in or are based in the brain, particularly PGRN-associated neurodegenerative diseases or disorders, including frontotemporal degenerative diseases or disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease. The present 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 for use in the treatment of brain metastases originating from any form of breast cancer, lung cancer, colon cancer, testicular cancer, renal cancer, and melanoma or any other solid tumor, as well as any hematological tumor, including all forms of leukemia and lymphoma.

Description

[Technical Field]

[0001] The present invention provides novel viral vectors for use in human gene therapy, particularly for use in the treatment of diseases or disorders that have their origin in or are based in the brain, particularly PGRN-associated neurodegenerative diseases or disorders, including frontotemporal degenerative diseases or disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease. The present 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 metastases originating from any form of breast cancer, lung cancer, colon cancer, testicular cancer, renal cancer, and melanoma or any other solid tumor, and / or any hematological tumor, including all forms of leukemia and lymphoma. [Background technology]

[0002] Background of the Invention Gene therapy for the treatment of human disease includes any method of genetic manipulation of isolated cells ex vivo or of cells and tissues in vivo. The first clinically successful gene therapy studies were published in 2000 and addressed hematopoietic stem cells (HSCs) to treat children with life-threatening congenital immune system defects (Cavazzana-Calvo et al. (2000) Science 288: 669-72). These studies were based on ex vivo manipulation of HSCs within CD34+ bone marrow cell populations using gammaretroviral gene therapy vectors.

[0003] Retroviral gene therapy vectors are viral vectors in which single-stranded RNA containing a viral vector RNA sequence and an RNA sequence encoding a therapeutic protein sequence (i.e., a healthy copy of the patient's affected gene) is incorporated into and transported by retroviral particles. Within a single gene therapy retroviral particle, two types of RNA molecules, along with viral proteins required for reverse transcription into double-stranded DNA, are encapsulated in a capsid structure made of viral proteins. The viral capsid is enclosed in a viral envelope capable of fusing with the cell membrane of target cells during the transduction process. The retroviral proteins enable reverse transcription of the transported therapeutic RNA sequence into double-stranded DNA, which is then transported to the nucleus of the transduced cell and integrated into the genome of the transduced target cell.

[0004] Neurodegenerative dementia is an important cause of disability in middle-aged and elderly patients, resulting in loss of physical and social independence. Treatment as well as daily care at home or in nursing homes poses major challenges for families, medical staff, and society. The prevalence of dementia in people over 60 years of age is estimated to be 5-7%, affecting more than 35 million people worldwide in 2010.

[0005] Overall, up to 20% of all patients developing dementia before age 65 suffer from frontotemporal dementia (FTD). Studies have estimated prevalence rates ranging between 15 and 22 / 100,000 (Onyike & Diehl-Schmid (2013) Int Rev Psychiatry 25: 130-137), with an overall incidence of 2.7 to 4.1 new cases per 100,000 (Onyike & Diehl-Schmid (2013) Int Rev Psychiatry 25: 130-137). In two UK counties, prevalence peaked at 42.6 / 100,000 between the ages of 65 and 69. There are currently no curative treatment options for neurodegenerative dementias, including FTD.

[0006] Various estimates exist regarding the proportion of all FTD cases that are mutated in the GRN gene, which encodes the granulin precursor protein or progranulin (PGRN). These estimates range from roughly 5% (Gass et al. (2006) Hum Mol Genet. 15: 2988-3001; Le Ber et al. (2007) Hum Mutat. 28: 846-55) to 30% (Bunessi et al. (2009) Neurobiology of Disease 33: 379-385), with a penetrance of one-third in individuals under 65 years of age and two-thirds in individuals over 65 years of age.

[0007] All GRN mutations identified in patients were associated with loss-of-function and haploinsufficiency, resulting in lower levels of PGRN, making PGRN-deficient FTD suitable targets for therapeutic approaches aimed at restoring physiological levels of PGRN.

[0008] PGRN is primarily expressed in microglia, the brain-resident counterpart of tissue-resident macrophages. In three reported animal studies (Arrant et al. (2018) J Neurosci. 38: 2341-58; Arrant et al. (2017) Brain 140: 1447-65; Amado et al. (2019) Mol Ther. 27: 465-478), gene therapy approaches failed to restore PGRN expression in microglia. In a prior study, an AAV viral gene therapy vector was injected into mouse brains, resulting in PGRN expression in neurons, but not in microglia. Additionally, in a recent animal study, strong PGRN overexpression was associated with signs of neuronal toxicity.

[0009] Therefore, there is a need for alternative treatment strategies that target physiological PGRN expression in brain microglia, which differ from the earlier attempts using AAV viral vectors described above, which resulted in neuronal PGRN overexpression and neuronal toxicity. Furthermore, there is a need in the art for safer strategies to express transgenes in peripheral blood, peripheral tissues and the brain / CNS, particularly in myeloid cells after transduction of HSCs. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Cavazzana-Calvo et al. (2000) Science 288: 669-72 [Non-patent document 2] Onyike & Diehl-Schmid (2013) Int Rev Psychiatry 25: 130-137 [Non-patent document 3] Onyike & Diehl-Schmid (2013) Int Rev Psychiatry 25: 130-137 [Non-patent document 4] Gass et al. (2006) Hum Mol Genet. 15: 2988-3001 [Non-patent document 5] Le Ber et al. (2007) Hum Mutat. 28: 846-55 [Non-patent document 6] Bunessi et al. (2009) Neurobiology of Disease 33: 379-385 Summary of the Invention [Means for solving the problem]

[0011] Summary of the Invention The present invention provides such alternative and improved strategies, as defined in the various embodiments and claims described herein. DETAILED DESCRIPTION OF THE INVENTION

[0012] In certain embodiments, the present invention relates to a viral vector comprising a nucleic acid molecule encoding a therapeutic polypeptide or combination of therapeutic polypeptides under the control of a promoter or promoter fragment, wherein the promoter or promoter fragment drives expression of the therapeutic protein or combination of therapeutic proteins in myeloid cells and microglia, and the promoter or promoter fragment is inactive in hematopoietic progenitor cells and / or hematopoietic stem cells.

[0013] Specifically, the present invention is based on the surprising identification of a promoter capable of driving transgene expression in myeloid cells and microglia but silent in stem cells, particularly hematopoietic stem and progenitor cells. Such cell-specific promoters are advantageous in cell and gene therapy applications because they restrict vector activity, resulting in expression of the accompanying transgene, to differentiated target cells, i.e., myeloid cells and microglia. This is particularly important because promoter / enhancer activity in undifferentiated stem cells can result in complications such as oncogene transactivation, clonal dominance, chromosomal instability, monosomy 7, or leukemic transformation, and because transgene expression in undifferentiated stem cells can result in impaired cell function or immune responses. Therefore, the promoters of the present invention are advantageous over ubiquitous promoters because they can significantly increase the accuracy and safety of cell and gene therapy applications.

[0014] Gene therapy in hematopoietic stem cells with busulfan-mediated bone marrow conditioning has been shown to result in at least partial reconstitution of the myeloid compartment in the brain with cells derived from genetically modified hematopoietic stem cell transplants (Biffi et al. (2013) Science 341:1233158). Thus, there is a need in the art for promoters that facilitate transgene expression and restrict it to hematopoietic phagocytes and brain myeloid cells, i.e., microglia. The present inventors have surprisingly identified promoters that drive transgene expression in myeloid cells and microglia. The term "myeloid cells," as used herein, refers to a range of bone marrow-derived cell lineages, including granulocytes (neutrophils, eosinophils, and basophils), monocytes, macrophages, Kupffer cells, and mast cells. Additionally, peripheral blood dendritic cells of myeloid origin, as well as dendritic cells and macrophages derived from monocytes in vitro under appropriate culture conditions, are also included.

[0015] The term "microglial cells" or "microglia," as used herein, refers to a class of glial cells involved in mediating immune responses within the central nervous system by acting as macrophages. Microglial cells can produce exosomes, cytokines, chemokines, and neurotrophic factors, and further include different forms of microglial cells, including amoeboid microglial cells, ramified microglial cells, and reactive microglial cells. Microglial cells include reactive microglia, defined as quiescent ramified microglia that transform into a reactive, macrophage-like state and accumulate at sites of brain injury and inflammation to support tissue repair and neural regeneration. It is known in the art that hematopoietic stem cells can migrate to the brain and differentiate into macrophages, which possess many of the characteristics of microglia. Because the promoters of the present invention have been demonstrated to be active in macrophages and microglia, it is at least plausible that such promoters would also be active in hematopoietic stem cell (HSC)-derived microglia-like cells.

[0016] Myeloid cells in peripheral blood are derived exclusively from HSCs, whereas tissue-resident macrophages and microglia are thought to arise exclusively from yolk sac erythroid myeloid precursors under normal conditions. Based on this distinct origin of peripheral blood myeloid cells and microglia, it may be considered surprising that the promoters of the present invention are able to drive expression in both cell types.

[0017] Importantly, the promoters of the present invention do not drive expression in stem or progenitor cells, particularly hematopoietic stem and progenitor cells (HSPCs) (see Figure 21).

[0018] As used herein, the term "hematopoietic stem and progenitor cells" or "HSPCs" refers to cells identified by the presence of the antigenic marker CD34 (CD34+), and thus characterized as CD34+ cells and populations of such cells. In certain embodiments, the term "HSPCs" refers to cells identified by the presence of the antigenic marker CD34 (CD34+) and the absence of lineage (lin) markers, and thus characterized as CD34+ / Lin(-) cells and populations of such cells. It is recognized that populations of cells comprising CD34+ and / or Lin(-) cells also include hematopoietic progenitor cells, and therefore, for purposes of this application, the term "HSPCs" includes hematopoietic stem cells and hematopoietic progenitor cells.

[0019] Those skilled in the art will recognize methods for determining whether a promoter is active in a specific cell type. For example, to determine whether a promoter is active in a specific cell type, cells of each cell type can be transduced with a viral vector containing a fluorescent marker under the control of the promoter of interest. Whether a promoter drives the expression of a fluorescent marker can be detected, for example, by flow cytometry. That is, if the fluorescent marker can be detected in sufficient amounts in transduced cells, the promoter is said to drive the expression of a transgene in this cell type. However, if the fluorescent marker cannot be detected at all or only in very small amounts in transduced cells, the promoter is said to not drive expression in this cell type. Those skilled in the art will also recognize that cells can differentiate into other cell types during the transduction procedure. However, those skilled in the art will recognize specific combinations of cell surface markers for determining cell types before and after the transduction procedure. Those skilled in the art recognize that the statement of no promoter activity is limited by the sensitivity of promoter-driven transgene product detection, and that fluorescent proteins as transgene products, particularly EGFP, for example, with high quantum yield, can be detected with high sensitivity. Therefore, the absence of fluorescent protein detection in these types of expression experiments is accepted as an indication of a promoter below the detection limit and is most likely not biologically relevant.

[0020] The promoter of the present invention can drive the expression of a transgene encoding a therapeutic protein or a combination of therapeutic proteins in myeloid cells and microglia. That is, the promoter of the present invention is operably linked to the transgene. As used herein, the term "operably linked" refers to the functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, this refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, a promoter transcriptional regulatory sequence operably linked to a transcribed sequence is physically adjacent to the transcribed sequence, i.e., is cis-acting.

[0021] A transgene can be any nucleic acid that encodes a protein or functional RNA. Preferred examples of transgenes are discussed below.

[0022] In certain embodiments, the present invention provides a method for producing a promoter comprising: a) the miR223 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; or b) a TMEM119 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:3, SEQ ID NO:23 or SEQ ID NO:24, or a functional fragment thereof; or c) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 2, SEQ ID NO: 21 or SEQ ID NO: 22, or a functional fragment thereof; or d) the OLFML3 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, 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 set forth in SEQ ID NO: 6, or a functional fragment thereof; or f) the AIF1 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5, or a functional fragment thereof; or g) i) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:3, SEQ ID NO:23 or SEQ ID NO:24, or a functional fragment thereof; and / or ii) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 2, SEQ ID NO: 21 or SEQ ID NO: 22, or a functional fragment thereof; and / or iii) the OLFML3 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth 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 set forth in SEQ ID NO: 6, or a functional fragment thereof; and / or v) AIF1 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5, or a functional fragment thereof. a fusion promoter comprising the miR233 promoter operably linked to, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof. The present invention relates to a viral vector according to the present invention, which is

[0023] That is, in certain embodiments, the promoter is the promoter miR223 or a functional fragment thereof. The term "miR223 promoter" refers to the sequence of SEQ ID NO: 1 and / or any fragment thereof of at least 200 nucleotides, and / or any sequence having more than 95% sequence identity or a fragment thereof of at least 200 nucleotides. [ka]

[0024] Herein, the miR223 promoter was shown to drive expression in various myeloid cell types (Figures 11 and 18), but not in microglia, according to literature. Surprisingly, we detected miR223 promoter activity in an immortalized microglial cell line (Figure 13).

[0025] The miR223 promoter can have the sequence of SEQ ID NO: 1. However, those skilled in the art will recognize that fragments and / or sequence variants of SEQ ID NO: 1 can have the same characteristics as the miR223 promoter.

[0026] Thus, the term "miR223 promoter" also extends to functional fragments of the miR223 promoter. A functional fragment of the miR223 promoter is a nucleotide sequence comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, or at least 700 consecutive nucleotides of SEQ ID NO: 1. A functional fragment of the miR223 promoter is defined as driving expression in the same cell types and at comparable levels as the promoter set forth in SEQ ID NO: 1.

[0027] It should be further understood that the present invention encompasses promoters comprising two or more functional fragments of the miR223 promoter. That is, in certain embodiments, a promoter can comprise two different nucleotide sequences comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, or at least 300 consecutive nucleotides of SEQ ID NO:1. In certain embodiments, a promoter can comprise three different nucleotide sequences comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, or at least 200 consecutive nucleotides of SEQ ID NO:1. In certain embodiments, a promoter can comprise four different nucleotide sequences comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 consecutive nucleotides of SEQ ID NO:1.

[0028] The term "miR223 promoter" also extends to promoters that have the promoter functionality of the miR223 promoter. A promoter is said to have the functionality of the miR223 promoter if it drives expression in the same cell type and at comparable levels, and if it contains at least a certain degree of sequence similarity to the miR223 promoter.

[0029] A promoter is said to have a certain degree of similarity to the miR223 promoter if it comprises a contiguous stretch of at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600 or at least 700 nucleotides of SEQ ID NO:1.

[0030] Alternatively, a promoter is said to have a degree of similarity to the miR223 promoter if the promoter has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO:1.

[0031] Furthermore, a promoter can be determined to have a certain degree of similarity to the miR223 promoter if the promoter comprises a contiguous stretch of at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600 or at least 700 nucleotides of SEQ ID NO:1, said contiguous stretch having at least 95% sequence identity to the corresponding fragment of SEQ ID NO:1.

[0032] That is, in certain embodiments, a functional fragment of the miR223 promoter is a nucleic acid sequence of at least 100, 150, 200, 300, 400, 500, 600, or 700 base pairs having at least 95% identity to SEQ ID NO: 1, wherein the nucleic acid sequence has miR223 promoter activity.

[0033] The term "sequence identity" as used herein is determined by comparing two optimally aligned sequences over a comparison window, and the polynucleotide fragments in the comparison window may contain additions or deletions (e.g., gaps or overhangs) compared to a reference sequence that does not contain additions or deletions due to optimal alignment of the two sequences. The percentage of sequence identity is calculated by determining the number of positions where the same nucleic acid base or amino acid residue is present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Optimal alignment of sequences for comparison can be achieved by the local homology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (USA) 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by inspection.

[0034] In certain embodiments, the promoter is the ITGAM promoter or a functional fragment thereof. The term "ITGAM promoter" refers to the sequence of SEQ ID NO: 6 and / or any fragment thereof of at least 200 nucleotides, and / or any sequence having more than 95% sequence identity or a fragment thereof of at least 200 nucleotides. [ka]

[0035] Here, the promoter ITGAM drives expression in various myeloid cell types (FIGS. 11 and 18) and microglia (FIG. 13).

[0036] The ITGAM promoter can have the sequence of SEQ ID NO: 6. However, one of skill in the art will recognize that fragments and / or sequence variants of SEQ ID NO: 6 can have the same characteristics as the ITGAM promoter.

[0037] Thus, the term "ITGAM promoter" also extends to functional fragments of the ITGAM promoter. A functional fragment of the ITGAM promoter is a nucleotide sequence comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, or at least 700 consecutive nucleotides of SEQ ID NO: 6. A functional fragment of the ITGAM promoter drives expression in the same cell types and at comparable levels as the promoter set forth in SEQ ID NO: 6.

[0038] It should be further understood that the present invention encompasses promoters comprising two or more functional fragments of an ITGAM promoter. That is, in certain embodiments, a promoter can comprise two different nucleotide sequences comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, or at least 300 consecutive nucleotides of SEQ ID NO:6. In certain embodiments, a promoter can comprise three different nucleotide sequences comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, or at least 200 consecutive nucleotides of SEQ ID NO:6. In certain embodiments, a promoter can comprise four different nucleotide sequences comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 consecutive nucleotides of SEQ ID NO:6.

[0039] The term "ITGAM promoter" also extends to promoters that have the promoter functionality of the ITGAM promoter. A promoter is said to have the functionality of the ITGAM promoter if it drives expression in the same cell type and at comparable levels and if it contains at least a certain degree of sequence similarity to the ITGAM promoter.

[0040] A promoter is said to have a certain degree of similarity to an ITGAM promoter if the promoter comprises a contiguous stretch of at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600 or at least 700 nucleotides of SEQ ID NO:6.

[0041] Alternatively, a promoter is said to have a degree of similarity to an ITGAM promoter if the promoter has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequence shown in SEQ ID NO:6.

[0042] Furthermore, a promoter can be determined to have a certain degree of similarity to an ITGAM promoter if the promoter comprises a contiguous stretch of at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600 or at least 700 nucleotides of SEQ ID NO:6, said contiguous stretch having at least 95% sequence identity to the corresponding fragment of SEQ ID NO:6.

[0043] That is, in certain embodiments, a functional fragment of an ITGAM promoter is a nucleic acid sequence of at least 100, 150, 200, 300, 400, 500, 600, or 700 base pairs having at least 95% identity to SEQ ID NO: 6, wherein the nucleic acid sequence has ITGAM promoter activity.

[0044] In certain embodiments, the promoter is the promoter AIF1 or a functional fragment thereof. The term "AIF1 promoter" refers to the sequence of SEQ ID NO: 5 and / or any fragment thereof of at least 200 nucleotides, and / or any sequence having more than 95% sequence identity or a fragment thereof of at least 200 nucleotides. [ka]

[0045] The AIF1 promoter can have the sequence of SEQ ID NO: 5. However, those skilled in the art will recognize that fragments and / or sequence variants of SEQ ID NO: 5 can have the same characteristics as the AIF1 promoter.

[0046] Thus, the term "AIF1 promoter" also extends to functional fragments of the AIF1 promoter. A functional fragment of the AIF1 promoter is a nucleotide sequence comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, or at least 700 consecutive nucleotides of SEQ ID NO: 5. A functional fragment of the AIF1 promoter drives expression in the same cell types and at comparable levels as the promoter set forth in SEQ ID NO: 5.

[0047] It should be further understood that the present invention encompasses promoters comprising two or more functional fragments of the AIF1 promoter. That is, in certain embodiments, a promoter can comprise two different nucleotide sequences comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, or at least 300 consecutive nucleotides of SEQ ID NO:5. In certain embodiments, a promoter can comprise three different nucleotide sequences comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, or at least 200 consecutive nucleotides of SEQ ID NO:5. In certain embodiments, a promoter can comprise four different nucleotide sequences comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 consecutive nucleotides of SEQ ID NO:5.

[0048] The term "AIF1 promoter" also extends to promoters that have the promoter functionality of the AIF1 promoter. A promoter is said to have the functionality of the AIF1 promoter if it drives expression in the same cell type and at comparable levels, and if it contains at least a certain degree of sequence similarity to the AIF1 promoter.

[0049] A promoter is said to have a certain degree of similarity to the AIF1 promoter if it comprises a contiguous stretch of at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600 or at least 700 nucleotides of SEQ ID NO:5.

[0050] Alternatively, a promoter is said to have a degree of similarity to the AIF1 promoter if the promoter has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequence shown in SEQ ID NO:5.

[0051] Furthermore, a promoter can be determined to have a certain degree of similarity to the AIF1 promoter if the promoter comprises a contiguous stretch of at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600 or at least 700 nucleotides of SEQ ID NO:5, said contiguous stretch having at least 95% sequence identity to the corresponding fragment of SEQ ID NO:5.

[0052] That is, in certain embodiments, a functional fragment of the AIF1 promoter is a nucleic acid sequence of at least 100, 150, 200, 300, 400, 500, 600 or 700 base pairs having at least 95% identity to SEQ ID NO: 5, wherein the nucleic acid sequence has AIF1 promoter activity.

[0053] In certain embodiments, the promoter is promoter P2RY12 (also known as P2Y12, see https: / / www.genenames.org / data / gene-symbol-report / #! / hgnc_id / 18124) or a functional fragment thereof. The term "P2RY12 promoter" refers to the sequence of SEQ ID NO: 2 and / or any fragment thereof of at least 200 nucleotides, and / or any sequence having greater than 95% sequence identity or a fragment thereof of at least 200 nucleotides. [ka]

[0054] The P2RY12 promoter can have the sequence of SEQ ID NO: 2. However, one of skill in the art will recognize that fragments and / or sequence variants of SEQ ID NO: 2 can have the same characteristics as the P2RY12 promoter.

[0055] Thus, the term "P2RY12 promoter" also extends to functional fragments of the P2RY12 promoter. A functional fragment of the P2RY12 promoter is a nucleotide sequence comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, or at least 700 consecutive nucleotides of SEQ ID NO: 1. A functional fragment of the P2RY12 promoter drives expression in the same cell types and at comparable levels as the promoter set forth in SEQ ID NO: 2. In certain embodiments, a functional fragment of the P2RY12 promoter has the sequence of SEQ ID NO: 21. In certain embodiments, a functional fragment of the P2RY12 promoter has the sequence of SEQ ID NO: 22.

[0056] It should be further understood that the present invention encompasses promoters comprising two or more functional fragments of the P2RY12 promoter. That is, in certain embodiments, a promoter can comprise two different nucleotide sequences comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, or at least 300 consecutive nucleotides of SEQ ID NO:2. In certain embodiments, a promoter can comprise three different nucleotide sequences comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, or at least 200 consecutive nucleotides of SEQ ID NO:2. In certain embodiments, a promoter can comprise four different nucleotide sequences comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 consecutive nucleotides of SEQ ID NO:2.

[0057] The term "P2RY12 promoter" also extends to promoters that have the promoter functionality of the P2RY12 promoter. A promoter is said to have the functionality of the P2RY12 promoter if it drives expression in the same cell type and at comparable levels and if it contains at least a certain degree of sequence similarity to the P2RY12 promoter.

[0058] A promoter is said to have a degree of similarity to the P2RY12 promoter if it comprises a contiguous stretch of at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600 or at least 700 nucleotides of SEQ ID NO:2.

[0059] Alternatively, a promoter is said to have a degree of similarity to the P2RY12 promoter if the promoter has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21, or SEQ ID NO:22.

[0060] Furthermore, a promoter can be determined to have a certain degree of similarity to the P2RY12 promoter if the promoter comprises a contiguous stretch of at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600 or at least 700 nucleotides of SEQ ID NO:2, wherein said contiguous stretch has at least 95% sequence identity to the corresponding fragment of SEQ ID NO:2.

[0061] That is, in certain embodiments, a functional fragment of the P2RY12 promoter is a nucleic acid sequence of at least 100, 150, 200, 300, 400, 500, 600, or 700 base pairs having at least 95% identity to SEQ ID NO: 1, wherein the nucleic acid sequence has P2RY12 promoter activity.

[0062] That is, in certain embodiments, the promoter is the promoter TMEM119 or a functional fragment thereof. The term "TMEM119 promoter" refers to the sequence of SEQ ID NO: 3 and / or any fragment thereof of at least 200 nucleotides, and / or any sequence having greater than 95% sequence identity or a fragment thereof of at least 200 nucleotides. [ka]

[0063] The TMEM119 promoter can have the sequence of SEQ ID NO: 3. However, one of skill in the art will recognize that fragments and / or sequence variants of SEQ ID NO: 3 can have the same characteristics as the TMEM119 promoter.

[0064] Thus, the term "TMEM119 promoter" also extends to functional fragments of the TMEM119 promoter. A functional fragment of the TMEM119 promoter is a nucleotide sequence comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, or at least 700 consecutive nucleotides of SEQ ID NO:3. A functional fragment of the TMEM119 promoter drives expression in the same cell types and at comparable levels as the promoter set forth in SEQ ID NO:3. In certain embodiments, a functional fragment of the TMEM119 promoter has the sequence of SEQ ID NO:23. In certain embodiments, a functional fragment of the TMEM119 promoter has the sequence of SEQ ID NO:24.

[0065] It should be further understood that the present invention encompasses promoters comprising two or more functional fragments of the TMEM119 promoter. That is, in certain embodiments, a promoter can comprise two different nucleotide sequences comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, or at least 300 contiguous nucleotides of SEQ ID NO:3. In certain embodiments, a promoter can comprise three different nucleotide sequences comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, or at least 200 contiguous nucleotides of SEQ ID NO:3. In certain embodiments, a promoter can comprise four different nucleotide sequences comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 contiguous nucleotides of SEQ ID NO:3.

[0066] The term "TMEM119 promoter" also extends to promoters that have the promoter functionality of the TMEM119 promoter. A promoter is said to have the functionality of the TMEM119 promoter if it drives expression in the same cell type and at comparable levels, and if it contains at least a certain degree of sequence similarity to the TMEM119 promoter.

[0067] A promoter is said to have a certain degree of similarity to the TMEM119 promoter if it comprises a contiguous stretch of at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600 or at least 700 nucleotides of SEQ ID NO:3.

[0068] Alternatively, a promoter is said to have a certain degree of similarity to the TMEM119 promoter if the promoter has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO:3, SEQ ID NO:23, or SEQ ID NO:24.

[0069] Furthermore, a promoter can be determined to have a certain degree of similarity to the TMEM119 promoter if the promoter comprises a contiguous stretch of at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600 or at least 700 nucleotides of SEQ ID NO:3, said contiguous stretch having at least 95% sequence identity to the corresponding fragment of SEQ ID NO:3.

[0070] That is, in certain embodiments, a functional fragment of the TMEM119 promoter is a nucleic acid sequence of at least 100, 150, 200, 300, 400, 500, 600, or 700 base pairs having at least 95% identity to SEQ ID NO: 3, wherein the nucleic acid sequence has TMEM119 promoter activity.

[0071] In certain embodiments, the promoter is the promoter OLFML3 or a functional fragment thereof. The term "OLFML3 promoter" refers to the sequence of SEQ ID NO: 4 and / or any fragment thereof of at least 200 nucleotides, and / or any sequence having more than 95% sequence identity or a fragment thereof of at least 200 nucleotides. [ka]

[0072] The OLFML3 promoter may have the sequence of SEQ ID NO: 4. However, those skilled in the art will recognize that fragments and / or sequence variants of SEQ ID NO: 4 may have the same characteristics as the OLFML3 promoter.

[0073] Thus, the term "OLFML3 promoter" also extends to functional fragments of the OLFML3 promoter. A functional fragment of the OLFML3 promoter is a nucleotide sequence comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, or at least 700 consecutive nucleotides of SEQ ID NO:4. A functional fragment of the OLFML3 promoter drives expression in the same cell types and at comparable levels as the promoter set forth in SEQ ID NO:4. In certain embodiments, a functional fragment of the OLFML3 promoter has the sequence of SEQ ID NO:25.

[0074] It should be further understood that the present invention encompasses promoters comprising two or more functional fragments of the OLFML3 promoter. That is, in certain embodiments, a promoter can comprise two different nucleotide sequences comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, or at least 300 contiguous nucleotides of SEQ ID NO:4. In certain embodiments, a promoter can comprise three different nucleotide sequences comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, or at least 200 contiguous nucleotides of SEQ ID NO:4. In certain embodiments, a promoter can comprise four different nucleotide sequences comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 contiguous nucleotides of SEQ ID NO:4.

[0075] The term "OLFML3 promoter" also extends to promoters that have the promoter functionality of the OLFML3 promoter. A promoter is said to have the functionality of the OLFML3 promoter if it drives expression in the same cell type and at comparable levels and if it contains at least a certain degree of sequence similarity to the OLFML3 promoter.

[0076] A promoter is said to have a certain degree of similarity to the OLFML3 promoter if it comprises a contiguous stretch of at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600 or at least 700 nucleotides of SEQ ID NO:4.

[0077] Alternatively, a promoter is said to have a certain degree of similarity to the OLFML3 promoter if the promoter has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequence shown in SEQ ID NO:4 or SEQ ID NO:25.

[0078] Furthermore, a promoter can be determined to have a certain degree of similarity to the OLFML3 promoter if the promoter comprises a contiguous stretch of at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600 or at least 700 nucleotides of SEQ ID NO:4, said contiguous stretch having at least 95% sequence identity to the corresponding fragment of SEQ ID NO:4.

[0079] That is, in certain embodiments, a functional fragment of the OLFML3 promoter is a nucleic acid sequence of at least 100, 150, 200, 300, 400, 500, 600 or 700 base pairs having at least 95% identity to SEQ ID NO: 4, wherein the nucleic acid sequence has OLFML3 promoter activity.

[0080] In certain embodiments, the promoter is a fusion promoter comprising (a) the miR223 promoter, its fragment, or a promoter with miR223 functionality, and (b) a second promoter.In addition to its specific activity in myeloid cells and microglia, the miR223 promoter is attractive for use in cell and gene therapy applications due to its resistance to DNA methylation.This is important because it is known that differentiation of stem cells into myeloid cells or microglia-like cells results in extensive methylation of promoter sequences, which typically results in the silencing of transgenes in differentiated cells.Therefore, the miR223 promoter has the advantage of enabling stable transgene expression in differentiated cells originating from HSCs.

[0081] Preferably, the fusion promoter comprises a miR223 promoter, a fragment thereof, or a promoter having miR223 functionality, (a) a TMEM119 promoter, a functional fragment thereof, or a promoter having TMEM119 functionality; (b) a P2RY12 promoter, a functional fragment thereof, or a promoter having P2RY12 functionality; (c) an OLFML3 promoter, a functional fragment thereof, or a promoter having OLFML3 functionality; (d) an ITGAM promoter, a functional fragment thereof, or a promoter having ITGAM functionality; or (e) an AIF1 promoter, a functional fragment thereof, or a promoter having AIF1 functionality.

[0082] The term "miR223 fusion construct" or "miR223 fusion promoter" refers to (i) fused to the P2Y12 promoter or a promoter fragment derived from the P2Y12 promoter, the promoter fragment consisting of at least 200 nucleotides of the P2Y12 promoter sequence; or (ii) fused to the TMEM119 promoter or a promoter fragment derived from the TMEM119 promoter consisting of at least 200 nucleotides of the TMEM119 promoter sequence; or (iii) fused to the OLFML3 promoter or a promoter fragment derived from the OLFML3 promoter consisting of at least 200 nucleotides of the OLFML3 promoter sequence; or (iv) fused to the AIF1 promoter or a promoter fragment derived from the AIF1 promoter, consisting of at least 200 nucleotides of the AIF1 promoter sequence; or (v) fused to the ITGAM promoter or a promoter fragment derived from the ITGAM promoter, comprising at least 200 nucleotides of the ITGAM promoter sequence; This refers to a promoter construct consisting of the miR223 promoter.

[0083] That is, in certain embodiments, the fusion promoter comprises a miR223 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof, and a P2RY12 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 2, SEQ ID NO: 21, or SEQ ID NO: 22, or a functional fragment thereof. It should be understood that the functional fragment is preferably a promoter having miR223 and / or P2RY12 functionality, as defined above.

[0084] In certain embodiments, a fusion promoter comprising the miR223 promoter and the P2RY12 promoter can comprise the nucleotide sequence SEQ ID NO:26 or SEQ ID NO:27, or a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:26 or SEQ ID NO:27.

[0085] That is, in certain embodiments, the fusion promoter comprises a miR223 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof, and a TMEM119 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23 or SEQ ID NO: 24, or a functional fragment thereof. It is understood that the functional fragment is preferably a promoter having miR223 and / or TMEM119 functionality, as defined above.

[0086] In certain embodiments, a fusion promoter comprising the miR223 promoter and the TMEM119 promoter can comprise the nucleotide sequence SEQ ID NO:28, or a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:28.

[0087] In certain embodiments, the fusion promoter comprises a miR223 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof, and an OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. It is understood that the functional fragment is preferably a promoter having miR223 and / or OLFML3 functionality, as defined above.

[0088] In certain embodiments, a fusion promoter comprising the miR223 promoter and the OLFML3 promoter can comprise the nucleotide sequence SEQ ID NO:29, or a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:29.

[0089] In certain embodiments, the fusion promoter comprises a miR223 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof, and an ITGAM promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 6, or a functional fragment thereof. It is understood that the functional fragment is preferably a promoter having miR223 and / or ITGAM functionality, as defined above.

[0090] In certain embodiments, the fusion promoter comprises the miR223 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof, and the AIF1 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 5, or a functional fragment thereof. It is understood that the functional fragment is preferably a promoter having miR223 and / or AIF1 functionality, as defined above.

[0091] In certain embodiments, the present invention provides a method for producing a promoter comprising: a) the miR223 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth 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 set forth in SEQ ID NO: 6, or a functional fragment thereof; or c) a fusion promoter comprising the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof, operably linked to the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23 or SEQ ID NO: 24, or a functional fragment thereof. The present invention relates to a vector according to the present invention, which is

[0092] In certain embodiments, the present invention provides a method for producing a promoter comprising: a) the miR223 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; or b) a fusion promoter comprising the miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof. The present invention relates to a vector according to the present invention, which is

[0093] In certain embodiments, the present invention provides a method for producing a promoter comprising: a) the miR223 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; or b) a fusion promoter comprising the miR233 promoter operably linked to the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23 or SEQ ID NO: 24; or a functional fragment thereof; or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof. The present invention relates to a vector according to the present invention, which is

[0094] In certain embodiments, the present invention provides a method for producing a promoter comprising: a) the miR223 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; or b) a fusion promoter comprising the miR233 promoter operably linked to the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 23; or a functional fragment thereof; or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof. The present invention relates to a vector according to the present invention, which is

[0095] In certain embodiments, the present invention relates to a vector of the present invention, wherein the promoter is a fusion promoter comprising (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof, operably linked to (b) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 23; or a functional fragment thereof.

[0096] In certain embodiments, the present invention relates to a vector of the present invention, wherein the promoter is a fusion promoter comprising (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, operably linked to (b) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 23.

[0097] Within the present invention, a promoter is said to have the functionality of a particular promoter (reference promoter, for example, miR223 according to SEQ ID NO: 1) if, in addition to the sequence similarity disclosed above, it drives expression in the same cell type and at a comparable level. A promoter is said to drive expression at a comparable level if the expression level of a reporter gene from said promoter is at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the expression level of the same reporter gene from the reference promoter under comparable conditions. A large number of reporter genes suitable for determining whether two promoters have comparable activity and cell specificity are known in the art.

[0098] In a particular embodiment, the present invention relates to a viral vector according to the present invention, wherein the viral vector comprises at least one transcriptional regulatory element, wherein the at least one transcriptional regulatory element is positioned to inhibit or activate the transcriptional activity of a promoter.

[0099] That is, viral vectors can further comprise regulatory elements that allow more precise control of the expression of transgenes.The term "transcriptional regulatory element" as used herein refers to a nucleic acid fragment that can regulate the expression of one or more genes, preferably transgenes.The transcriptional regulatory element can activate or inhibit the expression of transgenes.Therefore, the transcriptional regulatory element, transgene and promoter are operably linked to each other.

[0100] It should be understood that the transcriptional regulatory element is a nucleic acid sequence adjacent to the promoter of the present invention. Preferably, the transcriptional regulatory element constitutes a binding site for a transcriptional activator or repressor. A transcriptional activator is a protein that activates the expression of a transgene when bound to a transcriptional regulatory element. A transcriptional repressor is a protein that prevents the expression of a transgene when bound to a transcriptional regulatory element.

[0101] In certain embodiments, transcriptional activators or repressors can undergo a conformational change that determines their binding strength to transcriptional regulatory elements. For example, if the activator is specifically bound by an inducer molecule, the transcriptional activator can only bind to the transcriptional regulatory element, thereby activating the expression of the transgene. Alternatively, if the activator is specifically bound by a repressor molecule, the transcriptional repressor can only bind to the transcriptional regulatory element, thereby inactivating the expression of the transgene.

[0102] Those skilled in the art will recognize various systems that can be used to control the expression of a transgene from a promoter of the invention. In a particular embodiment, the invention provides a method for the production of a transcriptional regulatory element comprising: 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 an RU486-binding domain or an abscisic acid-binding domain; iii) a steroid-binding domain, in particular an ecdysone-binding domain; or iv) Dimerization factor systems, in particular rapamycin-based or rapalog-based dimerization factor systems. The present invention relates to a viral vector according to the present invention, comprising:

[0103] That is, in certain embodiments, the inducer or repressor molecule is an antibiotic or antibiotic derivative. Specific binding of the antibiotic or antibiotic derivative to a transcriptional activator or repressor protein can induce or repress transgene expression, respectively. Well-known examples of regulatory proteins that function as transcriptional activators or repressors are proteins containing tetracycline / doxycycline binding domains, macrolide binding domains, or pristinamycin binding domains.

[0104] Alternatively, the transcriptional activator or repressor may contain a binding site for a hormone. In this case, the binding of the transcriptional activator or repressor to the transcriptional regulatory element contained in the viral vector is controlled by the binding of the hormone to the transcriptional activator or repressor. Well-known examples of regulatory proteins that function as transcriptional activators or repressors are proteins containing an RU486-binding domain or an abscisic acid-binding domain.

[0105] Alternatively, the transcriptional activator or repressor may contain a binding site for a steroid. In this case, binding of the transcriptional activator or repressor to the transcriptional regulatory element contained in the viral vector is controlled by binding of the steroid to the transcriptional activator or repressor. A well-known example of a regulatory protein that functions as a transcriptional activator or repressor is a protein containing an ecdysone-binding domain.

[0106] In certain embodiments, transgene expression can be controlled by a dimerization factor system. A dimerization factor system is a transcriptional activator consisting of two separate proteins. The first protein contains a binding site for a transcriptional regulatory element contained in a viral vector and also contains a drug-binding domain. The second protein contains another drug-binding domain and an activator or repressor domain that can induce or repress transgene expression, respectively. Activation or repression by the dimerization factor system only occurs in the presence of a dimerization factor molecule that can be specifically bound by the drug-binding domains of both proteins, thereby bringing the two proteins into close proximity so that transgene expression can be induced or repressed. Well-known examples of dimerization factor systems are rapamycin-based or rapalog-based dimerization factor systems.

[0107] In certain embodiments, the invention relates to a viral vector according to the invention, wherein the viral vector encodes a riboswitch, which controls translation of an mRNA encoding a therapeutic protein or combination of therapeutic proteins.

[0108] Instead of or in addition to a transcriptional regulatory element, the viral vector of the invention can encode a riboswitch that controls translation of the mRNA encoded by the transgene.

[0109] The term "riboswitch," as used herein, refers to a regulatory segment of an RNA polynucleotide (or DNA encoding a riboswitch). A riboswitch in the context of the present invention contains a sensor region (e.g., an aptamer) and an effector stem-loop, which together are responsible for sensing the presence of a ligand (e.g., a small molecule) and modulating the accessibility of a polyadenylation sequence located in the effector stem-loop.

[0110] In certain embodiments, the present invention provides a method for treating a cancer, comprising administering to a patient a therapeutic polypeptide comprising: i) a polypeptide that restores cellular function and / or elicits a cellular response in a cell or tissue; or ii) Polypeptides that enable and / or increase cellular targeting specificity The present invention relates to a viral vector, which is

[0111] The transgene preferably encodes one or more therapeutic proteins. Within the present invention, two main types of therapeutic proteins are envisaged.

[0112] The first type of therapeutic protein is a protein that restores the cellular function of target cells or induces a cellular response in target cells. For example, it is known that certain diseases are caused by an unnaturally low level of a specific protein or an inactive mutant variant of a specific protein. Normal protein function in such cells can be restored by delivering a transgene encoding a functional variant of the protein to such cells. Alternatively, the transgene can encode a protein that induces a cellular response in the cells expressing the transgene or in the surrounding tissues. For example, the transgene can encode a cytokine that induces a specific response in target cells. In addition, the cytokine can be secreted from the target cell so that it can induce a response not only in the target cell but also in the surrounding tissues.

[0113] That is, in certain embodiments, the present invention relates to a polypeptide that restores cellular function and / or induces a cellular response in a cell, and is selected from the group consisting of PGRN, presenilin 1, presenilin 2, IL-2, IL-12, IL-15, IL-21, IFN-alpha, IFN-alpha receptor, IFN-gamma, IFN-gamma receptor, FasL / Fas, CD11b, a selectin such as L-selectin or P-selectin, PSGL (P-selectin ligand), TRAIL, TRAIL-R, lymphotoxin beta (LT-β), LT-βR, decoy receptors 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-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-50, IL-51, IL-52, IL-53, IL- The present invention relates to a viral vector comprising at least a fragment of one or more polypeptides selected from the group consisting of CXCR3 ligands such as I-23, CXCL9 and CXCL-10, PD-1, PD-1L, PD-2 (PDC2), PD-2L, granzyme B, granulysin, CD11b, TIGIT, CD112, CD155, nitric oxide synthase, DNA methyltransferase 3b (DNMT3b), Jumonji domain-containing protein 1A (JMJD1A), somatostatin, histone deacetylase (HDAC) such as HDAC3 or HDAC9, CSF1 receptor (CSF1R), IL-34, TAM, any chemokine and chemokine receptor, and any cytokine and cytokine receptor.

[0114] In certain embodiments, the polypeptide that restores cellular function and / or induces a cellular response in a cell comprises at least a fragment of one or more polypeptides encoded by the genes MAPT, C9orf72, TDP-43, FUS, CHMP2B, VCP, SQSTM1, UBQLN2, TBK1, OPTN, SOD1, SYT11, FGF20, PM20D1, BST1, GPNMB, APP, PSEN1 and / or PSEN2.

[0115] Alternatively, the therapeutic protein may be a protein that directs target cells to a specific location. For example, the therapeutic protein may be an antigen-binding molecule that directs transduced cells to a specific cell type or tissue. For example, expression of a protein that specifically binds to a tumor antigen can direct transduced cells, such as immune cells, to tumors. In certain embodiments, the antigen-binding molecule may be or comprise an antibody. In certain embodiments, the antigen-binding molecule may be or comprise a fragment of an antibody. In certain embodiments, the antigen-binding molecule may be a chimeric antigen receptor (CAR).

[0116] Thus, in a particular embodiment, the present invention relates to a viral vector according to the present invention, wherein the polypeptide enabling and / or increasing cellular targeting specificity enables and / or increases specificity for a tumor antigen, in particular the tumor antigen is VEGF, a VEGF receptor, an antagonist of a metalloproteinase (e.g., MMP-9), CD40 / CD40L, EGFR, Annexin 1, FGFR-1, Her2, St6galnac5, MMP1-28, TIMPS1-4, melanotransferrin, alpha4beta1 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 associated antigen 72-4 (CA72-4), cancer associated antigen 19-9 (CA19-9), WT1, CD11b, L-selectin, NY-ESO-1, or a fragment thereof.

[0117] In a specific embodiment, the present invention provides a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene: 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 set forth in SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, or a functional fragment thereof. The present invention relates to a viral vector encoding the

[0118] That is, in certain embodiments, the present invention relates to a viral vector encoding Progranulin (PGRN). The terms "Progranulin," "PGRN," "Granulin," and "GRN" refer to a protein comprising the protein sequence of SEQ ID NO: 7 and / or the protein sequence of SEQ ID NO: 8 and / or the protein sequence of SEQ ID NO: 9, or any protein fragment derived from the protein sequence of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 having a length of at least 50 amino acids, or any protein sequence having more than 95% homology thereto. Nucleic acid sequences encoding said proteins are also provided herein. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0119] Progranulin is the precursor protein of granulin. Cleavage of progranulin produces various active 6-kDa granulin peptides. These smaller cleavage products are named granulin A, granulin B, granulin C, etc. Epithelin 1 and 2 are synonymous with granulin A and B, respectively. Cleavage of progranulin into granulin occurs either in the extracellular matrix or in lysosomes. Elastase, proteinase 3, and matrix metalloproteinases are proteases that can cleave progranulin into individual granulin peptides. Progranulin and granulin can be further distinguished by their hypothesized opposing roles in cells. Progranulin is associated with anti-inflammation, while cleaved granulin peptides have been implicated in pro-inflammatory behavior. Mutations in the progranulin (GRN) gene are the primary cause of familial frontotemporal dementia. This mutation results in haploinsufficiency and therefore reduced levels of progranulin and GRN-associated brain degenerative changes that manifest over years, if not decades. In such cases, progranulin levels can be restored by the viral vectors of the present invention.

[0120] A functional fragment of Progranulin is a fragment of at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids that has at least 95% sequence identity to SEQ ID NO:7, SEQ ID NO:8, and / or SEQ ID NO:9, and that has Progranulin activity. A protein is said to have Progranulin activity if it can be cleaved into at least one granulin. In certain embodiments, a protein is said to have Progranulin activity if it can be cleaved into at least one of granulin A, granulin B, and / or granulin C. In certain embodiments, a protein is said to have Progranulin activity if it can be cleaved into granulin A, granulin B, and granulin C.

[0121] In a specific embodiment, the present invention provides a viral vector encoding PGRN or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, or a functional fragment thereof, wherein one or more promoters a) a myeloid-specific promoter or a functional fragment thereof; and / or b) a microglia-specific promoter or a functional fragment thereof; and / or c) i) a first promoter that is a myeloid-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) a second promoter A fusion promoter comprising or consisting of The present invention relates to a viral vector comprising:

[0122] That is, in certain embodiments, the PGRN or functional fragment or mutant variant thereof disclosed above can be expressed from a myeloid-specific promoter or functional fragment thereof. In other embodiments, the PGRN or functional fragment or mutant variant thereof disclosed above can be expressed from a microglia-specific promoter or functional fragment thereof. In other embodiments, the PGRN or functional fragment or mutant variant thereof disclosed above can be expressed from a fusion promoter, preferably the fusion promoter comprises a myeloid-specific or microglia-specific promoter or functional fragment thereof.

[0123] The term "myeloid-specific promoter" as used herein refers to any promoter that can drive expression in myeloid cells.Those skilled in the art will recognize how to identify whether a promoter can drive expression in myeloid cells.For example, myeloid cells, such as the monocytic cell line THP-1, can be transduced with a viral vector encoding a fluorescent marker under the control of the promoter in question.If the expression of the fluorescent marker can be detected in myeloid cells after the viral vector is integrated into the genome of myeloid cells, the promoter is determined to be a myeloid-specific promoter.Myeloid-specific promoters within the meaning of the present invention include, but are not limited to, miR223 promoter, AIF1 promoter, and ITGAM promoter.

[0124] That is, in certain embodiments, the present invention provides a viral vector encoding PGRN or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, or a functional fragment thereof, wherein the myeloid-specific promoter is a) the 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 set forth in SEQ ID NO: 1, or a functional fragment thereof; or b) 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 set forth in SEQ ID NO:6, or a functional fragment thereof; or c) 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 set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a viral vector,

[0125] The term "microglia-specific promoter" as used herein refers to any promoter that can drive expression in microglia. Those skilled in the art will recognize how to identify whether a promoter can drive expression in microglia. For example, microglia, such as immortalized microglia cell lines, can be transduced with a viral vector encoding a fluorescent marker under the control of the promoter in question. If expression of the fluorescent marker can be detected in microglia after integration of the viral vector into the microglia genome, the promoter is determined to be a microglia-specific promoter. Microglia-specific promoters within the meaning of the present invention include, but are not limited to, P2RY12 promoter, TMEM119 promoter, OLFML3 promoter, ITGAM promoter, and AIF1 promoter.

[0126] That is, in certain embodiments, the present invention provides a viral vector encoding PGRN or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, or a functional fragment thereof, wherein the microglia-specific promoter is a) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:3, SEQ ID NO:23 or SEQ ID NO:24, or a functional fragment thereof; or b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; or c) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. The present invention relates to a viral vector,

[0127] In certain embodiments, the present invention relates to a viral vector encoding PGRN or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, or a functional fragment thereof, wherein the first promoter is a myeloid-specific promoter and the second promoter is a microglia-specific promoter, or vice versa.

[0128] The second promoter can be any promoter known in the art.However, in certain embodiments, the above-disclosed PGRN or its functional fragment or mutant variant can be expressed from a fusion promoter comprising a myeloid-specific promoter and a microglia-specific promoter.That is, any of the above-disclosed myeloid-specific promoters can be combined with any of the above-disclosed microglia-specific promoters in any order.

[0129] In certain embodiments, PGRN or a functional fragment or mutant variant thereof disclosed above can be expressed from a fusion promoter comprising miR223, a functional fragment thereof or a promoter having miR223 functionality, and a microglia-specific promoter.

[0130] That is, in certain embodiments, the present invention provides a viral vector encoding PGRN or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, or a functional fragment thereof, 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 set forth in SEQ ID NO:1, or a functional fragment thereof, and wherein the first promoter is i) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23 or SEQ ID NO: 24, or a functional fragment thereof. ii) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; iii) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, 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 set forth in SEQ ID NO: 6, or a functional fragment thereof; and / or v) AIF1 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a viral vector in which the vector is operably linked to

[0131] In certain embodiments, the present invention relates to a viral vector encoding PGRN or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, or a functional fragment thereof, wherein the promoter is the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:1, or a functional fragment thereof.

[0132] In certain embodiments, the present invention relates to a viral vector encoding PGRN or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, or a functional fragment thereof, wherein the first promoter is an ITGAM promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:6, or a functional fragment thereof.

[0133] In certain embodiments, the present invention relates to a viral vector encoding PGRN or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:1, or a functional fragment thereof; and (b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21, or SEQ ID NO:22, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:26 or SEQ ID NO:27.

[0134] In certain embodiments, the present invention relates to a viral vector encoding PGRN or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:1, or a functional fragment thereof; and (b) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:3, SEQ ID NO:23, or SEQ ID NO:24, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:28.

[0135] In certain embodiments, the present invention relates to a viral vector encoding PGRN or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:1, or a functional fragment thereof; and (b) the OLFML3 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:4 or SEQ ID NO:25, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:29.

[0136] In a specific embodiment, the present invention provides a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene: 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 set forth 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 set forth in SEQ ID NO: 12, or a functional fragment thereof. The present invention relates to a viral vector encoding the

[0137] That is, in certain embodiments, the present invention relates to a viral vector encoding interleukin-12 (IL-12). The term "interleukin-12" or "IL-12" refers to a protein comprising the protein sequence of SEQ ID NO: 11 and the protein sequence of SEQ ID NO: 12, or any protein fragment derived from the protein sequence of SEQ ID NO: 11 and / or SEQ ID NO: 12 having a length of at least 50 amino acids, or any protein sequence having more than 95% homology thereto. Nucleic acid sequences encoding said proteins are also provided herein. [ka] [ka] [ka]

[0138] Preferably, the viral vector of the present invention encodes both a polypeptide according to SEQ ID NO: 11 and a polypeptide according to SEQ ID NO: 12. In certain embodiments, the two IL-12 subunits, alpha and beta, can be connected via a linker. In certain embodiments, the linker has the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 34). In certain embodiments, the IL-12 encoded in the viral vector of the present invention is a single-chain IL-12 variant. Single-chain IL-12 variants have been disclosed in the art.

[0139] Interleukin-12 (IL-12) is an interleukin naturally produced by dendritic cells, macrophages, neutrophils, and human B-lymphoblastoid cells (NC-37) in response to antigen stimulation. IL-12 is composed of a bundle of four alpha helices. It is a heterodimeric cytokine encoded by two separate genes, IL-12A (p35) and IL-12B (p40). The active heterodimer (referred to as "p70") and the p40 homodimer are formed after protein synthesis. Therefore, the viral vector of the present invention preferably encodes both the alpha subunit (SEQ ID NO: 11) and beta subunit (SEQ ID NO: 12) of IL-12. Interleukin-12 (IL-12) has emerged as one of the most potent agents for antitumor immunotherapy. However, the potentially fatal toxicity associated with systemic administration of IL-12 precludes its clinical application in the form of a pure cytokine.

[0140] A functional fragment of IL-12 is a fragment of at least 50, at least 100, at least 150, or at least 200 amino acids that has at least 95% sequence identity with SEQ ID NO: 11 or SEQ ID NO: 12 and that has IL-12 activity. Assays for determining whether a protein has IL-12 activity have been described in the art, for example, by Peng et al., A single-chain IL-12 IgG3 antibody fusion protein retains antibody specificity and IL-12 bioactivity and demonstrates antitumor activity; J Immunol. 1999 Jul 1;163(1):250-8.

[0141] In a specific embodiment, the present invention provides a viral vector encoding IL-12 or a functional fragment thereof, including single chain variants thereof; or a polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:11 and / or SEQ ID NO:12, or a functional fragment thereof, wherein one or more promoters a) a myeloid-specific promoter or a functional fragment thereof; and / or b) a microglia-specific promoter or a functional fragment thereof; and / or c) i) a first promoter that is a myeloid-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) a second promoter A fusion promoter comprising or consisting of The present invention relates to a viral vector comprising:

[0142] That is, in certain embodiments, the IL-12 or functional fragment or mutant variant thereof disclosed above can be expressed from a myeloid-specific promoter or functional fragment thereof. In other embodiments, the IL-12 or functional fragment or mutant variant thereof disclosed above can be expressed from a microglia-specific promoter or functional fragment thereof. In other embodiments, the IL-12 or functional fragment or mutant variant thereof disclosed above can be expressed from a fusion promoter, preferably the fusion promoter comprises a myeloid-specific or microglia-specific promoter or functional fragment thereof.

[0143] That is, in certain embodiments, the present invention provides a viral vector encoding IL-12 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:11 and / or SEQ ID NO:12, or a functional fragment thereof, wherein the myeloid-specific promoter is a) the 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 set forth in SEQ ID NO: 1, or a functional fragment thereof; b) 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 set forth in SEQ ID NO:6, or a functional fragment thereof; c) the 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 set forth in SEQ ID NO: 5, or a functional fragment thereof; or The present invention relates to a viral vector,

[0144] In certain embodiments, the present invention provides a viral vector encoding IL-12 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:11 and / or SEQ ID NO:12, or a functional fragment thereof, wherein the microglia-specific promoter is a) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:3, SEQ ID NO:23 or SEQ ID NO:24, or a functional fragment thereof; or b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; c) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. The present invention relates to a viral vector,

[0145] In certain embodiments, the present invention relates to a viral vector encoding IL-12 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:11 and / or SEQ ID NO:12, or a functional fragment thereof, wherein the first promoter is a myeloid-specific promoter and the second promoter is a microglia-specific promoter, or vice versa.

[0146] The second promoter can be any promoter known in the art. However, in certain embodiments, the IL-12 or its functional fragment or mutant variant disclosed above can be expressed from a fusion promoter comprising a myeloid-specific promoter and a microglia-specific promoter. That is, any of the myeloid-specific promoters disclosed above can be combined with any of the microglia-specific promoters disclosed above in any order.

[0147] In certain embodiments, IL-12 or a functional fragment or mutant variant thereof disclosed above may be expressed from a fusion promoter comprising miR223, a functional fragment thereof, or a promoter having miR223 functionality, and a microglia-specific promoter.

[0148] That is, in certain embodiments, the present invention provides a viral vector encoding IL-12 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:11 and / or SEQ ID NO:12, or a functional fragment thereof, 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 set forth in SEQ ID NO:1, or a functional fragment thereof, and wherein the first promoter is i) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23 or SEQ ID NO: 24, or a functional fragment thereof. ii) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; iii) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, 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 set forth in SEQ ID NO: 6, or a functional fragment thereof; and / or v) AIF1 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a viral vector in which the vector is operably linked to

[0149] In certain embodiments, the present invention relates to a viral vector encoding IL-12 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:11 and / or SEQ ID NO:12, or a functional fragment thereof, wherein the promoter is the miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:1, or a functional fragment thereof.

[0150] In certain embodiments, the present invention relates to a viral vector encoding IL-12 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:11 and / or SEQ ID NO:12, or a functional fragment thereof, wherein the first promoter is an ITGAM promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:6, or a functional fragment thereof.

[0151] In certain embodiments, the present invention relates to a viral vector encoding IL-12 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:11 and / or SEQ ID NO:12, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:1, or a functional fragment thereof; and (b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21, or SEQ ID NO:22, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:26 or SEQ ID NO:27.

[0152] In certain embodiments, the present invention relates to a viral vector encoding IL-12 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:11 and / or SEQ ID NO:12, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:1, or a functional fragment thereof; and (b) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:3, SEQ ID NO:23, or SEQ ID NO:24, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:28.

[0153] In certain embodiments, the present invention relates to a viral vector encoding IL-12 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:11 and / or SEQ ID NO:12, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:1, or a functional fragment thereof; and (b) the OLFML3 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:4 or SEQ ID NO:25, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:29.

[0154] In a specific embodiment, the present invention provides a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene: a) interferon-gamma (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 set forth in SEQ ID NO: 10, or a functional fragment thereof. The present invention relates to a viral vector encoding the

[0155] That is, in certain embodiments, the present invention relates to a viral vector encoding interferon-gamma (IFN-gamma). The term "interferon-gamma" or "IFN-gamma" or "IFN-γ" refers to the protein sequence of SEQ ID NO: 10 and / or any sequence having greater than 95% homologous sequence identity thereto. Nucleic acid sequences encoding said proteins are also provided herein. [ka] [ka]

[0156] IFN-gamma is a dimerizing soluble cytokine that is the only member of the type II class of interferons. In humans, the IFN-gamma protein is encoded by the IFNG gene. IFN-gamma, or type II interferon, is a cytokine critical for innate and adaptive immunity against viral, some bacterial, and protozoan infections. IFN-gamma is a key activator of macrophages and an inducer of major histocompatibility complex class II molecule expression. Abnormal IFN-gamma expression is associated with numerous autoinflammatory and autoimmune diseases. The importance of IFN-gamma in the immune system arises, in part, from its ability to directly inhibit viral replication and, most importantly, from its immunostimulatory and immunomodulatory effects. IFN-gamma is primarily produced by natural killer cells (NK) and natural killer T cells (NKT) as part of the innate immune response, and by CD4 Th1 and CD8 cytotoxic T lymphocyte (CTL) effector T cells after antigen-specific immunity develops as part of the adaptive immune response. IFN-gamma is also produced by non-cytotoxic innate lymphoid cells (ILCs), a family of immune cells first discovered in the early 2010s.

[0157] IFN-gamma 1b has been approved by the U.S. Food and Drug Administration for the treatment of chronic granulomatous disease and osteopetrosis. It is under investigation for the treatment of Friedreich's ataxia. Although not officially approved, IFN-gamma has also been shown to be effective in treating patients with moderate to severe atopic dermatitis. IFN-gamma has not yet been approved for the treatment of any cancer immunotherapy. However, improved survival has been observed when IFN-gamma was administered to patients with bladder cancer and melanoma cancer. The most promising results were achieved in patients with stage 2 and 3 ovarian cancer.

[0158] A functional fragment of IFN-gamma is a fragment of at least 50, at least 100, or at least 150 amino acids that has at least 95% sequence identity with SEQ ID NO: 10 and that has IFN-gamma activity. Assays for determining whether a protein has IFN-gamma activity have been described in the art, for example, by Corstjens et al., A user-friendly, highly sensitive assay to detect the IFN-gamma secretion by T cells; Clin Biochem. 2008 Apr; 41(6): 440-444.

[0159] In certain embodiments, the present invention provides a viral vector encoding IFN-gamma or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, wherein one or more promoters are a) a myeloid-specific promoter or a functional fragment thereof; and / or b) a microglia-specific promoter or a functional fragment thereof; and / or c) i) a first promoter that is a myeloid-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) a second promoter A fusion promoter comprising or consisting of The present invention relates to a viral vector comprising:

[0160] That is, in certain embodiments, the IFN-gamma or functional fragment or mutant variant thereof disclosed above can be expressed from a myeloid-specific promoter or functional fragment thereof. In other embodiments, the IFN-gamma or functional fragment or mutant variant thereof disclosed above can be expressed from a microglia-specific promoter or functional fragment thereof. In other embodiments, the IFN-gamma or functional fragment or mutant variant thereof disclosed above can be expressed from a fusion promoter, preferably the fusion promoter comprises a myeloid-specific or microglia-specific promoter or functional fragment thereof.

[0161] That is, in certain embodiments, the present invention provides a viral vector encoding IFN-gamma or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, wherein the myeloid-specific promoter is a) the 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 set forth in SEQ ID NO: 1, or a functional fragment thereof; b) 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 set forth in SEQ ID NO:6, or a functional fragment thereof; c) 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 set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a viral vector,

[0162] In certain embodiments, the present invention provides a viral vector encoding IFN-gamma or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, wherein the microglia-specific promoter is a) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:3, SEQ ID NO:23 or SEQ ID NO:24, or a functional fragment thereof; or b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; or c) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. The present invention relates to a viral vector,

[0163] In certain embodiments, the present invention relates to a viral vector encoding IFN-gamma or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, wherein the first promoter is a myeloid-specific promoter and the second promoter is a microglia-specific promoter, or vice versa.

[0164] The second promoter can be any promoter known in the art.However, in certain embodiments, the IFN-gamma or its functional fragment or mutant variant disclosed above can be expressed from a fusion promoter comprising a myeloid-specific promoter and a microglia-specific promoter.That is, any of the myeloid-specific promoters disclosed above can be combined with any of the microglia-specific promoters disclosed above in any order.

[0165] In certain embodiments, IFN-gamma or a functional fragment or mutant variant thereof disclosed above can be expressed from a fusion promoter comprising miR223, a functional fragment thereof, or a promoter having miR223 functionality, and a microglia-specific promoter.

[0166] That is, in certain embodiments, the present invention provides a viral vector encoding IFN-gamma or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, wherein the first promoter is the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof, and wherein the first promoter is ii) the TMEM119 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:3, SEQ ID NO:23 or SEQ ID NO:24, or a functional fragment thereof. i) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; iii) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, 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 set forth in SEQ ID NO: 6, or a functional fragment thereof; and / or v) AIF1 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a viral vector in which the vector is operably linked to

[0167] In certain embodiments, the present invention relates to a viral vector encoding IFN-gamma or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, wherein the promoter is the miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof.

[0168] In certain embodiments, the present invention relates to a viral vector encoding IFN-gamma or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, wherein the first promoter is an ITGAM promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 6, or a functional fragment thereof.

[0169] In certain embodiments, the present invention relates to a viral vector encoding IFN-gamma or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 2, SEQ ID NO: 21, or SEQ ID NO: 22, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26 or SEQ ID NO: 27.

[0170] In certain embodiments, the present invention relates to a viral vector encoding IFN-gamma or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28.

[0171] In certain embodiments, the present invention relates to a viral vector encoding IFN-gamma or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the OLFML3 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29.

[0172] In a specific embodiment, the present invention provides a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene: 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 set forth in SEQ ID NO: 13, or a functional fragment thereof. The present invention relates to a viral vector encoding the

[0173] That is, in certain embodiments, the present invention relates to a viral vector encoding granulocyte-macrophage colony-stimulating factor (GM-CSF). The term "GM-CSF" refers to the protein sequence of SEQ ID NO: 13 and / or any sequence having greater than 95% homologous sequence identity thereto. Nucleic acid sequences encoding said proteins are also provided herein. [ka]

[0174] Granulocyte-macrophage colony-stimulating factor (GM-CSF), also known as colony-stimulating factor 2 (CSF2), is a monomeric glycoprotein secreted by macrophages, T cells, mast cells, natural killer cells, endothelial cells, and fibroblasts, where it functions as a cytokine. Naturally occurring pharmaceutical analogs of GM-CSF are called sargramostim and molgramostim. Unlike granulocyte-colony-stimulating factor (GCSF), which specifically promotes neutrophil proliferation and maturation, GM-CSF affects many more cell types, particularly macrophages and eosinophils. GM-CSF is a monomeric glycoprotein that functions as a cytokine—it is a leukocyte growth factor. GM-CSF stimulates stem cells to produce granulocytes (neutrophils, eosinophils, and basophils) and monocytes. Monocytes exit the circulation and migrate to tissues, where they mature into macrophages and dendritic cells. It is thus part of the immune / inflammatory cascade in which activation of small numbers of macrophages can rapidly lead to an increase in their numbers, a process crucial for fighting infection.

[0175] A functional fragment of GM-CSF is a fragment of at least 50, at least 100, at least 110, at least 120, at least 130, or at least 140 amino acids that has at least 95% sequence identity with SEQ ID NO: 13 and that has GM-CSF activity. Assays for determining whether a protein has GM-CSF activity have been described in the art, for example, by Singh et al., GM-CSF Enhances Macrophage Glycolytic Activity In Vitro and Improves Detection of Inflammation In Vivo; J Nucl Med. 2016 Sep;57(9):1428-35. doi: 10.2967 / jnumed.115.167387. Epub 2016 Apr 14.

[0176] In a specific embodiment, the present invention provides a viral vector encoding GM-CSF or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 13, or a functional fragment thereof, wherein one or more promoters are a) a myeloid-specific promoter or a functional fragment thereof; and / or b) a microglia-specific promoter or a functional fragment thereof; and / or c) i) a first promoter that is a myeloid-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) a second promoter A fusion promoter comprising or consisting of The present invention relates to a viral vector comprising:

[0177] That is, in certain embodiments, the GM-CSF or functional fragment or mutant variant thereof disclosed above can be expressed from a myeloid-specific promoter or functional fragment thereof. In other embodiments, the GM-CSF or functional fragment or mutant variant thereof disclosed above can be expressed from a microglia-specific promoter or functional fragment thereof. In other embodiments, the GM-CSF or functional fragment or mutant variant thereof disclosed above can be expressed from a fusion promoter, preferably the fusion promoter comprises a myeloid-specific or microglia-specific promoter or functional fragment thereof.

[0178] That is, in certain embodiments, the present invention provides a viral vector encoding GM-CSF or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 13, or a functional fragment thereof, wherein the myeloid-specific promoter is a) the 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 set forth in SEQ ID NO: 1, or a functional fragment thereof; b) 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 set forth in SEQ ID NO:5, or a functional fragment thereof; b) 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 set forth in SEQ ID NO:6, or a functional fragment thereof. The present invention relates to a viral vector,

[0179] In certain embodiments, the present invention provides a viral vector encoding GM-CSF or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 13, or a functional fragment thereof, wherein the microglia-specific promoter is a) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:3, SEQ ID NO:23 or SEQ ID NO:24, or a functional fragment thereof; or b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; c) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. The present invention relates to a viral vector,

[0180] In certain embodiments, the present invention relates to a viral vector encoding GM-CSF or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 13, or a functional fragment thereof, wherein the first promoter is a myeloid-specific promoter and the second promoter is a microglia-specific promoter, or vice versa.

[0181] The second promoter can be any promoter known in the art.However, in certain embodiments, the GM-CSF or its functional fragment or mutant variant disclosed above can be expressed from a fusion promoter comprising a myeloid-specific promoter and a microglia-specific promoter.That is, any of the myeloid-specific promoters disclosed above can be combined with any of the microglia-specific promoters disclosed above in any order.

[0182] In certain embodiments, GM-CSF or a functional fragment or mutant variant thereof disclosed above can be expressed from a fusion promoter comprising miR223, a functional fragment thereof, or a promoter having miR223 functionality, and a microglia-specific promoter.

[0183] That is, in certain embodiments, the present invention provides a viral vector encoding GM-CSF or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 13, or a functional fragment thereof, wherein the first promoter is the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof, and wherein the first promoter is i) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23 or SEQ ID NO: 24, or a functional fragment thereof. ii) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; iii) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, 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 set forth in SEQ ID NO: 6, or a functional fragment thereof; and / or v) AIF1 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a viral vector in which the vector is operably linked to

[0184] In certain embodiments, the present invention relates to a viral vector encoding GM-CSF or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 13, or a functional fragment thereof, wherein the promoter is the miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof.

[0185] In certain embodiments, the present invention relates to a viral vector encoding GM-CSF or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 13, or a functional fragment thereof, wherein the first promoter is an ITGAM promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 6, or a functional fragment thereof.

[0186] In certain embodiments, the present invention relates to a viral vector encoding GM-CSF or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 13, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 2, SEQ ID NO: 21, or SEQ ID NO: 22, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26 or SEQ ID NO: 27.

[0187] In certain embodiments, the present invention relates to a viral vector encoding GM-CSF or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 13, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28.

[0188] In certain embodiments, the present invention relates to a viral vector encoding GM-CSF or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 13, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the OLFML3 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29.

[0189] In a specific embodiment, the present invention provides a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene: 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 set forth in SEQ ID NO: 14, or a functional fragment thereof. The present invention relates to a viral vector encoding the

[0190] That is, in certain embodiments, the present invention relates to a viral vector encoding granulocyte colony-stimulating factor (G-CSF). The term "G-CSF" refers to the protein sequence of SEQ ID NO: 14 and / or any sequence having a sequence identity of greater than 95% homology thereto. Nucleic acid sequences encoding said proteins are also provided herein. [ka]

[0191] Granulocyte colony-stimulating factor (G-CSF or GCSF), also known as colony-stimulating factor 3 (CSF3), is a glycoprotein that stimulates the bone marrow to produce and release granulocytes and stem cells into the bloodstream. Functionally, it is a type of colony-stimulating factor, a cytokine and hormone, produced by many different tissues. Naturally occurring pharmaceutical analogs of G-CSF are called filgrastim and lenograstim. G-CSF also stimulates the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils.

[0192] Chemotherapy can cause bone marrow suppression and unacceptably low levels of white blood cells (leukopenia), making patients more susceptible to infection and sepsis. G-CSF stimulates the production of granulocytes, a type of white blood cell. In oncology and hematology, recombinant forms of G-CSF are used by certain cancer patients to accelerate recovery from post-chemotherapy neutropenia, reduce mortality, and allow for more intensive treatment regimens. G-CSF has been shown to reduce inflammation, lower amyloid beta load, and reverse cognitive impairment in mouse models of Alzheimer's disease. Due to its neuroprotective properties, G-CSF is currently being investigated in phase IIb clinical trials for cerebral ischemia, and several clinical pilot studies have been published for other neurological diseases, such as amyotrophic lateral sclerosis.

[0193] A functional fragment of G-CSF is a fragment of at least 50, at least 100, at least 120, at least 140, at least 160, or at least 180 amino acids that has at least 95% sequence identity to SEQ ID NO: 14 and that has G-CSF activity. Assays for determining whether a protein has G-CSF activity have been described in the art, for example, by Mickiene et al., Human granulocyte-colony stimulating factor (G-CSF) / stem cell factor (SCF) fusion proteins: design, characterization and activity; Peer J. 2020; 8: e9788.

[0194] In a specific embodiment, the present invention provides a viral vector encoding G-CSF or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 14, or a functional fragment thereof, wherein one or more promoters a) a myeloid-specific promoter or a functional fragment thereof; and / or b) a microglia-specific promoter or a functional fragment thereof; and / or c) i) a first promoter that is a myeloid-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) a second promoter A fusion promoter comprising or consisting of The present invention relates to a viral vector comprising:

[0195] That is, in certain embodiments, the G-CSF or functional fragment or mutant variant thereof disclosed above can be expressed from a myeloid-specific promoter or functional fragment thereof. In other embodiments, the G-CSF or functional fragment or mutant variant thereof disclosed above can be expressed from a microglia-specific promoter or functional fragment thereof. In other embodiments, the G-CSF or functional fragment or mutant variant thereof disclosed above can be expressed from a fusion promoter, preferably the fusion promoter comprises a myeloid-specific or microglia-specific promoter or functional fragment thereof.

[0196] That is, in certain embodiments, the present invention provides a viral vector encoding G-CSF or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 14, or a functional fragment thereof, wherein the myeloid-specific promoter is a) the 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 set forth in SEQ ID NO: 1, or a functional fragment thereof; 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 set forth in SEQ ID NO:6, or a functional fragment thereof; or b) 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 set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a viral vector,

[0197] In certain embodiments, the present invention provides a viral vector encoding G-CSF or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 14, or a functional fragment thereof, wherein the microglia-specific promoter is a) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:3, SEQ ID NO:23 or SEQ ID NO:24, or a functional fragment thereof; b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; or c) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. The present invention relates to a viral vector,

[0198] In certain embodiments, the present invention relates to a viral vector encoding G-CSF or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 14, or a functional fragment thereof, wherein the first promoter is a myeloid-specific promoter and the second promoter is a microglia-specific promoter, or vice versa.

[0199] The second promoter can be any promoter known in the art.However, in certain embodiments, the G-CSF or its functional fragment or mutant variant disclosed above can be expressed from a fusion promoter comprising a myeloid-specific promoter and a microglia-specific promoter.That is, any of the myeloid-specific promoters disclosed above can be combined with any of the microglia-specific promoters disclosed above in any order.

[0200] In certain embodiments, G-CSF or a functional fragment or mutant variant thereof disclosed above can be expressed from a fusion promoter comprising miR223, a functional fragment thereof, or a promoter having miR223 functionality, and a microglia-specific promoter.

[0201] That is, in certain embodiments, the present invention provides a viral vector encoding G-CSF or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 14, or a functional fragment thereof, wherein the first promoter is the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof, and wherein the first promoter is i) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23 or SEQ ID NO: 24, or a functional fragment thereof. ii) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; iii) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, 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 set forth in SEQ ID NO: 6, or a functional fragment thereof; and / or v) AIF1 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a viral vector in which the vector is operably linked to

[0202] In certain embodiments, the present invention relates to a viral vector encoding G-CSF or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 14, or a functional fragment thereof, wherein the promoter is the miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof.

[0203] In certain embodiments, the present invention relates to a viral vector encoding G-CSF or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 14, or a functional fragment thereof, wherein the first promoter is an ITGAM promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 6, or a functional fragment thereof.

[0204] In certain embodiments, the present invention relates to a viral vector encoding G-CSF or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 14, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 2, SEQ ID NO: 21, or SEQ ID NO: 22, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26 or SEQ ID NO: 27.

[0205] In certain embodiments, the present invention relates to a viral vector encoding G-CSF or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 14, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28.

[0206] In certain embodiments, the present invention relates to a viral vector encoding G-CSF or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 14, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the OLFML3 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29.

[0207] In a specific embodiment, the present invention provides a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene: a) GM-CSF and IFN-gamma, or functional fragments thereof; or b) a first polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, and a second polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 13, or a functional fragment thereof; or c) a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 15 The present invention relates to a viral vector encoding the

[0208] That is, in certain embodiments, the present invention relates to a viral vector encoding a GM-CSF-IFN-gamma co-expression construct. The co-expression construct can encode GM-CSF as defined above, or any functional fragment or variant thereof. The co-expression construct can further encode IFN-gamma as defined above, or any functional fragment or variant thereof. GM-CSF and IFN-gamma, and their functional fragments or variants, can be expressed as separate polypeptides from the viral vector of the present invention. In certain embodiments, GM-CSF and IFN-gamma can be expressed as a fusion protein.

[0209] Exemplary nucleic acid sequences for GM-CSF - INF-gamma co-expression can include the following nucleic acid sequences: [ka] [ka]

[0210] In certain embodiments, the present invention provides a viral vector encoding a GM-CSF - INF-gamma co-expression construct or a functional fragment thereof; or a first polypeptide encoding a sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 15, or having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, and a second polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 13, or a functional fragment thereof, wherein the one or more promoters a) a myeloid-specific promoter or a functional fragment thereof; and / or b) a microglia-specific promoter or a functional fragment thereof; and / or c) i) a first promoter that is a myeloid-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) a second promoter A fusion promoter comprising or consisting of The present invention relates to a viral vector comprising:

[0211] That is, in certain embodiments, the GM-CSF-IFN-gamma co-expression constructs, or functional fragments or mutant variants thereof, disclosed above, can be expressed from a myeloid-specific promoter or functional fragment thereof. In other embodiments, the GM-CSF-IFN-gamma co-expression constructs, or functional fragments or mutant variants thereof, disclosed above, can be expressed from a microglia-specific promoter or functional fragment thereof. In other embodiments, the GM-CSF-IFN-gamma co-expression constructs, or functional fragments or mutant variants thereof, disclosed above, can be expressed from a fusion promoter, preferably comprising a myeloid-specific or microglia-specific promoter or functional fragment thereof.

[0212] That is, in certain embodiments, the present invention provides a viral vector encoding a GM-CSF - INF-gamma co-expression construct or a functional fragment thereof; or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 15, or encoding a first polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, and a second polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 13, or a functional fragment thereof, wherein the myeloid specific promoter is a) the 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 set forth in SEQ ID NO: 1, or a functional fragment thereof; b) 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 set forth in SEQ ID NO:6, or a functional fragment thereof; c) the 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 set forth in SEQ ID NO: 5, or a functional fragment thereof; or The present invention relates to a viral vector,

[0213] In certain embodiments, the present invention provides a viral vector encoding a GM-CSF - INF-gamma co-expression construct or a functional fragment thereof; or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 15, or encoding a first polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, and a second polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 13, or a functional fragment thereof, wherein the microglia-specific promoter is a) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:3, SEQ ID NO:23 or SEQ ID NO:24, or a functional fragment thereof; b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; or c) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. The present invention relates to a viral vector,

[0214] In certain embodiments, the present invention relates to a viral vector encoding a GM-CSF - INF-gamma co-expression construct or a functional fragment thereof; or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 15, or encoding a first polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, and a second polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 13, wherein the first promoter is a myeloid-specific promoter and the second promoter is a microglia-specific promoter, or vice versa.

[0215] The second promoter can be any promoter known in the art. However, in certain embodiments, the GM-CSF-IFN-gamma co-expression constructs disclosed above, or functional fragments or mutant variants thereof, can be expressed from a fusion promoter comprising a myeloid-specific promoter and a microglia-specific promoter. That is, any of the myeloid-specific promoters disclosed above can be combined with any of the microglia-specific promoters disclosed above, in any order.

[0216] In certain embodiments, the GM-CSF - INF-gamma co-expression constructs disclosed above, or functional fragments or mutant variants thereof, can be expressed from a fusion promoter comprising miR223, a functional fragment thereof, or a promoter having miR223 functionality, and a microglia-specific promoter.

[0217] That is, in certain embodiments, the present invention provides a viral vector encoding a GM-CSF - INF-gamma co-expression construct or a functional fragment thereof; or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 15, or a first polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, and a second polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 13, or a functional fragment thereof, wherein the first promoter is the miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof, i) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23 or SEQ ID NO: 24, or a functional fragment thereof. ii) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; iii) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, 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 set forth in SEQ ID NO: 6, or a functional fragment thereof; and / or v) AIF1 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a viral vector in which the vector is operably linked to

[0218] In certain embodiments, the present invention relates to a viral vector encoding a GM-CSF-INF-gamma co-expression construct or a functional fragment thereof; or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 15, or a first polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, and a second polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 13, wherein the promoter is the miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof.

[0219] In certain embodiments, the present invention relates to a viral vector encoding a GM-CSF-INF-gamma co-expression construct or a functional fragment thereof; or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 15, or a first polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, and a second polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 13, wherein the first promoter is an ITGAM promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 6, or a functional fragment thereof.

[0220] In certain embodiments, the present invention provides a virus encoding a GM-CSF - INF-gamma co-expression construct or a functional fragment thereof; or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 15, or a first polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, and a second polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 13, or a functional fragment thereof. The present invention relates to a viral vector, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 2, SEQ ID NO: 21, or SEQ ID NO: 22, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26 or SEQ ID NO: 27.

[0221] In certain embodiments, the present invention provides a virus encoding a GM-CSF - INF-gamma co-expression construct or a functional fragment thereof; or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 15, or a first polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, and a second polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 13, or a functional fragment thereof. The present invention relates to a viral vector, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28.

[0222] In certain embodiments, the present invention provides a GM-CSF - INF-gamma co-expression construct; or a viral vector encoding a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 15, or encoding a first polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, and a second polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 13, or a functional fragment thereof. The present invention relates to a viral vector, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the OLFML3 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29.

[0223] In a specific embodiment, the present invention provides a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene: a) G-CSF and IFN-gamma, or functional fragments thereof; or b) a first polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, and a second polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 14, or a functional fragment thereof; or c) a polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 16 The present invention relates to a viral vector encoding the

[0224] That is, in certain embodiments, the present invention relates to a viral vector encoding a G-CSF-IFN-gamma co-expression construct. The co-expression construct can encode G-CSF as defined above, or any functional fragment or variant thereof. The co-expression construct can further encode IFN-gamma as defined above, or any functional fragment or variant thereof. G-CSF and IFN-gamma, and their functional fragments or variants, can be expressed as separate polypeptides from the viral vector of the present invention. In certain embodiments, G-CSF and IFN-gamma can be expressed as a fusion protein.

[0225] Nucleic acid sequences encoding the co-expression constructs are also provided herein.

[0226] An exemplary G-CSF-IFN-gamma co-expression construct can include the following nucleic acid sequence: [ka]

[0227] In a specific embodiment, the present invention provides a viral vector encoding a G-CSF - INF-gamma co-expression construct or a functional fragment thereof; or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 16, or a functional fragment thereof, or encoding a first polypeptide comprising having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, and a second polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 14, or a functional fragment thereof, wherein the one or more promoters a) a myeloid-specific promoter or a functional fragment thereof; and / or b) a microglia-specific promoter or a functional fragment thereof; and / or c) i) a first promoter that is a myeloid-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) a second promoter A fusion promoter comprising or consisting of The present invention relates to a viral vector comprising:

[0228] That is, in certain embodiments, the G-CSF-IFN-gamma co-expression constructs or functional fragments or mutant variants thereof disclosed above can be expressed from a myeloid-specific promoter or functional fragments thereof. In other embodiments, the G-CSF-IFN-gamma co-expression constructs or functional fragments or mutant variants thereof disclosed above can be expressed from a microglia-specific promoter or functional fragments thereof. In other embodiments, the G-CSF-IFN-gamma co-expression constructs or functional fragments or mutant variants thereof disclosed above can be expressed from a fusion promoter, preferably comprising a myeloid-specific or microglia-specific promoter or functional fragments thereof.

[0229] That is, in certain embodiments, the present invention provides a viral vector encoding a G-CSF - INF-gamma co-expression construct or a functional fragment thereof; or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 16, or a functional fragment thereof, or encoding a first polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, and a second polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 14, or a functional fragment thereof, wherein the myeloid specific promoter is a) the 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 set forth in SEQ ID NO: 1, or a functional fragment thereof; b) 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 set forth in SEQ ID NO:6, or a functional fragment thereof; or c) 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 set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a viral vector,

[0230] In a particular embodiment, the present invention provides a viral vector encoding a G-CSF - INF-gamma co-expression construct or a functional fragment thereof; or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 16 or a functional fragment thereof, or encoding a first polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, and a second polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 14, or a functional fragment thereof, wherein the microglia-specific promoter is a) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:3, SEQ ID NO:23 or SEQ ID NO:24, or a functional fragment thereof; b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; or c) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. The present invention relates to a viral vector,

[0231] In a specific embodiment, the present invention relates to a viral vector encoding a G-CSF - INF-gamma co-expression construct or a functional fragment thereof; or a nucleic acid sequence or functional fragment thereof having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 16, or a first polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, and a second polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 14, wherein the first promoter is a myeloid-specific promoter and the second promoter is a microglia-specific promoter, or vice versa.

[0232] The second promoter can be any promoter known in the art. However, in certain embodiments, the G-CSF-IFN-gamma co-expression construct disclosed above, or its functional fragment or mutant variant, can be expressed from a fusion promoter comprising a myeloid-specific promoter and a microglia-specific promoter. That is, any of the myeloid-specific promoters disclosed above can be combined with any of the microglia-specific promoters disclosed above, in any order.

[0233] In certain embodiments, the G-CSF-IFN-gamma co-expression constructs disclosed above, or functional fragments or mutant variants thereof, can be expressed from a fusion promoter comprising miR223, a functional fragment thereof, or a promoter having miR223 functionality, and a microglia-specific promoter.

[0234] That is, in a specific embodiment, the present invention provides a viral vector encoding a G-CSF - INF-gamma co-expression construct or a functional fragment thereof; or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 16 or a functional fragment thereof, or encoding a first polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, and a second polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 14, or a functional fragment thereof, wherein the first promoter is the miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof, i) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23 or SEQ ID NO: 24, or a functional fragment thereof. ii) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; iii) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, 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 set forth in SEQ ID NO: 6, or a functional fragment thereof; and / or v) AIF1 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a viral vector in which the vector is operably linked to

[0235] In certain embodiments, the present invention relates to a viral vector encoding a G-CSF-INF-gamma co-expression construct or a functional fragment thereof; or a nucleic acid sequence or functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 16, or a first polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, and a second polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 14, wherein the promoter is the miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof.

[0236] In certain embodiments, the present invention relates to a viral vector encoding a G-CSF-INF-gamma co-expression construct or a functional fragment thereof; or a nucleic acid sequence or functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 16, or a first polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, and a second polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 14, wherein the first promoter is an ITGAM promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 6, or a functional fragment thereof.

[0237] In certain embodiments, the present invention provides a G-CSF - INF-gamma co-expression construct or a functional fragment thereof; or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 16 or a functional fragment thereof, encoding a first polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, and a second polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 14, or a functional fragment thereof. The present invention relates to a viral vector for encoding a miR233 promoter, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 2, SEQ ID NO: 21, or SEQ ID NO: 22, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26 or SEQ ID NO: 27.

[0238] In certain embodiments, the present invention provides a G-CSF - INF-gamma co-expression construct or a functional fragment thereof; or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 16 or a functional fragment thereof, encoding a first polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, and a second polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 14, or a functional fragment thereof. The present invention relates to a viral vector for encoding a miR233 promoter, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28.

[0239] In certain embodiments, the present invention provides a G-CSF - INF-gamma co-expression construct or a functional fragment thereof; or a first polypeptide encoding a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 16 or a functional fragment thereof, or having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, and a second polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 14, or a functional fragment thereof. The present invention relates to a viral vector encoding a fragment, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the OLFML3 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29.

[0240] In a specific embodiment, the present invention provides a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene: 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 set forth in SEQ ID NO: 17, or a functional fragment thereof. The present invention relates to a viral vector encoding the

[0241] That is, in certain embodiments, the present invention relates to a viral vector encoding interleukin-2 (IL-2). The term "IL-2" refers to the protein sequence of SEQ ID NO: 17 and / or any sequence having greater than 95% homologous sequence identity thereto. Nucleic acid sequences encoding said proteins are also provided herein. [ka]

[0242] Interleukin-2 (IL-2) is an interleukin, a type of cytokine signaling molecule in the immune system. It is a 15.5-16 kDa protein that regulates the activity of immune-responsible white blood cells (leukocytes, often lymphocytes). IL-2 is part of the body's natural response to microbial infection, distinguishing between foreign ("non-self") and "self." IL-2 mediates its effects by binding to the IL-2 receptor expressed by lymphocytes. The primary source of IL-2 is activated CD4+ and CD8+ T cells.

[0243] Aldesleukin is a form of recombinant interleukin-2. It is produced using recombinant DNA technology and sold as a protein therapeutic under the trademark Proleukin. It has been approved by the Food and Drug Administration (FDA) and in several European countries for the treatment of cancer (malignant melanoma, renal cell carcinoma) in large intermittent doses and has been used on a large scale in continuous doses.

[0244] A functional fragment of IL-2 is a fragment of at least 50, at least 100, at least 110, at least 120, at least 130, or at least 140 amino acids that has at least 95% sequence identity with SEQ ID NO: 17 and has IL-2 activity. Assays for determining whether a protein has IL-2 activity have been described in the art, for example, by Leivestad et al., "A simple and sensitive bioassay for the detection of IL-2 activity; J Immunol Methods. 1988 Nov 10;114(1-2):95-9. doi: 10.1016 / 0022-1759(88)90159-7."

[0245] In a specific embodiment, the present invention provides a viral vector encoding IL-2 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 17, or a functional fragment thereof, wherein one or more promoters are a) a myeloid-specific promoter or a functional fragment thereof; and / or b) a microglia-specific promoter or a functional fragment thereof; and / or c) i) a first promoter that is a myeloid-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) a second promoter A fusion promoter comprising or consisting of The present invention relates to a viral vector comprising:

[0246] That is, in certain embodiments, the IL-2 or functional fragment or mutant variant thereof disclosed above can be expressed from a myeloid-specific promoter or functional fragment thereof. In other embodiments, the IL-2 or functional fragment or mutant variant thereof disclosed above can be expressed from a microglia-specific promoter or functional fragment thereof. In other embodiments, the IL-2 or functional fragment or mutant variant thereof disclosed above can be expressed from a fusion promoter, preferably the fusion promoter comprises a myeloid-specific or microglia-specific promoter or functional fragment thereof.

[0247] That is, in certain embodiments, the present invention provides a viral vector encoding IL-2 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 17, or a functional fragment thereof, wherein the myeloid-specific promoter is a) the 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 set forth in SEQ ID NO: 1, or a functional fragment thereof; b) 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 set forth in SEQ ID NO:6, or a functional fragment thereof; or c) 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 set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a viral vector,

[0248] In certain embodiments, the present invention provides a viral vector encoding IL-2 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 17, or a functional fragment thereof, wherein the microglia-specific promoter is a) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:3, SEQ ID NO:23 or SEQ ID NO:24, or a functional fragment thereof; b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; or c) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. The present invention relates to a viral vector,

[0249] In certain embodiments, the present invention relates to a viral vector encoding IL-2 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 17, or a functional fragment thereof, wherein the first promoter is a myeloid-specific promoter and the second promoter is a microglia-specific promoter, or vice versa.

[0250] The second promoter can be any promoter known in the art.However, in certain embodiments, the IL-2 or its functional fragment or mutant variant disclosed above can be expressed from a fusion promoter comprising a myeloid-specific promoter and a microglia-specific promoter.That is, any of the myeloid-specific promoters disclosed above can be combined with any of the microglia-specific promoters disclosed above in any order.

[0251] In certain embodiments, IL-2 or a functional fragment or mutant variant thereof disclosed above can be expressed from a fusion promoter comprising miR223, a functional fragment thereof, or a promoter having miR223 functionality, and a microglia-specific promoter.

[0252] That is, in certain embodiments, the present invention provides a viral vector encoding IL-2 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 17, or a functional fragment thereof, wherein the first promoter is the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof, and wherein the first promoter is i) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23 or SEQ ID NO: 24, or a functional fragment thereof. ii) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; iii) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, 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 set forth in SEQ ID NO: 6, or a functional fragment thereof; and / or v) AIF1 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a viral vector in which the vector is operably linked to

[0253] In certain embodiments, the present invention relates to a viral vector encoding IL-2 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 17, or a functional fragment thereof, wherein the promoter is the miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof.

[0254] In certain embodiments, the present invention relates to a viral vector encoding IL-2 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 17, or a functional fragment thereof, wherein the first promoter is an ITGAM promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 6, or a functional fragment thereof.

[0255] In certain embodiments, the present invention relates to a viral vector encoding IL-2 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 17, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 2, SEQ ID NO: 21, or SEQ ID NO: 22, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26 or SEQ ID NO: 27.

[0256] In certain embodiments, the present invention relates to a viral vector encoding IL-2 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 17, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28.

[0257] In certain embodiments, the present invention relates to a viral vector encoding IL-2 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 17, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the OLFML3 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29.

[0258] In a specific embodiment, the present invention provides a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene: 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 set forth in SEQ ID NO: 18, or a functional fragment thereof. The present invention relates to a viral vector encoding the

[0259] That is, in certain embodiments, the present invention relates to a viral vector encoding interleukin-15 (IL-15). The term "IL-15" refers to the protein sequence of SEQ ID NO: 18 and / or any sequence having greater than 95% homologous sequence identity thereto. Nucleic acid sequences encoding said protein are also provided herein. [ka]

[0260] Interleukin-15 (IL-15) is a cytokine with structural similarity to interleukin-2 (IL-2). Like IL-2, IL-15 binds to and signals through a complex composed of the IL-2 / IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). IL-15 is secreted by mononuclear phagocytes (and some other cells) after infection with a virus or viruses. This cytokine induces the proliferation of natural killer cells, cells of the innate immune system whose primary role is to kill virus-infected cells. IL-15 regulates the activation and proliferation of T cells and natural killer (NK) cells. IL-15 provides a survival signal that maintains memory T cells in the absence of antigen. This cytokine is also involved in NK cell development. In rodent lymphocytes, IL-15 prevents apoptosis by inducing BCL2L1 / BCL-x(L), an inhibitor of the apoptotic pathway. In humans with celiac disease, IL-15 similarly suppresses apoptosis in T-lymphocytes by inducing Bcl-2 and / or Bcl-xL.

[0261] IL-15 has been shown to enhance CD8+ T cell antitumor immunity in preclinical models. A phase I clinical trial to evaluate the safety, dosing, and antitumor efficacy of IL-15 in patients with metastatic melanoma and renal cell carcinoma (kidney cancer) has begun enrolling patients at the National Institutes of Health.

[0262] A functional fragment of IL-15 is a fragment of at least 50, at least 100, at least 110, at least 120, at least 130, or at least 140 amino acids that has at least 95% sequence identity to SEQ ID NO: 18 and that has IL-15 activity. Assays for determining whether a protein has IL-15 activity have been described in the art, for example, by Hu et al., Discovery of a novel IL-15-based protein with improved developability and efficacy for cancer immunotherapy; Sci Rep. 2018 May 16;8(1):7675. doi: 10.1038 / s41598-018-25987-4.

[0263] In a specific embodiment, the present invention provides a viral vector encoding IL-15 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 18, or a functional fragment thereof, wherein one or more promoters a) a myeloid-specific promoter or a functional fragment thereof; and / or b) a microglia-specific promoter or a functional fragment thereof; and / or c) i) a first promoter that is a myeloid-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) a second promoter A fusion promoter comprising or consisting of The present invention relates to a viral vector comprising:

[0264] That is, in certain embodiments, the IL-15 or functional fragment or mutant variant thereof disclosed above can be expressed from a myeloid-specific promoter or functional fragment thereof. In other embodiments, the IL-15 or functional fragment or mutant variant thereof disclosed above can be expressed from a microglia-specific promoter or functional fragment thereof. In other embodiments, the IL-15 or functional fragment or mutant variant thereof disclosed above can be expressed from a fusion promoter, preferably the fusion promoter comprises a myeloid-specific or microglia-specific promoter or functional fragment thereof.

[0265] That is, in certain embodiments, the present invention provides a viral vector encoding IL-15 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 18, or a functional fragment thereof, wherein the myeloid-specific promoter is a) the 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 set forth in SEQ ID NO: 1, or a functional fragment thereof; b) 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 set forth in SEQ ID NO:6, or a functional fragment thereof; or c) 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 set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a viral vector,

[0266] In certain embodiments, the present invention provides a viral vector encoding IL-15 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 18, or a functional fragment thereof, wherein the microglia-specific promoter is a) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:3, SEQ ID NO:23 or SEQ ID NO:24, or a functional fragment thereof; b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; or c) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. The present invention relates to a viral vector,

[0267] In certain embodiments, the present invention relates to a viral vector encoding IL-15 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 18, or a functional fragment thereof, wherein the first promoter is a myeloid-specific promoter and the second promoter is a microglia-specific promoter, or vice versa.

[0268] The second promoter can be any promoter known in the art. However, in certain embodiments, the above-disclosed IL-15 or its functional fragment or mutant variant can be expressed from a fusion promoter comprising a myeloid-specific promoter and a microglia-specific promoter. That is, any of the above-disclosed myeloid-specific promoters can be combined with any of the above-disclosed microglia-specific promoters in any order.

[0269] In certain embodiments, IL-15 or a functional fragment or mutant variant thereof disclosed above may be expressed from a fusion promoter comprising miR223, a functional fragment thereof, or a promoter having miR223 functionality, and a microglia-specific promoter.

[0270] That is, in a specific embodiment, the present invention provides a viral vector encoding IL-15 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 18, or a functional fragment thereof, 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 set forth in SEQ ID NO: 1, or a functional fragment thereof, and wherein the first promoter is i) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23 or SEQ ID NO: 24, or a functional fragment thereof. ii) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; iii) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, 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 set forth in SEQ ID NO: 6, or a functional fragment thereof; and / or v) AIF1 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a viral vector in which the vector is operably linked to

[0271] In certain embodiments, the present invention relates to a viral vector encoding IL-15 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 18, or a functional fragment thereof, wherein the promoter is the miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof.

[0272] In certain embodiments, the present invention relates to a viral vector encoding IL-15 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 18, or a functional fragment thereof, wherein the first promoter is an ITGAM promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 6, or a functional fragment thereof.

[0273] In certain embodiments, the present invention relates to a viral vector encoding IL-15 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 18, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 2, SEQ ID NO: 21, or SEQ ID NO: 22, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26 or SEQ ID NO: 27.

[0274] In certain embodiments, the present invention relates to a viral vector encoding IL-15 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 18, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28.

[0275] In certain embodiments, the present invention relates to a viral vector encoding IL-15 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 18, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the OLFML3 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29.

[0276] In a specific embodiment, the present invention provides a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene: 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 set forth in SEQ ID NO: 19, or a functional fragment thereof. The present invention relates to a viral vector encoding the

[0277] That is, in certain embodiments, the present invention relates to a viral vector encoding interleukin-21 (IL-21). The term "IL-21" refers to the protein sequence of SEQ ID NO: 19 and / or any sequence having greater than 95% sequence identity thereto. Nucleic acid sequences encoding said protein are also provided herein. [ka]

[0278] Interleukin-21 (IL-21) is a cytokine that has potent regulatory effects on cells of the immune system, including natural killer (NK) cells and cytotoxic T cells, which can destroy virus-infected or cancerous cells. This cytokine induces cell division / proliferation in its target cells.

[0279] The role of IL-21 in modulating the differentiation programming of human T cells has been reported, which showed that it enriched a population of central memory CTLs with a unique CD28+CD127hiCD45RO+ phenotype and the ability to produce IL-2. Tumor-reactive antigen-specific CTLs generated by priming in the presence of IL-21 resulted in a stable "helper-independent" phenotype. IL-21 has also been described to have antitumor effects through continuous and increased CD8+ cell responses to achieve durable tumor immunity.

[0280] IL-21 has been approved for phase 1 clinical trials in patients with metastatic melanoma (MM) and renal cell carcinoma (RCC). It has been shown to be safe, although flu-like symptoms are a side effect. Dose-limiting toxicities included low lymphocyte, neutrophil, and platelet counts, as well as hepatotoxicity. According to the Response Evaluation Criteria in Solid Tumors (RECIST) response scale, two of 47 MM patients and four of 19 RCC patients demonstrated complete and partial responses, respectively. Additionally, increased perforin, granzyme B, IFN-gamma, and CXCR3 mRNA levels were observed in peripheral NK cells and CD8+ T cells. These results suggest that IL-21 enhances CD8+ effector function, thereby resulting in an antitumor response. IL-21 has progressed to phase 2 clinical trials, where it has been administered alone or in combination with drugs such as sorafenib and rituximab.

[0281] A functional fragment of IL-21 is a fragment of at least 50, at least 100, at least 110, at least 120, at least 130, or at least 140 amino acids that has at least 95% sequence identity to SEQ ID NO: 19 and that has IL-21 activity. Assays for determining whether a protein has IL-21 activity have been described in the art, for example, by Maurer et al., Generation and characterization of human anti-human IL-21 neutralizing monoclonal antibodies; MAbs. 2012 Jan-Feb; 4(1): 69-83.

[0282] In a specific embodiment, the present invention provides a viral vector encoding IL-21 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 19, or a functional fragment thereof, wherein one or more promoters a) a myeloid-specific promoter or a functional fragment thereof; and / or b) a microglia-specific promoter or a functional fragment thereof; and / or c) i) a first promoter that is a myeloid-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) a second promoter A fusion promoter comprising or consisting of The present invention relates to a viral vector comprising:

[0283] That is, in certain embodiments, the above-disclosed IL-21 or functional fragment or mutant variant thereof may be expressed from a myeloid-specific promoter or functional fragment thereof. In other embodiments, the above-disclosed IL-21 or functional fragment or mutant variant thereof may be expressed from a microglia-specific promoter or functional fragment thereof. In other embodiments, the above-disclosed IL-21 or functional fragment or mutant variant thereof may be expressed from a fusion promoter, preferably the fusion promoter comprises a myeloid-specific or microglia-specific promoter or functional fragment thereof.

[0284] That is, in certain embodiments, the present invention provides a viral vector encoding IL-21 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 19, or a functional fragment thereof, wherein the myeloid-specific promoter is a) the 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 set forth in SEQ ID NO: 1, or a functional fragment thereof; b) 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 set forth in SEQ ID NO:6, or a functional fragment thereof; or c) 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 set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a viral vector,

[0285] In certain embodiments, the present invention provides a viral vector encoding IL-21 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 19, or a functional fragment thereof, wherein the microglia-specific promoter is

[0286] a) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:3, SEQ ID NO:23 or SEQ ID NO:24, or a functional fragment thereof; b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; or c) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. The present invention relates to a viral vector,

[0287] In certain embodiments, the present invention relates to a viral vector encoding IL-21 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 19, or a functional fragment thereof, wherein the first promoter is a myeloid-specific promoter and the second promoter is a microglia-specific promoter, or vice versa.

[0288] The second promoter can be any promoter known in the art. However, in certain embodiments, the above-disclosed IL-21 or its functional fragment or mutant variant can be expressed from a fusion promoter comprising a myeloid-specific promoter and a microglia-specific promoter. That is, any of the above-disclosed myeloid-specific promoters can be combined with any of the above-disclosed microglia-specific promoters in any order.

[0289] In certain embodiments, IL-21 or a functional fragment or mutant variant thereof disclosed above may be expressed from a fusion promoter comprising miR223, a functional fragment thereof, or a promoter having miR223 functionality, and a microglia-specific promoter.

[0290] That is, in certain embodiments, the present invention provides a viral vector encoding IL-21 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 19, or a functional fragment thereof, 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 set forth in SEQ ID NO: 1, or a functional fragment thereof, and wherein the first promoter is i) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23 or SEQ ID NO: 24, or a functional fragment thereof. ii) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; iii) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, 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 set forth in SEQ ID NO: 6, or a functional fragment thereof; and / or v) AIF1 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a viral vector in which the vector is operably linked to

[0291] In certain embodiments, the present invention relates to a viral vector encoding IL-21 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 19, or a functional fragment thereof, wherein the promoter is the miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof.

[0292] In certain embodiments, the present invention relates to a viral vector encoding IL-21 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 19, or a functional fragment thereof, wherein the first promoter is an ITGAM promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 6, or a functional fragment thereof.

[0293] In certain embodiments, the present invention relates to a viral vector encoding IL-21 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 19, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 2, SEQ ID NO: 21, or SEQ ID NO: 22, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26 or SEQ ID NO: 27.

[0294] In certain embodiments, the present invention relates to a viral vector encoding IL-21 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 19, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28.

[0295] In certain embodiments, the present invention relates to a viral vector encoding IL-21 or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 19, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) the OLFML3 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29.

[0296] In a specific embodiment, the present invention provides a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene: 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 set forth in SEQ ID NO: 20, or a functional fragment thereof. The present invention relates to a viral vector encoding the

[0297] That is, in certain embodiments, the present invention relates to a viral vector encoding interferon-alpha (IFN-alpha). The term "IFN-alpha" refers to the protein sequence of SEQ ID NO: 20 and / or any sequence having greater than 95% homologous sequence identity thereto. Nucleic acid sequences encoding said proteins are also provided herein. [ka]

[0298] Human interferon alpha-2 (IFNα2) is a cytokine that belongs to the type I IFN family. IFNα2 is a protein secreted by virus-infected cells and acts on other cells to inhibit viral infection.

[0299] When given orally, IFNα2 is degraded by digestive enzymes and becomes inactive. Therefore, IFNα2 is primarily administered by subcutaneous or intramuscular injection. Once in the blood, IFNα2 is rapidly eliminated by the kidney. Due to the short lifespan of IFNα2 in the organism, multiple injections per week are required. Peginterferon alpha-2a and peginterferon alpha-2b (polyethylene glycol linked to IFNα2) are long-lasting IFNα2 formulations that allow for a single injection per week.

[0300] Recombinant IFNα2 (α2a and α2b) has demonstrated efficiency in treating patients diagnosed with some viral infections (such as chronic viral hepatitis B and C) or some types of cancer (melanoma, renal cell carcinoma, and various hematological malignancies).

[0301] A functional fragment of IFN-alpha is a fragment of at least 50, at least 100, at least 110, at least 120, at least 130, or at least 140 amino acids that has at least 95% sequence identity to SEQ ID NO: 20 and that has IFN-alpha activity. Assays for determining whether a protein has IFN-alpha activity have been described in the art, for example, by Moll et al., The differential activity of interferon-α subtypes is consistent among distinct target genes and cell types; Cytokine. 2011 Jan; 53(1): 52-59.

[0302] In a specific embodiment, the present invention provides a viral vector encoding IFN-alpha or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:20, or a functional fragment thereof, wherein one or more promoters a) a myeloid-specific promoter or a functional fragment thereof; and / or b) a microglia-specific promoter or a functional fragment thereof; and / or c) i) a first promoter that is a myeloid-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) a second promoter A fusion promoter comprising or consisting of The present invention relates to a viral vector comprising:

[0303] That is, in certain embodiments, the IFN-alpha or functional fragment or mutant variant thereof disclosed above can be expressed from a myeloid-specific promoter or functional fragment thereof. In other embodiments, the IFN-alpha or functional fragment or mutant variant thereof disclosed above can be expressed from a microglia-specific promoter or functional fragment thereof. In other embodiments, the IFN-alpha or functional fragment or mutant variant thereof disclosed above can be expressed from a fusion promoter, preferably the fusion promoter comprises a myeloid-specific or microglia-specific promoter or functional fragment thereof.

[0304] That is, in certain embodiments, the present invention provides a viral vector encoding IFN-alpha or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:20, or a functional fragment thereof, wherein the myeloid-specific promoter is a) the 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 set forth in SEQ ID NO: 1, or a functional fragment thereof; b) 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 set forth in SEQ ID NO:6, or a functional fragment thereof; or c) 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 set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a viral vector,

[0305] In certain embodiments, the present invention provides a viral vector encoding IFN-alpha or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 20, or a functional fragment thereof, wherein the microglia-specific promoter is a) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:3, SEQ ID NO:23 or SEQ ID NO:24, or a functional fragment thereof; b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; or c) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. The present invention relates to a viral vector,

[0306] In certain embodiments, the present invention relates to a viral vector encoding IFN-alpha or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 20, or a functional fragment thereof, wherein the first promoter is a myeloid-specific promoter and the second promoter is a microglia-specific promoter, or vice versa.

[0307] The second promoter can be any promoter known in the art.However, in certain embodiments, the above-disclosed IFN-alpha or its functional fragment or mutant variant can be expressed from a fusion promoter comprising a myeloid-specific promoter and a microglia-specific promoter.That is, any of the above-disclosed myeloid-specific promoters can be combined with any of the above-disclosed microglia-specific promoters in any order.

[0308] In certain embodiments, the IFN-alpha or functional fragment or mutant variant thereof disclosed above may be expressed from a fusion promoter comprising miR223, a functional fragment thereof or a promoter having miR223 functionality, and a microglia-specific promoter.

[0309] That is, in certain embodiments, the present invention provides a viral vector encoding IFN-alpha or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:20, or a functional fragment thereof, wherein the first promoter is the miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:1, or a functional fragment thereof, and wherein the first promoter is i) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23 or SEQ ID NO: 24, or a functional fragment thereof. ii) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; iii) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, 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 set forth in SEQ ID NO: 6, or a functional fragment thereof; and / or v) AIF1 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a viral vector in which the vector is operably linked to

[0310] In certain embodiments, the present invention relates to a viral vector encoding IFN-alpha or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 20, or a functional fragment thereof, wherein the promoter is the miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof.

[0311] In certain embodiments, the present invention relates to a viral vector encoding IFN-alpha or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 20, or a functional fragment thereof, wherein the first promoter is an ITGAM promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 6, or a functional fragment thereof.

[0312] In certain embodiments, the present invention relates to a viral vector encoding IFN-alpha or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:20, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:1, or a functional fragment thereof; and (b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21, or SEQ ID NO:22, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:26 or SEQ ID NO:27.

[0313] In certain embodiments, the present invention relates to a viral vector encoding IFN-alpha or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:20, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:1, or a functional fragment thereof; and (b) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:3, SEQ ID NO:23, or SEQ ID NO:24, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:28.

[0314] In certain embodiments, the present invention relates to a viral vector encoding IFN-alpha or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:20, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising: (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:1, or a functional fragment thereof; and (b) the OLFML3 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:4 or SEQ ID NO:25, or a functional fragment thereof. In certain embodiments, the fusion promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:29.

[0315] In a specific embodiment, the present invention provides a viral vector according to the present invention, wherein the one or more promoters are: a) a myeloid-specific promoter or a functional fragment thereof; and / or b) a microglia-specific promoter or a functional fragment thereof; and / or c) i) a first promoter that is a myeloid-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) a second promoter A fusion promoter comprising or consisting of The present invention relates to a viral vector comprising:

[0316] That is, any of the transgenes disclosed above, or functional fragments or variants thereof, may be operably linked to one or more promoters. In certain embodiments, the transgenes disclosed above, or functional fragments or variants thereof, may be operably linked to a myeloid-specific promoter or functional fragments thereof. In certain embodiments, the transgenes disclosed above, or functional fragments or variants thereof, may be operably linked to a microglia-specific promoter or functional fragments thereof. In certain embodiments, the transgenes disclosed above, or functional fragments or variants thereof, may be operably linked to a fusion promoter comprising a myeloid-specific or microglia-specific promoter or functional fragments thereof and a second promoter. In certain embodiments, the present invention provides a viral vector according to the present invention, wherein the myeloid-specific promoter is a) the 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 set forth in SEQ ID NO: 1, or a functional fragment thereof; or b) 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 set forth in SEQ ID NO:6, or a functional fragment thereof; or c) 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 set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a viral vector,

[0317] The term "myeloid-specific promoter" as used herein refers to any promoter that can drive expression in myeloid cells.Those skilled in the art will recognize how to identify whether a promoter can drive expression in myeloid cells.For example, myeloid cells, such as the monocytic cell line THP-1, can be transduced with a viral vector encoding a fluorescent marker under the control of the promoter in question.If the expression of the fluorescent marker can be detected in myeloid cells after the viral vector is integrated into the genome of myeloid cells, the promoter is determined to be a myeloid-specific promoter.Myeloid-specific promoters within the meaning of the present invention include, but are not limited to, miR223 promoter, AIF1 promoter, and ITGAM promoter.

[0318] That is, in a specific embodiment, the present invention provides a viral vector according to the present invention, wherein the microglia-specific promoter is a) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:3, SEQ ID NO:23 or SEQ ID NO:24, or a functional fragment thereof; or b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; or c) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. The present invention relates to a viral vector,

[0319] The term "microglia-specific promoter" as used herein refers to any promoter that can drive expression in microglia. Those skilled in the art will recognize how to identify whether a promoter can drive expression in microglia. For example, a viral vector encoding a fluorescent marker under the control of the promoter in question can be transduced into microglia, such as an immortalized microglia cell line. If expression of the fluorescent marker can be detected in microglia after integration of the viral vector into the microglia genome, the promoter is determined to be a microglia-specific promoter. Microglia-specific promoters within the meaning of the present invention include, but are not limited to, P2RY12 promoter, TMEM119 promoter, OLFML3 promoter, ITGAM promoter, and AIF1 promoter.

[0320] In certain embodiments, the present invention relates to a viral vector according to the invention, wherein the first promoter is a myeloid-specific promoter and the second promoter is a microglia-specific promoter, or vice versa.

[0321] That is, the fusion promoter can preferably comprise a myeloid-specific promoter and a microglia-specific promoter. In certain embodiments, the microglia-specific promoter is fused to the 5' end of the myeloid-specific promoter. In certain embodiments, the microglia-specific promoter is fused to the 3' end of the myeloid-specific promoter.

[0322] In a specific embodiment, the invention relates to a viral vector of the invention, wherein the first promoter is the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof, and wherein the first promoter is i) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23 or SEQ ID NO: 24, or a functional fragment thereof. ii) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:23, or a functional fragment thereof; iii) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, 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 set forth in SEQ ID NO: 6, or a functional fragment thereof; and / or v) AIF1 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a viral vector in which the vector is operably linked to

[0323] In certain embodiments, the present invention relates to a viral vector of the present invention, 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 set forth in SEQ ID NO: 1, or a functional fragment thereof, and the first promoter is operably linked to a TMEM119 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23 or SEQ ID NO: 24, or a functional fragment thereof.

[0324] In a particular embodiment, the present invention relates to a viral vector according to the invention, wherein the viral vector comprises at least one transcriptional regulatory element, wherein the at least one transcriptional regulatory element is positioned so as to inhibit or activate the transcriptional activity of the promoter.

[0325] In a particular embodiment, the present invention provides a method for the production of a transcriptional regulatory element comprising the steps of: 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 an RU486-binding domain or an abscisic acid-binding domain; iii) steroid-binding domains, in particular ecdysone-binding domains; iv) Dimerization factor systems, in particular rapamycin-based or rapalog-based dimerization factor systems. The present invention relates to a viral vector according to the present invention, comprising:

[0326] In certain embodiments, the invention relates to a viral vector according to the invention, wherein the viral vector encodes a riboswitch, which controls translation of an mRNA encoding a therapeutic protein or a combination of said therapeutic proteins.

[0327] That is, a viral vector encoding any one of the transgenes disclosed above, or functional fragments or variants thereof, can comprise a regulatory element that allows for control of expression of the transgene. Preferably, the regulatory element is any of the regulatory elements disclosed elsewhere herein.

[0328] In certain embodiments, the present invention provides a method for producing a pharmaceutical composition comprising: a) retroviral vectors, in particular lentiviral vectors, more particularly lentiviral SIN vectors; or b) a foamy viral vector; or c) a viral vector selected from the group consisting of an adenovirus vector, an adeno-associated virus vector, a herpesvirus vector, a parvovirus vector, a coronavirus vector, and an alpha-retrovirus vector. The present invention relates to a viral vector, which is

[0329] The viral vector according to the present invention can be any type of viral vector that allows the delivery of a transgene into mammalian, or preferably human, cells.

[0330] In certain embodiments, viral vector is retroviral vector.As used herein, the term " retrovirus " refers to the virus that consists of the outer envelope glycoprotein shell of viral origin, including but not limited to the vesicular stomatitis virus (VSV) glycoprotein (VSVG) with membrane fusion activity, that encapsulates viral RNA, and the viral proteins that are required for the reverse transcription of its genomic RNA into linear double-stranded DNA copy, and then the covalent integration of its genomic DNA into host genome.

[0331] Retroviruses are common tools for gene delivery (Miller, 2000, Nature. 357: 455-460). Once a virus integrates into the host genome, it is called a "provirus." The provirus serves as a template for RNA polymerase II, directing the expression of RNA molecules encoding the structural proteins and enzymes required to produce new viral particles. Illustrative retroviruses include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentiviruses.

[0332] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV types 1 and 2); Visna-Maedi virus (VMV); Caprine Arthritis-Encephalitis Virus (CAEV); Equine Infectious Anemia Virus (EIAV); Feline Immunodeficiency Virus (FIV); Bovine Immunodeficiency Virus (BIV); and Simian Immunodeficiency Virus (SIV). In one embodiment, HIV-based vector backbones (i.e., HIV cis-acting sequence elements) are preferred.

[0333] The term "vector" is used herein to refer to a nucleic acid molecule that can transfer or transport another nucleic acid molecule. The nucleic acid to be transferred is generally linked to, i.e., inserted into, the vector nucleic acid molecule. A vector can contain a sequence that directs autonomous replication in a cell, or can contain a sequence sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-defective retroviruses and lentiviruses.

[0334] Within the present invention, viral vectors are used to transduce target cells. The term "transduction" refers to the creation of conditions that aim to bring the viral vector into physical contact with the target cell, followed by the transfer of viral nucleic acid into the target cell, which, in the case of retroviruses, is reverse transcribed into DNA and integrated into the genome of the target cell.

[0335] The term "lentiviral vector" can be used to refer to a lentiviral infectious particle consisting of a biological membrane studded with viral envelope glycoproteins, or simply a biological membrane lacking the viral envelope protein shell, that has membrane fusion capabilities and encloses a lentiviral capsid structure formed by lentiviral proteins, the capsid structure enclosing the lentiviral RNA and the lentiviral proteins required for reverse transcription and stable integration into the genome of the target cell.

[0336] Lentiviral vectors allow the delivery of nucleic acid molecules encoding therapeutic polypeptides to dividing and / or non-dividing cells. Lentiviral vectors can be used for in vivo injection in conjunction with in vitro transduction, while AAV infectious particles can be used to deliver DNA to non-dividing cells by in vivo injection.

[0337] Preferably, the viral vector of the present invention is a self-inactivating lentiviral vector. A "self-inactivating" (SIN) vector is a replication-deficient vector, e.g., a viral or lentiviral vector in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion and / or substitution) to prevent viral transcription beyond the first round of viral replication. As a result, the vector can infect and then integrate into the host genome only once and is unable to transmit further. This is because the right (3') LTR U3 region is used as a template for the left (5') LTR U3 region during viral replication, and therefore, viral transcripts cannot be generated without the U3 enhancer-promoter. Without viral transcripts, they cannot be processed or packaged into virions, thus ending the viral life cycle.

[0338] In certain embodiments, the viral vector can be a foamy viral vector. The term "foamy viral vector" as used herein refers to a viral vector that uses a part derived from a foamy virus. Methods for developing viral vectors are known to those skilled in the art (e.g., Mergia, A, and M Heinkelein, 2003, Current topics in microbiology and immunology vol. 277: 131-59).

[0339] In certain embodiments, the viral vector is selected from the group consisting of an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, a parvovirus vector, a coronavirus vector, and an alpha-retrovirus vector.

[0340] The term "adenoviral vector," as used herein, refers to a viral vector or plasmid that contains structural and functional genetic elements or portions thereof derived primarily from an adenovirus.

[0341] The term "adenovirus," as used herein, refers to members of the family Adenoviridae, which are typically medium-sized (90-100 nm), non-enveloped (without an outer lipid bilayer) viruses with an icosahedral nucleocapsid containing a double-stranded DNA genome.

[0342] Methods for obtaining adenoviral vectors are known to those skilled in the art (see, for example, Kamen, A., and Henry, O., 2004, The Journal of Gene Medicine: A cross-disciplinary journal for research on the science of gene transfer and its clinical applications, 6(S1), S184-S192; Volpers, C. and Kochanek, S., 2004, The Journal of Gene Medicine: A cross-disciplinary journal for research on the science of gene transfer and its clinical applications, 6(S1), S164-S171).

[0343] The term "herpesvirus vector," as used herein, refers to a viral vector or plasmid containing structural and functional genetic elements or portions thereof derived primarily from a herpesvirus. The term "herpesvirus," as used herein, refers to any virus from the Simplexvirus genus. Methods for obtaining herpesvirus vectors are known to those skilled in the art (see, e.g., Logvinoff, Carine, and Alberto L. Epstein, 2001, Human gene therapy 12.2: 161-167).

[0344] The term "alpharetroviral vector," as used herein, refers to a viral vector or plasmid that contains structural and functional genetic elements, or portions thereof, primarily derived from an alpharetrovirus. The term "alpharetrovirus," as used herein, refers to any virus from the genus Alpharetrovirus. Methods for obtaining alpharetroviral vectors are known to those skilled in the art (see, e.g., Garoff, Henrik, and Kejun Li, 1998, Gene Therapy. 61-69).

[0345] In certain embodiments, the viral vector can be an adeno-associated viral (AAV) vector. Currently, there are two classes of recombinant AAV (rAAV) in use: single-stranded AAV (ssAAV) and self-complementary AAV (scAAV). ssAAV is packaged as either a sense (plus strand) or antisense (minus strand) genome.

[0346] That is, in certain embodiments, the viral vector is a DNA-based viral vector, in which the viral DNA can directly integrate into the genome of the target cell without reverse transcription of the viral DNA.

[0347] In certain embodiments, the present invention provides a method for producing a pharmaceutical composition comprising: a) the miR223 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth 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 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or the functional fragment thereof, is operably linked to the microglia-specific promoter, or the functional fragment thereof.

[0348] Due to its resistance to methylation during cell differentiation, the miR223 promoter shows great potential for use in cell and gene therapy applications targeting HSCs or keratinocytes. In certain embodiments, the miR223 promoter or a functional fragment or variant thereof can be fused to a second promoter, preferably a microglia-specific promoter.

[0349] Thus, in certain embodiments, the present invention provides a method for producing a microglia-specific promoter comprising: a) the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:3, SEQ ID NO:23 or SEQ ID NO:24, or a functional fragment thereof; b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:21 or SEQ ID NO:22, or a functional fragment thereof; c) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof; d) an ITGAM promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:6, or a functional fragment thereof; or e) AIF1 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5, or a functional fragment thereof. The fusion promoter of the present invention is

[0350] In certain embodiments, the fusion promoter comprises a miR223 promoter and a P2RY12 promoter. a) the miR223 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and b) the P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 2, SEQ ID NO: 21 or SEQ ID NO: 22, or a functional fragment thereof. The present invention relates to a fusion promoter comprising:

[0351] In certain embodiments, a miR223-P2RY12 fusion promoter of the present invention comprises a nucleotide sequence set forth in SEQ ID NO: 26 or SEQ ID NO: 27. In certain embodiments, a miR223-P2RY12 fusion promoter of the present invention comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 26 or SEQ ID NO: 27.

[0352] In certain embodiments, the fusion promoter comprises a miR223 promoter and a TMEM119 promoter. a) the miR223 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and b) the TMEM119 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23 or SEQ ID NO: 24, or a functional fragment thereof. The present invention relates to a fusion promoter comprising:

[0353] In certain embodiments, a miR223-TMEM119 fusion promoter of the invention comprises the nucleotide sequence set forth in SEQ ID NO: 28. In certain embodiments, a miR223-TMEM119 fusion promoter of the invention comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 28.

[0354] In certain embodiments, the fusion promoter comprises the miR223 promoter and the OLFML3 promoter. a) the miR223 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and b) the OLFML3 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9, or a functional fragment thereof. The present invention relates to a fusion promoter comprising:

[0355] In certain embodiments, the miR223-OLFML3 fusion promoter of the present invention comprises the nucleotide sequence set forth in SEQ ID NO: 29. In certain embodiments, the miR223-OLFML3 fusion promoter of the present invention comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 29.

[0356] In certain embodiments, the fusion promoter comprises the miR223 promoter and the AIF1 promoter. a) the miR223 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and b) AIF1 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5, or a functional fragment thereof. The present invention relates to a fusion promoter comprising:

[0357] In certain embodiments, the fusion promoter comprises a miR223 promoter and an ITGAM promoter. a) the miR223 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and b) an ITGAM promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 6, or a functional fragment thereof; The present invention relates to a fusion promoter comprising:

[0358] In a particular embodiment, the present invention relates to a fusion promoter according to the present invention, wherein the fusion promoter comprises at least one transcriptional regulatory element, wherein the at least one transcriptional regulatory element is positioned to inhibit or activate the transcriptional activity of the promoter.

[0359] In a particular embodiment, the present invention provides a method for the production of a transcriptional regulatory element comprising the steps of: 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 an RU486-binding domain or an abscisic acid-binding domain; iii) steroid-binding domains, in particular ecdysone-binding domains; iv) Dimerization factor systems, in particular rapamycin-based or rapalog-based dimerization factor systems. The present invention relates to a fusion promoter according to the present invention, comprising:

[0360] In certain embodiments, the invention relates to a fusion promoter of the invention, wherein the viral vector encodes a riboswitch, and the riboswitch controls translation of an mRNA encoding a therapeutic protein or combination of therapeutic proteins.

[0361] That is, the fusion promoters of the present invention can include regulatory elements that allow for control of transgene expression in a more precise manner. Preferably, the regulatory elements are any of the regulatory elements disclosed elsewhere herein.

[0362] In certain embodiments, the present invention provides a fusion promoter comprising: a) comprising any one of the sequences set forth in SEQ ID NOs: 26 to 29, or b) The fusion promoter of the present invention comprises a sequence having 90%, 91%, 92%, 93%, 94%, or 95% sequence identity with any one of the sequences set forth in SEQ ID NOs: 26 to 29, and the promoter drives expression in microglia and / or myeloid cells.

[0363] In certain embodiments, the present invention provides a fusion promoter comprising: a) comprising the sequence set forth in SEQ ID NO: 28; or b) comprising a sequence having 90%, 91%, 92%, 93% or 95% sequence identity to the sequence set forth in SEQ ID NO: 28; The fusion promoter of the present invention, wherein the promoter drives expression in microglia and / or myeloid cells.

[0364] In a particular embodiment, the present invention relates to a host cell comprising a viral vector according to the present invention.

[0365] That is, the present invention further relates to a host cell comprising the viral vector of the present invention. In certain embodiments, the host cell may be a cell used to produce the viral vector of the present invention. For example, the host cell may be a HEK293T cell. In certain embodiments, the host cell may be a cell (e.g., HSC) or its progeny (e.g., macrophage) infected with an infectious viral particle containing viral nucleic acid, regardless of its virus production ability.

[0366] A host cell is also said to contain a viral vector of the invention when it is transfected with a plasmid encoding the genetic elements for the production of a viral vector, and the plasmid is integrated into the genome of the host cell in a "stable producer cell." Thus, the viral vector does not necessarily have to be in a circular form contained in the host cell.

[0367] In certain embodiments, the present invention relates to host cells of the present invention that are hematopoietic stem cells, preferably hematopoietic stem cells of a CD34-positive enriched cell population, or myeloid cells. That is, in certain embodiments, the host cells can be transduced hematopoietic stem cells, preferably hematopoietic stem cells of a CD34-positive enriched cell population, or transduced myeloid cells. In particular, the host cells used in the treatment and / or prevention of any of the diseases and / or disorders disclosed herein are preferably transduced hematopoietic stem cells, preferably hematopoietic stem cells of a CD34-positive enriched cell population, or transduced myeloid cells.

[0368] In certain embodiments, the host cell can be a hematopoietic stem cell, i.e., in certain embodiments, the present invention relates to a hematopoietic stem cell transduced with any of the viral vectors disclosed herein.

[0369] The term "hematopoietic stem cell", which is identical to the term "hematopoietic stem cell" or "HSC" or "HSPC", relates to any cell population obtained by, but not limited to, bone marrow aspiration, apheresis after stem cell mobilization, or obtained from umbilical cord blood, and / or any cell population enriched for CD34-positive or CD133-positive cells by any method (but not limited to CD34-positive and / or CD133-positive cell labeling and enrichment) or by depletion of lineage-positive cells by any method known in the art.

[0370] As used herein, " CD34 positive enrichment " refers to the population containing a higher number and / or a higher percentage of CD34 positive cells than those found in the cell population before the enrichment step. Various methods for CD34 positive cell enrichment are known to those skilled in the art (see, for example, Baldwin, K. et al., 2015, Stem cells, 33(5), 1532-1542; Wojciechowski, Joel C et al., 2008, British journal of hematology vol. 140,6 673-81; Gori, JL et al., 2012, Blood, The Journal of the American Society of Hematology, 120(13), e35-e44; Kilic, P. et al., 2019, Cells Tissues Organs, 207(1), 15-20).

[0371] In a preferred embodiment, the host cells are cells in an enriched population of CD34-positive bone marrow cells. In a more preferred embodiment, the host cells are hematopoietic stem / progenitor cells in an enriched population of CD34-positive bone marrow cells. In a most preferred embodiment, the host cells are hematopoietic stem cells in an enriched population of CD34-positive bone marrow cells.

[0372] In other embodiments, the host cell may be a myeloid cell. That is, in certain embodiments, the invention relates to granulocytes (neutrophils, eosinophils, and basophils), monocytes, macrophages, Kupffer cells, or mast cells transduced with any of the viral vectors disclosed herein. In certain embodiments, the host cell is a macrophage. In certain embodiments, the host cell is a monocyte. In additional embodiments, the host cell is a microglia.

[0373] Those skilled in the art are aware of methods for enriching and / or identifying the above-disclosed cell types, as well as methods for transducing them with viral vectors.

[0374] In a particular embodiment, the present invention relates to a pharmaceutical composition comprising a viral vector according to the invention and / or a host cell according to the invention.

[0375] That is, in certain embodiments, the present invention relates to a pharmaceutical composition comprising any one of the viral vectors disclosed herein and / or any one of the host cells disclosed herein.

[0376] In certain embodiments, a pharmaceutical composition comprises a viral vector according to the present invention. In such embodiments, the pharmaceutical composition is preferably used to transduce target cells, such as hematopoietic stem cells, ex vivo. Alternatively, the pharmaceutical composition can be administered directly to a subject in need thereof, such that the viral vector contained therein transduces the target cells in vivo. Those skilled in the art will recognize viral vectors suitable for targeting specific populations of target cells in vivo. Those skilled in the art will also recognize methods for formulating viral vectors in pharmaceutical compositions.

[0377] In another embodiment, the pharmaceutical composition comprises a host cell comprising a viral vector of the present invention. Such a host cell can be obtained by transducing a host cell with any one of the vectors of the present invention. The pharmaceutical composition comprising the transduced host cell can be administered to a subject in need thereof. Those skilled in the art will be aware of methods for formulating transduced host cells in pharmaceutical compositions.

[0378] The term "pharmaceutical composition" as used herein refers to a composition resulting from the combination of individual components that are themselves pharmaceutically acceptable. For example, if intravenous or intrathecal administration is envisaged, the components are suitable or acceptable (both in quality and content) for intravenous or intrathecal administration. Those skilled in the art will recognize the pharmaceutically acceptable components that are suitable for the formulation of viral vectors and host cells, respectively.

[0379] In certain embodiments, the present invention relates to a pharmaceutical composition comprising a viral vector according to the invention and / or a host cell according to the invention and at least one additional therapeutic agent.

[0380] The term "therapeutic agent," as used herein, refers to a compound of matter or composition of matter that provides a therapeutic benefit to a subject after administration to the subject in a therapeutically effective amount. A therapeutic agent can be any type of drug, agent, pharmaceutical, hormone, antibiotic, protein, gene, growth factor, and / or bioactive material used in the treatment, control, or prevention of a disease or medical condition.

[0381] In some embodiments, the pharmaceutical compositions of the invention (and any additional therapeutic agents) are formulated, dosed, and administered in a manner consistent with good clinical practice. Factors to consider in this context include the particular disorder being treated, the particular subject being treated, the clinical condition of the subject, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to a physician.

[0382] The viral vectors of the present invention and / or the host cells of the present invention need not be, but are optionally formulated in pharmaceutical compositions together with one or more additional therapeutic agents currently used to prevent or treat the disorder in question.

[0383] The vectors of the present invention can be administered to a subject parenterally, preferably intravascularly (including intravenously) and intrathecally. When administered parenterally, it is preferred that the vector be provided in a pharmaceutical vehicle suitable for injection, such as a sterile aqueous solution or dispersion. After administration, the subject is monitored to detect changes in gene expression. The dosage and duration of treatment are determined individually depending on the condition or disease to be treated. A wide variety of conditions or diseases can be treated based on the gene expression produced by administering a gene of interest in the vector of the present invention. The dosage of the vector delivered using the method of the present invention varies depending on the desired response by the host and the vector used.

[0384] It is contemplated within the present invention that the viral vector, host cell, or pharmaceutical composition of the present invention is administered into a subject's bloodstream or cerebrospinal fluid (or brain tissue). As used herein, "introducing" host cells "into a subject's bloodstream" includes, but is not limited to, introducing such cells into one of the subject's veins or arteries via injection. Such administration can be, for example, one time, multiple times, and / or one or more times over an extended period of time. While a single injection is preferred, in some cases, repeated injections over time (e.g., quarterly, semi-annually, or annually) may be necessary. Such administration is also preferably carried out using a mixture of host cells and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.01-0.1 M, preferably 0.05 M phosphate buffer or 0.8% saline. Moreover, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, and suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as Ringer's dextrose, Ringer's dextrose-based replenishers, and the like. Fluids commonly used for intravenous administration can be found, for example, in Remington: The Science and Practice of Pharmacy, 20th Ed., p. 808, Lippincott Williams & Wilkins (2000). Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.

[0385] As used herein, it is preferred that the viral vectors, host cells, or pharmaceutical compositions of the present invention be administered into the bloodstream of a subject. However, the viral vectors, host cells, or pharmaceutical compositions of the present invention can also be administered directly to target tissues. That is, in certain embodiments, the viral vectors, host cells, or pharmaceutical compositions of the present invention can be injected directly into the brain. Alternatively, the viral vectors, host cells, or pharmaceutical compositions of the present invention can be administered by direct CNS injection, injection into the CSF, intrathecal injection, and / or intravascular administration.

[0386] Alternatively, the viral vector, host cell or pharmaceutical composition of the invention can be administered directly into the tumor.

[0387] In a particular embodiment, the present invention relates to a viral vector according to the invention, a host cell according to the invention or a pharmaceutical composition according to the invention for use in medicine.

[0388] That is, the viral vectors, host cells, or pharmaceutical compositions of the present invention can be used to treat a subject in need thereof. The term "treatment," as used herein, includes preventative (e.g., prophylactic), curative, or palliative treatment, and "treating," as used herein, also includes preventative, curative, and palliative treatment. The term "subject," as used herein, relates to an animal, preferably a mammal, and more preferably a human.

[0389] In certain embodiments, the present invention relates to a viral vector according to the present invention, a host cell according to the present invention or a pharmaceutical composition according to the present invention for use in the treatment of a disease or disorder that has its origin or manifestation in or is based in the brain.

[0390] Targeting brain cells for therapeutic treatment is challenging due to the selective permeability of the blood-brain barrier. Within the present invention, the inventors target brain diseases or disorders through cell and gene therapy. To this end, hematopoietic stem cells or populations of cells containing hematopoietic stem cells can be transformed with any one of the viral vectors disclosed herein and administered to subjects suffering from brain diseases or disorders. Hematopoietic stem cells can circulate in the bloodstream and cross the blood-brain barrier, particularly during transient leakage of the blood-brain barrier following treatment-related radiation or chemotherapy, such as busulfan administration. Once inside the brain, hematopoietic stem cells can differentiate into macrophages that exhibit microglial characteristics and replace microglia in the brain (Speicher et al., Generating microglia from human pluripotent stem cells: novel in vitro models for the study of neurodegeneration; Molecular Neurodegeneration; 14, Article number 46 (2016)). The viral vectors of the present invention are particularly suitable for targeting the brain, as they have been demonstrated to be active in both macrophage and microglial cells.

[0391] Although it is preferred to administer hematopoietic stem cells comprising the viral vector of the present invention into the bloodstream of a subject in need thereof, the viral vector, host cell or pharmaceutical composition of the present invention can also be administered directly into the brain (intracranial).

[0392] In a particular embodiment, the present invention relates to a viral vector according to the invention, a host cell according to the invention or a pharmaceutical composition according to the invention for use in the prevention and / or treatment of a PGRN-related disease or disorder, in particular wherein the viral vector encodes PGRN or a functional fragment thereof.

[0393] Various diseases and disorders have been reported to be caused by abnormal expression of progranulin. In particular, mutations in the PGRN gene have been reported as the cause of various neurodegenerative diseases or disorders. Therefore, the viral vector of the present invention can be used to restore progranulin levels in the brain of a subject suffering from a PGRN-related disease or disorder. Therefore, it is preferred that the transgene encoded in the viral vector is a PGRN gene or a polynucleotide encoding a polypeptide having PGRN functionality and at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.

[0394] In a particular embodiment, the present invention relates to a viral vector, a host cell or a pharmaceutical composition for use according to the present invention, wherein the PGRN-associated disease or disorder is a neurodegenerative disease or disorder.

[0395] That is, the PGRN-associated disease may be a neurodegenerative disease or disorder. Within the present invention, the neurodegenerative disease or disorder is preferably a neurodegenerative disease or disorder associated with abnormal PGRN expression.

[0396] In certain embodiments, the present invention relates to a viral vector, a host cell, or a pharmaceutical composition for use according to the present invention, wherein the neurodegenerative disease or disorder is a frontotemporal degenerative disease or disorder, hi certain embodiments, the present invention relates to a viral vector, a host cell, or a pharmaceutical composition for use according to the present invention, 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.

[0397] In a particular embodiment, the present invention relates to a viral vector, a host cell or a pharmaceutical composition for use according to the present invention, wherein the frontotemporal degenerative disease or disorder is frontotemporal dementia. Preferably, the frontotemporal degenerative disease or disorder is frontotemporal dementia caused by a mutation in the PGRN gene.

[0398] In a particular embodiment, the present invention relates to a viral vector according to the invention, a host cell according to the invention or a pharmaceutical composition according to the invention 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.

[0399] That is, the viral vector of the present invention can be used in the treatment of cancer. Herein, it has been demonstrated that the promoter of the present invention is active in various myeloid cells and microglia. Therefore, the viral vector of the present invention or a host cell containing the viral vector of the present invention can be used in the treatment of cancer in the brain and other parts of the body.

[0400] For example, hematopoietic stem cells containing the viral vector of the present invention can be administered to a subject suffering from cancer. The hematopoietic stem cells can differentiate into myeloid cells, migrate to the site of the tumor, and elicit an immune response against the tumor. The myeloid cells can contain a transgene encoding one of the cytokines disclosed herein to increase the immune response against the tumor. Alternatively or additionally, the transgene can encode an antigen-binding protein that targets the myeloid cells to the tumor and elicits a more pronounced immune response against the tumor.

[0401] In certain embodiments, the present invention relates to a method for treating cancer, lymphoma, and / or sarcoma, comprising administering to a subject the method of the present invention ... or brain metastasis.

[0402] That is, the viral vectors or host cells of the present invention can be used to treat tumors in the brain. Brain tumors can be primary or secondary brain tumors. As described above, hematopoietic stem cells containing the viral vectors of the present invention can migrate to the brain and differentiate into macrophages that exhibit microglial characteristics and can replace microglia in the brain. Inside the brain, such microglia and microglia-like cells can secrete cytokines such as IL-12, IFN-gamma, G-CSF, GM-CSF, IL-2, IL-15, IL-21, IFN-alpha, or combinations or fusion variants thereof to mount an immune response in the brain against tumors.

[0403] In certain embodiments, the present invention relates to a viral vector, a host cell or a pharmaceutical composition for use according to the present invention, 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.

[0404] In certain embodiments, the invention relates to a viral vector, a host cell or a pharmaceutical composition for use according to the invention, wherein the brain tumor is a metastatic tumor originating from any form of breast cancer, lung cancer, colon cancer, testicular cancer, renal cancer, melanoma, ovarian cancer, prostate cancer, neuroendocrine tumor or any other solid tumor or any sarcoma, or any hematological tumor including all forms of leukemia and lymphoma.

[0405] The term "cancer," as used herein, refers to diseases characterized by dysregulated cell proliferation and / or growth. The term includes benign and malignant cancerous diseases, such as tumors, and can refer to invasive or non-invasive cancers. The term includes all types of cancer, including carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, and blastomas.

[0406] In certain embodiments, the present invention relates to a viral vector, a host cell or a pharmaceutical composition for use according to the present invention, 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, and 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.

[0407] As disclosed above, the present invention can be used in the prevention and / or treatment of diseases or disorders that originate or manifest in the brain or are based in the brain. Therefore, it is envisioned that hematopoietic stem cells containing the viral vector of the present invention are administered to a subject in need thereof. Once administered to a subject, the hematopoietic stem cells can migrate to the brain and differentiate into microglia-like macrophages or microglia.

[0408] Alternatively, the AAV-based viral vectors of the present invention, or pharmaceutical compositions comprising the AAV-based viral vectors of the present invention, can be applied directly to brain compartments for in vivo infection of cells in need thereof.

[0409] In order to more effectively replace microglia in the brain with the transduced cells of the present invention, it is preferred to deplete endogenous microglia before administering transduced cells.It has been reported that various treatment regimens that reduce the integrity of the blood-brain barrier result in the depletion of microglia.For example, Capotondo et al. demonstrated that brain pretreatment is helpful for the successful reconstitution of microglia after hematopoietic stem cell transplantation (Proc Natl Acad Sci US A. 2012 Sep 11; 109(37): 15018-15023).

[0410] In certain embodiments, the invention relates to a viral vector, a host cell, or a pharmaceutical composition for use according to the invention, wherein the bone marrow conditioning treatment comprises the use of a cytotoxic agent, an alkylating agent, busulfan, treosulfan, etoposide, lomustine, radiation therapy, targeted radiation therapy (e.g., an yttrium-90 labeled anti-CD45 antibody or an yttrium-90 labeled anti-CD66 antibody), ACK2 (anti-c-kit antibody), a CD117 antibody-drug conjugate, CD45-SAP, a colony-stimulating factor 1 (CSF1)-specific agent, PLX3397, BLZ9445, PLX5622, RG7155, PLX647, Ki20227, GW2580, IL-34, and / or dasatinib.

[0411] In certain embodiments, the invention relates to a viral vector, host cell or pharmaceutical composition for use according to the invention, wherein the CNS conditioning therapy comprises the use of busulfan.

[0412] In certain embodiments, the invention relates to a viral vector, host cell or pharmaceutical composition for use according to the invention, wherein the blood-brain barrier conditioning treatment comprises radiation therapy or targeted radiation therapy.

[0413] In a particular embodiment, the invention relates to a viral vector, a host cell or a pharmaceutical composition for use according to the invention, wherein the viral vector, the host cell or the pharmaceutical composition is administered after said treatment that reduces the integrity of the blood-brain barrier, in particular wherein the viral vector, the host cell or the pharmaceutical composition is administered at least half a day after the treatment that reduces the integrity of the blood-brain barrier.

[0414] That is, the viral vector, host cell or pharmaceutical composition of the present invention can be administered to a subject in need thereof 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days after the treatment that reduces the integrity of the blood-brain barrier.

[0415] It is important to understand that although the viral vectors of the present invention are particularly well suited for treating brain-based diseases or disorders due to the activity of their promoters in myeloid cells and microglia, they can also be used to target tumors in the CNS or any other part of the body. In principle, the viral vectors of the present invention can be used to treat cancer in any organ or tissue that is accessible to myeloid cells, such as macrophages or monocytes.

[0416] In a particular embodiment, the present invention relates to a viral vector according to the invention, a host cell according to the invention or a pharmaceutical composition according to the invention for use in the treatment of an autoimmune disease.

[0417] That is, the viral vector, host cell or pharmaceutical composition of the present invention can also be used in the treatment of autoimmune diseases.

[0418] The term "autoimmune disease" as used herein is defined as a disorder caused by an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive response to self-antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune bullous diseases other than pemphigus vulgaris, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, all types of vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis.

[0419] Transgenes that can be used to treat autoimmune diseases include IL-1, IL-1R antagonist, IL-2, IL-4, IL-10, TGF-beta, FOXP3, T-bet, GATA-3, CD36 family (CD36-L1, CD36-L2) binding CD1b, CD1c, CD1D, and T-cell receptor-recognized MHC-related protein 1 (MR1).

[0420] In a particular embodiment, the present invention relates to a viral vector according to the invention, a host cell according to the invention or a pharmaceutical composition according to the invention for use in the treatment of an autoinflammatory disease.

[0421] The term "autoinflammatory disease," as used herein, should be understood to encompass any autoinflammatory disease. Non-limiting examples of autoinflammatory diseases that can be treated with the viral vectors, host cells, or pharmaceutical compositions of the present invention include hypocomplementemic and normocomplementemic urticarial vasculitis, pericarditis, myositis, antisynthetase syndrome, scleritis, macrophage activation syndrome, Becet syndrome, PAPA syndrome, Blau syndrome, gout, adult and juvenile Still's disease, cryropyrinopathy, Muckle-Wells syndrome, familial cold-induced autoinflammatory syndrome, neonatal-onset multisystem inflammatory disease, familial Mediterranean fever, chronic infantile neurological cutaneous and articular syndrome, systemic juvenile idiopathic arthritis, hyper-IgD syndrome, Schnitzler syndrome, and TNF receptor-associated periodic syndrome (TRAPS).

[0422] Transgenes that can be used to treat autoinflammatory diseases include the IL-1 receptor antagonist, IL-1beta.

[0423] In a particular embodiment, the present invention relates to a viral vector according to the invention, a host cell according to the invention or a pharmaceutical composition according to the invention for use in the treatment of an allergic disease.

[0424] The term "allergic disease," as used herein, refers to any symptom, tissue damage, or loss of tissue function resulting from allergies, including, but not limited to, diseases such as atopic dermatitis, urticaria, contact dermatitis, allergic conjunctivitis, allergic rhinitis, allergic asthma, anaphylaxis, food allergies, and hay fever.

[0425] Transgenes that can be used to treat allergic diseases include genes encoding proteins including any portion of IgE, including Fc, Fab, including the variable and hypervariable regions of Fab; or antibodies and other receptor-binding proteins directed against any receptor on cells implicated in mediating allergic responses, including mast cells, eosinophils, B cells, and T cells. Additionally, soluble and potentially neutralizing binding proteins and peptides or antibodies should be induced by genes directed against IL-1, IL-4, IL-33, and any other cytokines, including all forms of interleukins and chemokines, implicated in allergic diseases.

[0426] In a particular embodiment, the present invention relates to a viral vector according to the invention, a host cell according to the invention or a pharmaceutical composition according to the invention for use in hematopoiesis and solid organ transplantation.

[0427] That is, the viral vector, host cell, or pharmaceutical composition of the present invention can be administered to a subject in need thereof prior to hematopoietic or solid organ transplantation. Transgenes that can be used in hematopoietic and solid organ transplantation include IL-1, IL-1R antagonist, IL-2, IL-4, IL-10, TGF-beta, FOXP3, T-bet, GATA-3, CD36 family (CD36-L1, CD36-L2) binding CD1b, CD1c, CD1D, and T cell receptor-recognized MHC-related protein 1 (MR1).

[0428] In certain embodiments, the present invention provides a method for treating a disease or disorder having its origin or manifestation in or based in the brain in a subject in need thereof, comprising: a) genetically modifying a population of hematopoietic stem cells and / or enriched CD34-positive bone marrow cells, comprising contacting the population of hematopoietic stem cells and / or enriched CD34-positive bone marrow cells with a viral vector of the present invention; or genetically modifying a population of myeloid cells and / or enriched myeloid cells, comprising contacting the population of myeloid cells and / or enriched myeloid cells with a viral vector of the present invention; b) intravenously administering the genetically modified cells obtained from step (a) to a subject in need thereof; c) treating a disease or disorder having its origin or manifestation in or based in the brain in a subject in need thereof; The present invention relates to a method comprising:

[0429] That is, the present invention further relates to a method for treating a disease or disorder in the brain. As described above, host cells containing the viral vector of the present invention can migrate to the brain of a subject suffering from a brain-based disease or disorder and replace microglia.

[0430] Thus, cells such as hematopoietic stem cells or myeloid cells can be transduced ex vivo with the viral vectors of the present invention. The population of transduced cells can then be administered to a subject in need. In certain embodiments, transduced hematopoietic stem cells are administered to a subject in need. This embodiment can be advantageous because stem cells have a higher potential to cross the blood-brain barrier than other cell types. However, the host cells can also be myeloid cells, such as monocytes and / or macrophages. However, monocytes and / or macrophages are preferably used in subjects with a compromised blood-brain barrier.

[0431] In a particular embodiment, the invention relates to a method according to the invention, wherein the population of hematopoietic stem cells and / or enriched CD34-positive bone marrow cells, or the population of myeloid cells and / or enriched myeloid cells, is obtained from a subject or an outpatient donor in need.

[0432] That is, in certain embodiments, the methods of the present invention involve the use of autologous cells. Those skilled in the art are aware of methods for enriching certain cell types from a subject's blood. Consequently, certain types of blood cells can be enriched from the blood of a subject in need, transduced with a viral vector of the present invention, and administered back to the subject in need. Autologous cells have the advantage of reducing the risk of an immunogenic response.

[0433] In other embodiments, the cells administered to a subject in need may originate from a foreign donor, and those skilled in the art will be aware of methods for identifying compatible donors or for manipulating the cells and / or the subject in need to reduce the risk of an immunogenic response.

[0434] In certain embodiments, the present invention provides a method for treating a disease or disorder having its origin or manifestation in or based in the brain in a subject in need thereof, comprising: a) mobilizing hematopoietic stem cells in a subject in need thereof; b) after mobilization of hematopoietic stem cells in step (a), intravenously administering the viral vector of the present invention to a subject in need thereof; c) treating a disease or disorder having its origin or manifestation in or based in the brain in a subject in need thereof; The present invention relates to a method comprising:

[0435] That is, the viral vector of the present invention or a pharmaceutical composition containing the viral vector of the present invention can be directly administered to a subject in need thereof. Preferably, the subject is pretreated with an agent that induces hematopoietic stem cell mobilization in the subject so that the mobilized hematopoietic stem cells can be infected in vivo with the viral vector of the present invention. Agents commonly used to stimulate hematopoietic stem cell mobilization from bone marrow are G-CSF and plerixafor. However, other agents that stimulate hematopoietic stem cell mobilization from bone marrow are known in the art and can be used as part of the claimed method.

[0436] In certain embodiments, hematopoietic stem cells transduced in vivo can migrate to the brain, where they differentiate into microglia or microglia-like cells. In such embodiments, the method can be used for the prevention and / or treatment of brain-based diseases and disorders. The microglia-like cells can express one or more transgenes required for the prevention and / or treatment of brain-based diseases or disorders. For example, when used for the prevention and / or treatment of one of the PGRN-associated diseases or disorders disclosed herein, the microglia-like cells can express PGRN. Alternatively, when used in the treatment of brain tumors, the microglia-like cells can express one of the cytokines disclosed herein.

[0437] In a particular embodiment, the present invention relates to a method according to the present invention, wherein mobilizing hematopoietic stem cells in a subject in need thereof comprises administering G-CSF and / or plerixafor (INN and USAN, trade name Mozobil), an immunostimulant used to mobilize hematopoietic stem cells into the bloodstream in cancer patients.

[0438] In a particular embodiment, the present invention relates to a method according to the invention, wherein the disease or disorder which has its origin or manifestation in 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 frontotemporal degenerative disease or disorder, in particular wherein the frontotemporal degenerative disease or disorder is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, neuronal ceroid lipofuscinosis and Parkinson's disease, and in particular wherein said viral vector encodes PGRN or a functional fragment thereof.

[0439] That is, a viral vector encoding a polypeptide that has progranulin or PGRN functionality and has at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9 can be used in the treatment of any of the PGRN-associated neurodegenerative diseases disclosed herein.

[0440] In a particular embodiment, the invention relates to a method according to the invention, wherein the disease or disorder having its origin or manifestation in or based in the brain 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 cancer, melanoma, prostate cancer or any other solid tumor or any sarcoma, or any hematological tumor including all forms of leukemia and lymphoma, in particular wherein the viral vector encodes IL-12, IFN-gamma, GM-CSF, G-CSF, IL-2, IL-15, IL-21 and / or IFN-alpha or functional fragments thereof.

[0441] In certain embodiments, the present invention relates to a method according to the present invention, wherein the method comprises an additional step of temporarily reducing the integrity of the blood-brain barrier, in particular, the step of 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.

[0442] In certain embodiments, the present invention relates to methods according to the present invention, wherein a treatment that reduces the integrity of the blood-brain barrier is performed before the administration of genetically modified cells to a subject in need thereof, and in particular, wherein the time interval between the treatment that reduces the integrity of the blood-brain barrier and the administration of the genetically modified cells is performed after the treatment that reduces the integrity of the blood-brain barrier.

[0443] The treatment for reducing blood-brain barrier integrity can be any one of the treatments disclosed herein, hi certain embodiments, the treatment for reducing blood-brain barrier integrity can be administered 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days prior to administration of the viral vector, host cell, or pharmaceutical composition of the present invention.

[0444] As mentioned above, the viral vectors, host cells or pharmaceutical compositions of the present invention can also be used to treat cancer in other parts of the body, i.e., in a particular embodiment, the present invention provides a method for treating cancer in a subject in need thereof, comprising: a) mobilizing hematopoietic stem cells in a subject in need thereof; b) after mobilization of hematopoietic stem cells in step (a), intravenously administering the viral vector of the present invention to a subject in need thereof; c) treating cancer in a subject in need thereof; The present invention relates to a method comprising:

[0445] For the treatment of cancer, it is preferred that the viral vector encodes at least one of IL-12, IFN-gamma, GM-CSF, G-CSF, IL-2, IL-15, IL-21 and / or IFN-alpha, or a functional fragment thereof.

[0446] In certain embodiments, the present invention provides a method for expressing a transgene in the brain and / or CNS of a subject, comprising: a) genetically modifying a population of hematopoietic stem cells and / or enriched CD34-positive bone marrow cells, comprising contacting the population of hematopoietic stem cells and / or enriched CD34-positive bone marrow cells with a viral vector according to the invention; or genetically modifying a population of myeloid cells and / or enriched myeloid cells, comprising contacting the population of myeloid cells and / or enriched myeloid cells with a viral vector according to the invention; b) administering the genetically modified cells obtained from step (a) intravenously or intrathecally to the subject in need thereof; c) expressing the transgene encoded by the viral vector in the brain and / or CNS of the subject; The present invention relates to a method comprising:

[0447] That is, the method can be used to express a transgene in the brain or central nervous system of a subject in need thereof. To do this, a population of cells can be transduced ex vivo with a viral vector of the present invention. In certain embodiments, the population of cells can be a population of hematopoietic stem cells or a population of enriched CD34-positive bone marrow cells. Preferably, the population of enriched CD34-positive bone marrow cells comprises hematopoietic stem cells and / or hematopoietic progenitor cells. In certain embodiments, the population of cells can be an enriched population of myeloid cells. The myeloid cells can be any of the myeloid cells disclosed herein. In certain embodiments, the myeloid cells can be macrophages.

[0448] The term "population of cells" is used to refer to a plurality of cells. For example, a population of hematopoietic stem cells refers to a plurality of stem cells. A population of hematopoietic stem cells can consist exclusively of hematopoietic stem cells. However, as used herein, "population of hematopoietic stem cells" is understood to preferably refer to a population of cells containing hematopoietic stem cells. That is, a "population of hematopoietic stem cells" can include other cell types, particularly CD34-positive cell types. Those skilled in the art are aware of methods for enriching hematopoietic stem cells from a mixture of cells, for example, from blood or bone marrow. For example, hematopoietic stem cells can be enriched based on the expression of the cell surface marker CD34, resulting in a population of enriched CD34-positive bone marrow cells. The enriched population of CD34-positive bone marrow cells can be a population of cells in which at least 70%, at least 80%, at least 90%, or at least 95% of the total cells in the population express the cell surface marker CD34.

[0449] An enriched population of myeloid cells is a population of cells in which at least 70%, at least 80%, at least 90%, or at least 95% of the total cells in the population are myeloid cells. Those skilled in the art will recognize combinations of cell surface markers that can be used to enrich for specific types or types of myeloid cells by flow cytometry.

[0450] The population of cells can be transduced with any of the viral vectors disclosed herein. The transduction step can be performed ex vivo. Those skilled in the art are aware of methods for transducing cells with viral vectors.

[0451] In a particular embodiment, the invention relates to a method according to the invention, wherein the population of hematopoietic stem cells and / or enriched CD34-positive bone marrow cells, or the population of myeloid cells and / or enriched myeloid cells, is obtained from the subject or an exogenous donor.

[0452] That is, the population of cells can include autologous or allogeneic cells, as described above.

[0453] In certain embodiments, the present invention provides a method for expressing a transgene in the brain and / or CNS of a subject, comprising: a) mobilizing hematopoietic stem cells in a subject; b) after mobilization of hematopoietic stem cells in step (a), intravenously administering the viral vector of the present invention to a subject in need thereof; c) expressing the transgene encoded by the viral vector in the brain and / or CNS of the subject; The present invention relates to a method comprising:

[0454] That is, in certain embodiments, the transgene can be delivered to a subject in need thereof in vivo. That is, preferably, the viral vector of the present invention can be administered directly to the subject after the subject has undergone stem cell mobilization therapy. Thus, in certain embodiments, the present invention relates to a method of the present invention, wherein mobilizing hematopoietic stem cells in a subject comprises administering G-CSF or plerixafor.

[0455] In certain embodiments, the present invention relates to a method according to the present invention, wherein the method comprises an additional step of reducing the integrity of the blood-brain barrier, in particular, the step of temporarily 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.

[0456] That is, migration of transduced hematopoietic stem cells to the brain may be more efficient if microglia are depleted in a subject in need thereof before the viral vector is administered. Methods and compounds for depleting microglia in a subject are known in the art and disclosed herein.

[0457] In certain embodiments, the present invention relates to methods according to the present invention, wherein a treatment that reduces the integrity of the blood-brain barrier is performed before the administration of genetically modified cells to a subject in need thereof, and in particular, wherein the time interval between the treatment that reduces the integrity of the blood-brain barrier and the administration of the genetically modified cells is performed after the treatment that reduces the integrity of the blood-brain barrier.

[0458] That is, the treatment for reducing the integrity of the blood-brain barrier can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days prior to administration of the viral vector of the present invention or a pharmaceutical composition comprising the viral vector of the present invention.

[0459] In certain embodiments, the present invention provides a method for treating a disease or disorder having its origin or manifestation in or based in the brain in a subject in need thereof, comprising: a) administering a viral vector of the present invention to the brain or intrathecally of a subject in need thereof; b) treating a disease or disorder having its origin or manifestation in or based in the brain in a subject in need thereof; The present invention relates to a method comprising:

[0460] That is, the viral vector of the present invention or a pharmaceutical composition comprising the viral vector of the present invention can be administered directly to the brain or spinal canal (intrathecally). In such an embodiment, it is preferred that the viral vector is an AAV-based viral vector. Thus, in a specific embodiment, the present invention relates to a viral vector of the present invention, wherein the viral vector is an AAV-based viral vector.

[0461] It is understood that the present methods can be used to treat or prevent any brain-based disease or disorder disclosed herein, particularly neurodegenerative diseases and disorders and cancer. The term "intrathecally," as used herein, means administration into or within the fluid-filled spaces between the thin layers of tissue that cover the brain and spinal cord.

[0462] The present invention provides novel retroviral vectors for use in human therapy, particularly for use in the treatment of diseases or disorders that have their origin in or are based in the brain, particularly PGRN-associated neurodegenerative diseases or disorders, including frontotemporal degenerative diseases or disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease. The present invention also provides retroviral 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 for use in the treatment of brain metastases originating from any form of breast cancer, lung cancer, colon cancer, testicular cancer, renal cancer, and melanoma, or any other solid tumor, and any hematological tumor, including all forms of leukemia and lymphoma.

[0463] In particular, the present invention provides retroviral gene therapy vectors, particularly lentiviral gene therapy vectors, comprising a nucleotide sequence encoding a therapeutic transgene, particularly a PGRN cDNA, under the control of a tissue-specific promoter, which can be used to transduce hematopoietic stem cells (HSCs). The specific vector architecture of the present invention results in exclusive expression of the therapeutic transgene in HSC-derived monocytes / macrophages, dendritic cells, and microglia-like and microglial cells in the brain, resulting in moderate levels of gene expression, avoiding hippocampal toxicity and neurodegeneration, and affecting neurons and glial cells, as seen with alternative constructs.

[0464] The vectors of the present invention include the safety features of a myeloid (myelo-) / microglia-specific promoter for phagocyte-specific expression, preferably, but not limited to, the miR223 gene promoter, or a fusion promoter construct containing the miR223 promoter, to drive transgene expression, particularly expression of the PGRN cDNA.

[0465] Thus, in a specific embodiment, the present invention relates to the introduction of a PGRN-encoding expression cassette comprising a myeloid / microglia-specific promoter, preferably, but not limited to, the miR223 promoter, into HSCs by a lentiviral self-inactivating (SIN) gene therapy vector.

[0466] In another specific embodiment, the present invention relates to the introduction of a PGRN-encoding expression cassette comprising a myeloid / microglia-specific promoter selected from the group consisting of the TMEM119 promoter, the P2RY12 promoter, the OLFML3 promoter, the AIF1 promoter, and the ITGAM promoter.

[0467] In a further specific embodiment, the present invention relates to the introduction of a PGRN-encoding expression cassette comprising a myeloid / microglia-specific promoter, preferably but not limited to, a miR223 fusion promoter, in particular a fusion promoter in which the miR223 promoter or a functional portion thereof is fused with the entire or functional portion of a promoter selected from the group consisting of the TMEM119 promoter, the P2RY12 promoter, the OLFML3 promoter, the AIF1 promoter, and the ITGAM promoter.

[0468] In one aspect, the present invention relates to the use of a TMEM119 promoter construct, a P2RY12 promoter construct, an OLFML3 promoter construct, or a fusion construct consisting of miR223 fused to the TMEM119, P2RY12, or OLFML3 promoter to drive PGRN expression in HSC-derived monocytes / macrophages, dendritic cells, and microglia-like cells or microglia following macrophage migration to the brain.

[0469] Transduction of the HSCs is followed by administration of the ex vivo treated HSCs to the patient. In a specific embodiment, the ex vivo treated HSCs are administered intravenously.

[0470] For HSC transplantation, the patient's bone marrow is pretreated with a suitable conditioning compound or treatment, particularly busulfan, treosulfan, radiation therapy, or a biological agent capable of depleting endogenous brain microglia, but preferably busulfan. This procedure allows the HSC-derived transgenic monocytes / macrophages or dendritic cells to enter the brain and achieve significant levels of chimerism of HSC-derived monocytes / macrophages, microglia-like macrophages, dendritic cells, and / or microglia cells in the brain, thereby delivering sufficient amounts of PGRN or other transgenes to the brain.

[0471] In specific embodiments of the invention, the patient is pre-treated with busulfan, treosulfan, radiation therapy or a biologic such as a monoclonal antibody-based or small molecule-based inhibitor of colony-stimulating factor 1 (CSF1) and a CSF1 receptor (CSF1R) inhibitor such as PLX3397, BLZ9445, PLX5622, RG7155, PLX647, Ki20227, GW2580 or the CSF1R-ligand IL-34, dasatinib, and any combination thereof, within a window of between the past 5 and the past 20 days prior to administration, but particularly within the last 8 days or last 15 days prior to induction.

[0472] The retroviral vectors of the present invention can also be used to target bone marrow-derived macrophages and microglia, which are involved in brain tumors and metastasis. The specific vector architecture of the present invention, including a myeloid / microglia-specific promoter, is the basis for the successful expression of proteins in bone marrow-derived monocytes / macrophages, dendritic cells, microglia-like cells, and microglia to reverse or slow tumor progression.

[0473] In particular, the present invention relates to the use of the retroviral vector constructs according to the present invention and described herein for the treatment of patients suffering from brain tumors, in particular 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.

[0474] In another specific embodiment, the present invention relates to the use of a retroviral vector construct according to the present invention and described herein for the treatment of patients suffering from brain metastases originating from any form of breast cancer, lung cancer, colon cancer, testicular cancer, renal cancer, and melanoma or any other solid tumor, as well as any hematological tumor, including all forms of leukemia and lymphoma.

[0475] In particular, the present invention provides the following embodiments: 1. A retroviral vector molecule comprising a nucleic acid molecule encoding a therapeutic polypeptide or combination of therapeutic polypeptides under the control of a myeloid / microglia-specific promoter, or a combination of myeloid-specific and microglia-specific promoters, particularly a fusion promoter, that drives expression of the therapeutic polypeptide or combination of therapeutic polypeptides in HSC-derived myeloid cells, HSC-derived blood monocytes / macrophages, dendritic cells, and in brain microglia or microglia-like cells following macrophage migration to the brain.

[0476] 2. The retroviral vector of embodiment 1, wherein the microglia-specific promoter is a promoter or promoter fragment having promoter functionality of a promoter selected from the group consisting of a TMEM119 promoter, a P2RY12 promoter, an OLFML3 promoter, an AIF1 promoter, and an ITGAM promoter.

[0477] 3. Myeloid / microglia-specific promoters (a) the AIF1 promoter or the ITGAM promoter; or (b) a fusion promoter comprising a promoter or a promoter fragment having the promoter functionality of the miR223 promoter and a promoter or a promoter fragment having the promoter functionality of a promoter selected from the group consisting of the TMEM119 promoter, the P2RY12 promoter, and the OLFML3 promoter. 2. The retroviral vector of embodiment 1, wherein the promoter or promoter fragment has promoter functionality of a promoter selected from:

[0478] 4. The retroviral vector of embodiment 3, wherein the promoter or promoter fragment having miR233 promoter functionality has at least 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or is a fragment thereof at least 200 nucleotides in length, and wherein this promoter or fragment still has the promoter functionality of the miR223 promoter.

[0479] 5. A retroviral vector according to any one of embodiments 1 to 3, wherein the promoter or promoter fragment having P2RY12 promoter functionality has at least 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the sequence set forth in SEQ ID NO: 2, or is a fragment thereof at least 200 nucleotides in length, and wherein the promoter or fragment still has the promoter functionality of the P2RY12 promoter.

[0480] 6. A retroviral vector according to any one of embodiments 1 to 3, wherein the promoter or promoter fragment having TMEM119 promoter functionality has at least 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the sequence set forth in SEQ ID NO: 3, or is a fragment thereof having a length of at least 200 nucleotides, and wherein the promoter or fragment still has the promoter functionality of the TMEM119 promoter.

[0481] 7. A retroviral vector described in any one of embodiments 1 to 3, wherein the promoter or promoter fragment having OLFML3 promoter functionality has at least 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the sequence set forth in SEQ ID NO: 4, or is a fragment thereof having a length of at least 200 nucleotides, and wherein this promoter or fragment still has the promoter functionality of the OLFML3 promoter.

[0482] 8. A retroviral vector according to any one of embodiments 1 to 3, wherein the promoter or promoter fragment having AIF1 promoter functionality has at least 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the sequence set forth in SEQ ID NO: 5, or is a fragment thereof having a length of at least 200 nucleotides, and wherein this promoter or fragment still has the promoter functionality of the AIF1 promoter.

[0483] 9. A retroviral vector described in any one of embodiments 1 to 3, wherein the promoter or promoter fragment having ITGAM promoter functionality has at least 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the sequence set forth in SEQ ID NO: 6, or is a fragment thereof having a length of at least 200 nucleotides, and wherein this promoter or fragment still has the promoter functionality of the ITGAM promoter.

[0484] 10. The retroviral vector of any one of embodiments 3 to 9, wherein the tissue-specific promoter is a miR223 promoter fusion promoter.

[0485] 11. The retroviral vector of any one of embodiments 1 to 10, wherein the therapeutic polypeptide is PGRN or a functional fragment thereof.

[0486] 12. The retroviral vector of embodiment 11, wherein the therapeutic polypeptide has at least 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the sequence set forth in SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, or a fragment thereof at least 50 amino acids in length, and wherein the polypeptide still provides PGRN functionality.

[0487] 13. The retroviral vector of embodiment 12, wherein the partial sequence of the PGRN polypeptide is at least 40 amino acids in length.

[0488] 14. The therapeutic polypeptide is selected from the group consisting of FasL / Fas, Trail / TRAIL-R, lymphotoxin beta, decoy receptors 1-3, TNF-alpha, TNF-alphaR, IFN-gamma, IFN-gamma receptor, IL-1-IL31, IL1R-IL31 receptor, IL-10, IL-12, IL-23, CXCL-10, PD-1L, PD-1, PD-2L, PD-2, granzyme B, granulysin, nitric oxide synthase, DNA methyltransferase, and the like. antagonists for various chemokines, chemokine receptors, VEGF, VEGF-receptors, metalloproteinases (e.g., MMP-9), tumor-specific ligands and receptors such as CD40 / CD40L, EGFR, annexin 1, FGFR- 11. The retroviral vector according to any one of embodiments 1 to 10, wherein the retroviral vector is selected from the group consisting of soluble and membrane-bound forms of: 1, Her2, St6galnac5, MMP1-28 and their counterparts TIMPS1-4 (tissue inhibitor of metalloproteinases), melanotransferrin, alpha4-beta1 integrin and its ligand endothelial cell VCAM-1, E-cadherin, alpha-v-beta3 integrin, alpha-v-beta5 integrin, alpha-v-beta6 integrin, alpha-v-beta8 integrin, mononucleotide variant neoantigens, INDEL frameshift neoantigens, splice variant antigens, fusion protein neoantigens, endogenous retroelement antigens, tumor-specific antigens, in particular tumor-specific antigens caused by cancer, in particular CCND1, BRCA, CEA, cancer-associated antigen 72-4 (CA72-4), cancer-associated antigen 19-9 (CA19-9), WT1 and NY-ESO-1.

[0489] 15. The retroviral vector of embodiment 14, wherein the therapeutic polypeptide is interferon gamma (IFN gamma) or a functional fragment thereof.

[0490] 16. The retroviral vector of embodiment 14, wherein the therapeutic polypeptide is P-selectin, MSH, GM-CSF, IL-12, TNF-alpha, or granzyme B.

[0491] 17. The retroviral vector of embodiment 15, wherein the therapeutic polypeptide has at least 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the sequence set forth in SEQ ID N...

Claims

1. A viral vector comprising a nucleic acid molecule encoding a therapeutic polypeptide or a combination of therapeutic polypeptides under the control of a promoter or a promoter fragment, wherein said promoter or promoter fragment drives expression of the therapeutic protein or the combination of therapeutic proteins in myeloid cells and microglia, and said promoter or promoter fragment is inactive in progenitor and / or stem cells; The promoter is (a) the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and (b) a TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:3, SEQ ID NO:23 or SEQ ID NO:24, or a functional fragment thereof. a fusion promoter comprising A viral vector in which the miR223 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof, is operably linked to the TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23 or SEQ ID NO: 24, or a functional fragment thereof.

2. The viral vector of claim 1, wherein the viral vector comprises at least one transcriptional regulatory element, and the at least one transcriptional regulatory element is positioned so as to inhibit or activate the transcriptional activity of the promoter.

3. the at least one transcriptional regulatory element comprises a binding site for a transcriptional activator or repressor, the transcriptional activator or repressor comprising: i) an antibiotic binding domain; ii) hormone-binding domain; iii) a steroid-binding domain; or iv) Dimerization factor system The viral vector of claim 2, comprising:

4. i) the antibiotic binding domain is a tetracycline / doxycycline binding domain, a macrolide binding domain, or a pristinamycin binding domain; ii) the hormone-binding domain is an RU486-binding domain or an abscisic acid-binding domain; iii) the steroid binding domain is an ecdysone binding domain; or iv) The viral vector of claim 3, wherein the dimerization factor system is a rapamycin-based or rapalog-based dimerization factor system.

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

6. the therapeutic polypeptide i) a polypeptide that restores cellular function and / or elicits a cellular response in a cell; or ii) Polypeptides that enable and / or increase cellular targeting specificity The viral vector according to any one of claims 1 to 5,

7. The polypeptide that restores cellular function and / or induces a cellular response in a cell is selected from the group consisting of PGRN, presenilin 1, presenilin 2, IL-2, IL-12, IL-15, IL-21, IFN-alpha, IFN-alpha receptor, IFN-gamma, IFN-gamma receptor, FasL / Fas, CD11b, selectin such as L-selectin or P-selectin, PSGL (P-selectin ligand), TRAIL, TRAIL-R, lymphotoxin beta (LT-β), LT-βR, decoy receptors 1 to 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, CXC 7. The viral vector of claim 6, comprising at least a fragment of one or more polypeptides selected from the group consisting of CXCR3 ligands such as CXCR3 ligands L9 and CXCL-10, PD-1, PD-1L, PD-2 (PDC2), PD-2L, granzyme B, granulysin, CD11b, TIGIT, CD112, CD155, nitric oxide synthase, DNA methyltransferase 3b (DNMT3b), Jumonji domain-containing protein 1A (JMJD1A), somatostatin, histone deacetylase (HDAC) such as HDAC3 or HDAC9, CSF1 receptor (CSF1R), IL-34, TAM, all chemokines and chemokine receptors, and all cytokines and cytokine receptors.

8. The polypeptide that enables and / or increases cellular targeting specificity enables and / or increases specificity for tumor antigens, and the tumor antigens are selected from the group consisting of VEGF, VEGF receptors, antagonists to metalloproteinases, CD40 / CD40L, EGFR, Annexin 1, FGFR-1, Her2, St6galnac5, MMP1-28, TIMPS1-4, melanotransferrin, alpha4-beta1in, and the like. The viral vector of claim 6, which is tegrin, VCAM-1, E-cadherin, alpha-v-beta3 integrin, alpha-v-beta5 integrin, alpha-v-beta6 integrin, alpha-v-beta8 integrin, CCND1, BRCA, CEA, cancer associated antigen 72-4 (CA72-4), cancer associated antigen 19-9 (CA19-9), WT1, CD11b, L-selectin, NY-ESO-1, or a fragment thereof.

9. a) a retroviral vector; or b) a foamy viral vector; or c) a viral vector selected from the group consisting of an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, a parvovirus vector, a coronavirus vector, and an alpha-retrovirus vector. The viral vector according to any one of claims 1 to 8,

10. The viral vector of claim 9, wherein the retroviral vector is a lentiviral vector or a lentiviral SIN vector.

11. a) the miR223 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or a functional fragment thereof; and b) a microglia-specific promoter that is the TMEM119 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 23 or SEQ ID NO: 24, or a functional fragment thereof. wherein the miR223 promoter, or the promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 1, or the functional fragment thereof, is operably linked to the microglia-specific promoter, or the functional fragment thereof.

12. 12. The fusion promoter of claim 11, wherein the fusion promoter comprises at least one transcriptional regulatory element, the at least one transcriptional regulatory element being positioned to inhibit or activate transcriptional activity of the promoter.

13. the at least one transcriptional regulatory element comprises a binding site for a transcriptional activator or repressor, the transcriptional activator or repressor comprising: i) an antibiotic binding domain; ii) hormone-binding domain; iii) a steroid-binding domain; iv) Dimerization factor system The fusion promoter of claim 12, comprising:

14. i) the antibiotic binding domain is a tetracycline / doxycycline binding domain, a macrolide binding domain, or a pristinamycin binding domain; ii) the hormone-binding domain is an RU486-binding domain or an abscisic acid-binding domain; iii) the steroid binding domain is an ecdysone binding domain; iv) The fusion promoter of claim 13, wherein the dimerization factor system is a rapamycin-based or rapalog-based dimerization factor system.

15. The fusion promoter is a) comprising the sequence set forth in SEQ ID NO: 28, or b) The fusion promoter of any one of claims 11 to 14, comprising a sequence having 90%, 91%, 92%, 93%, 94% or 95% sequence identity to the sequence set forth in SEQ ID NO: 28, wherein the promoter drives expression in microglia and / or myeloid cells.

16. A host cell comprising the viral vector of any one of claims 1 to 10.

17. The host cell of claim 16, which is a hematopoietic stem cell or a myeloid cell.

18. The host cell of claim 17 , wherein the hematopoietic stem cells are hematopoietic stem cells of a CD34-positive enriched cell population.

19. A pharmaceutical composition comprising a viral vector according to any one of claims 1 to 10 and / or a host cell according to any one of claims 16 or 18.

20. 20. A pharmaceutical composition according to claim 19 for use in medicine.

21. 20. The pharmaceutical composition of claim 19 for use in the treatment of a disease or disorder having its origin or manifestation in or based in the brain.

22. 20. The pharmaceutical composition of claim 19, wherein the viral vector encodes PGRN or a functional fragment thereof, for use in the prevention and / or treatment of a PGRN-related disease or disorder.

23. 23. The pharmaceutical composition for use according to claim 22, wherein the PGRN-related disease or disorder is a neurodegenerative disease or disorder.

24. 24. The pharmaceutical composition for use according to claim 23, wherein the neurodegenerative disease or disorder is a degenerative disease or disorder.

25. 25. The pharmaceutical composition for use according to claim 24, 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.

26. 20. The pharmaceutical composition of claim 19 for use in the treatment of cancer, lymphoma and / or sarcoma.

27. 27. The pharmaceutical composition for use according to claim 26, 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.

28. 28. The pharmaceutical composition for use according to claim 26 or 27, wherein the cancer, lymphoma and / or sarcoma is a brain tumor or brain metastasis.

29. 29. The pharmaceutical composition for use according to claim 28, 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.

30. 29. The pharmaceutical composition for use according to claim 28, wherein the brain tumor is a metastatic tumor originating from any form of breast cancer, lung cancer, colon cancer, testicular cancer, renal cancer, melanoma, ovarian cancer, prostate cancer, neuroendocrine tumor or any other solid tumor or any sarcoma, or any hematological tumor including all forms of leukemia and lymphoma.

31. 31. The pharmaceutical composition for use according to any one of claims 20 to 30, wherein said viral vector, said host cell or said pharmaceutical composition is administered in conjunction with a treatment that reduces the integrity of the blood-brain barrier.

32. 32. The pharmaceutical composition for use according to claim 31, wherein said treatment that reduces the integrity of the blood-brain barrier is a bone marrow conditioning treatment, a CNS conditioning treatment and / or a blood-brain barrier conditioning treatment.

33. 33. The pharmaceutical composition for use according to claim 32, wherein said bone marrow conditioning treatment comprises the use of cytotoxic agents, alkylating agents, busulfan, treosulfan, etoposide, lomustine, radiation therapy, targeted radiation therapy, 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 dasatinib.

34. 34. The pharmaceutical composition for use according to claim 32 or 33, wherein the CNS conditioning therapy comprises the use of busulfan.

35. The pharmaceutical composition for use according to any one of claims 32 to 34, wherein said blood-brain barrier conditioning treatment comprises radiation therapy or targeted radiation therapy.

36. 36. The pharmaceutical composition for use according to any one of claims 31 to 35, wherein said viral vector, said host cell or said pharmaceutical composition is administered after said treatment that reduces the integrity of the blood-brain barrier.

37. 37. The pharmaceutical composition for use according to claim 36, wherein the viral vector, the host cell or the pharmaceutical composition is administered no more than half a day after the treatment that reduces the integrity of the blood-brain barrier.

38. 20. The pharmaceutical composition of claim 19 for use in the treatment of an autoimmune disease.

39. 20. The pharmaceutical composition of claim 19 for use in the treatment of an autoinflammatory disease.

40. 20. The pharmaceutical composition of claim 19 for use in the treatment of an allergic disease.

41. 20. The pharmaceutical composition of claim 19 for use in hematopoiesis and solid organ transplantation.

42. 11. A composition for use in a method for treating a disease or disorder having its origin or manifestation in or based in the brain in a subject in need thereof, comprising the viral vector of any one of claims 1 to 10, said method comprising: a) genetically modifying a population of hematopoietic stem cells and / or enriched CD34-positive bone marrow cells, the step comprising contacting the population of hematopoietic stem cells and / or enriched CD34-positive bone marrow cells with the viral vector; or genetically modifying a population of myeloid cells and / or enriched myeloid cells, the step comprising contacting the population of myeloid cells and / or enriched myeloid cells with the viral vector; b) intravenously administering the genetically modified cells obtained from step (a) to the subject in need thereof; c) treating a disease or disorder having its origin or manifestation in or based in the brain in said subject in need thereof; A composition comprising:

43. 43. The composition of claim 42, wherein the population of hematopoietic stem cells and / or enriched CD34-positive bone marrow cells, or the population of myeloid cells and / or enriched myeloid cells, is obtained from the subject in need or an outpatient donor.

44. 11. A composition for use in a method for treating a disease or disorder having its origin or manifestation in or based in the brain in a subject in need thereof, comprising the viral vector of any one of claims 1 to 10, said method comprising: a) mobilizing hematopoietic stem cells in said subject in need; b) after said mobilization of hematopoietic stem cells in step (a), intravenously administering said composition to said subject in need thereof; c) treating a disease or disorder having its origin or manifestation in or based in the brain in said subject in need thereof; A composition comprising:

45. 45. The composition of claim 44, wherein said mobilization of hematopoietic stem cells in said subject in need thereof comprises administration of G-CSF and / or plerixafor.

46. 46. ​​The composition of any one of claims 42-45, wherein the disease or disorder that has its origin or manifestation in or is based in the brain is a PGRN-related disease or disorder.

47. 47. The composition of claim 46, wherein the PGRN-related disease or disorder is a neurodegenerative disease or disorder.

48. 48. The composition of claim 47, wherein the neurodegenerative disease or disorder is a degenerative disease or neurodegenerative disorder.

49. 49. The composition of claim 48, 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.

50. 50. The composition of any one of claims 46-49, wherein the viral vector encodes PGRN or a functional fragment thereof.

51. 46. ​​The composition of any one of claims 42 to 45, wherein the disease or disorder having its origin or manifestation in or based in the brain is a brain tumor.

52. 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 52. The composition of claim 51, wherein the brain tumor is a metastatic tumor originating from any form of breast cancer, lung cancer, colon cancer, testicular cancer, renal cancer, melanoma, prostate cancer or any other solid tumor or any sarcoma, or any hematological tumor including all forms of leukemia and lymphoma.

53. 53. The composition of claim 51 or 52, wherein the viral vector encodes IL-12, IFN-gamma, GM-CSF, G-CSF, IL-2, IL-15, IL-21 and / or IFN-alpha or functional fragments thereof.

54. 54. The composition of any one of claims 42 to 53, wherein the method comprises the additional step of reducing the integrity of the blood-brain barrier.

55. 55. The composition of claim 54, wherein the step of 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.

56. 56. The composition of claim 54 or 55, wherein a step of reducing the integrity of the blood-brain barrier is performed prior to said administration of said genetically modified cells to said subject in need thereof.

57. 57. The composition of claim 56, wherein the time interval between the step of reducing the integrity of the blood-brain barrier and the administration of the genetically modified cells is performed after the step of reducing the integrity of the blood-brain barrier.

58. 11. A composition for use in a method for treating cancer in a subject in need thereof, comprising the viral vector of any one of claims 1 to 10, said method comprising the steps of: a) mobilizing hematopoietic stem cells in said subject in need thereof; b) after said mobilization of hematopoietic stem cells in step (a), intravenously administering said composition to said subject in need thereof; c) treating cancer in said subject in need thereof; A composition comprising:

59. 59. The composition of claim 58, wherein said mobilization of hematopoietic stem cells in a subject in need thereof comprises administration of G-CSF and / or plerixafor.

60. 11. A composition for use in a method for expressing a transgene in the brain and / or CNS of a subject, comprising the viral vector of any one of claims 1 to 10, said method comprising: a) genetically modifying a population of hematopoietic stem cells and / or enriched CD34-positive bone marrow cells, the step comprising contacting the population of hematopoietic stem cells and / or enriched CD34-positive bone marrow cells with the viral vector; or genetically modifying a population of myeloid cells and / or enriched myeloid cells, the step comprising contacting the population of myeloid cells and / or enriched myeloid cells with the viral vector; b) intravenously administering the genetically modified cells obtained from step (a) to a subject in need thereof; c) expressing the transgene encoded by the viral vector in the brain and / or CNS of the subject; A composition comprising:

61. 61. The composition of claim 60, wherein the population of hematopoietic stem cells and / or enriched CD34-positive bone marrow cells, or the population of myeloid cells and / or enriched myeloid cells, is obtained from the subject or an outpatient donor.

62. 11. A composition for use in a method for expressing a transgene in the brain and / or CNS of a subject, comprising the viral vector of any one of claims 1 to 10, said method comprising: a) mobilizing hematopoietic stem cells in said subject; b) after said mobilization of hematopoietic stem cells in step (a), administering said composition intravenously to a subject in need thereof; c) expressing the transgene encoded by the viral vector in the brain and / or CNS of the subject; A composition comprising:

63. 63. The composition of claim 62, wherein said mobilization of hematopoietic stem cells in said subject comprises administration of G-CSF or plerixafor.

64. 64. The composition of any one of claims 60 to 63, wherein the method comprises the additional step of reducing the integrity of the blood-brain barrier.

65. 65. The composition of claim 64, wherein the step of 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.

66. 65. The composition of claim 64, wherein the step of reducing the integrity of the blood-brain barrier is performed prior to said administration of said genetically modified cells to said subject in need thereof.

67. 67. The composition of claim 66, wherein the time interval between the step of reducing the integrity of the blood-brain barrier and the administration of the genetically modified cells is performed after the step of reducing the integrity of the blood-brain barrier.

68. 11. A composition for use in a method for treating a disease or disorder having its origin or manifestation in or based in the brain in a subject in need thereof, comprising the viral vector of any one of claims 1 to 10, said method comprising: a) administering said composition to the brain or intrathecally of said subject in need thereof; and b) treating a disease or disorder having its origin or manifestation in or based in the brain in said subject in need thereof. A composition comprising:

69. 52. The composition of claim 51, wherein the viral vector is an AAV-based viral vector.

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