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

Abstract

The present invention provides a novel viral vector for use in the treatment of human diseases or disorders of brain origin or based on the brain, particularly PGRN-related neurodegenerative diseases or disorders, including frontotemporal degenerative diseases or disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease. [Solution] A viral vector is provided comprising a nucleic acid molecule encoding a therapeutic polypeptide or a combination of therapeutic polypeptides under the control of a promoter or promoter fragment, wherein the promoter or promoter fragment drives the expression of the therapeutic protein or combination of therapeutic proteins in myeloid cells and microglia, and the promoter or promoter fragment is inactive in progenitor cells and / or stem cells.

Description

Technical Field

[0001] The present invention provides a novel viral vector for use in human gene therapy, particularly for the treatment of PGRN-related neurodegenerative diseases or disorders, including frontotemporal degenerative diseases or disorders, particularly diseases or disorders having their origin in the brain or based on the brain, such as Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease. The present invention also provides a viral vector for use in the treatment of brain tumors, particularly brain tumors selected from the group consisting of glioblastoma, glioma, ganglioblastoma, astrocytoma, oligodendroglioma, PNET (primitive neuroectodermal), medulloblastoma, CNS lymphoma, and neuroblastoma or any other CNS tumor, and further for 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 any form of leukemia and lymphoma.

Background Art

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

[0003] A retroviral gene therapy vector is a viral vector 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 a retroviral particle and transported by it. Within a single gene therapy retroviral particle, two RNA molecules, along with a viral protein required for reverse transcription into double-stranded DNA, are encapsulated by a capsid structure made of viral protein. The viral capsid is encapsulated in a viral envelope that has the ability to fuse with the cell membrane of the target cell during the transduction process. The retroviral protein enables the 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 a significant cause of disability in middle-aged and older adults, resulting in the loss of physical and social independence. Treatment, as well as daily care at home or in nursing homes, presents major challenges for families, healthcare staff, and society. The prevalence of dementia in people over 60 is estimated at 5-7%, with over 35 million people affected worldwide in 2010.

[0005] Overall, up to 20% of all patients who develop dementia before the age of 65 have frontotemporal dementia (FTD). The study estimated the prevalence to be 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, the prevalence peaked at 42.6 / 100,000 between the ages of 65 and 69. There are currently no curative treatment options for neurodegenerative dementia, including FTD.

[0006] There are various estimates regarding the proportion of mutations in the GRN gene encoding granulin precursor protein or progranulin (PGRN) among all FTD cases. These estimates are roughly 5% (Gass et al. (2006) Hum Mol). Genet. 15: 2988-3001;Le Ber et al. (2007) Hum Mutat. 28: 846-55)~30%(Bunessi et al. This range extends to one-third of individuals under 65 years of age and two-thirds of individuals over 65 years of age (al. (2009) Neurobiology of Disease 33: 379-385) and represents a penetrant rate 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. This fact makes PGRN-deficient FTD a suitable target for therapeutic approaches aimed at restoring physiological levels of PGRN.

[0008] PGRNs are primarily expressed in microglia, the brain's resident counterparts of tissue-resident macrophages. In all 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 the previously reported studies, AAV virus gene therapy vectors were injected into the brains of mice, resulting in PGRN expression in neurons, but not in microglia. In addition, recent animal studies have shown that strong PGRN overexpression is associated with signs of neurotoxicity.

[0009] Therefore, there is a need for alternative treatment strategies that target physiological PGRN expression in brain microglia. This targeted strategy differs from the earlier attempts using AAV viral vectors, which resulted in neuronal PGRN overexpression and neurotoxicity. Furthermore, in this field, there is a need for safer strategies for expressing transgenes in myeloid cells, particularly after transduction of HSCs, in peripheral blood, peripheral tissues, and the brain / CNS. [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 [Overview of the project] [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. [Modes for carrying out the invention]

[0012] In certain embodiments, the present invention relates to 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 promoter fragment, wherein the promoter or promoter fragment drives the expression of a 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] In other words, the present invention is based on the remarkable identification of a promoter that can drive transgene expression in myeloid cells and microglia, but is silent in stem cells, particularly hematopoietic stem cells and hematopoietic stem / progenitor cells. Such a cell-specific promoter is advantageous in cell and gene therapy applications because it restricts vector activity, mediated by the expression of the associated transgene, to differentiated target cells, i.e., myeloid cells and microglia. This is particularly important because promoter / enhancer activity in undifferentiated stem cells can lead to troublesome problems 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 cellular function or immune responses. Therefore, the promoter of the present invention is advantageous over ubiquitous promoters because it 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 rearrangement of the myeloid compartment in the brain by cells derived from genetically modified hematopoietic stem cell transplants (Biffi et al. (2013) Science 341:1233158). Therefore, in the art, there is a need for promoters that facilitate transgene expression and restrict it to hematopoietic phagocytic cells and brain myeloid cells, i.e., microglia. Surprisingly, we have identified promoters that drive transgene expression in myeloid cells and microglia. The term "myeloid cells," as used herein, refers to a set of myeloid cell lineages, including granulocytes (neutrophils, eosinophils, and basophils), monocytes, macrophages, Kupffer cells, and mast cells. Furthermore, it also includes peripheral blood dendritic cells of myeloid origin, as well as dendritic cells and macrophages obtained in vitro from monocytes in the presence of appropriate culture conditions.

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

[0016] Myeloid cells in peripheral blood originate exclusively from hematopoietic stem cells (HSCs), while tissue-resident macrophages and microglia are thought to arise solely from yolk sac erythrocyte bone marrow precursors under normal conditions. Given these different origins of peripheral blood myeloid cells and microglia, it can be considered remarkable that the promoter of the present invention can drive expression in both cell types.

[0017] Importantly, the promoter of the present invention does not drive expression in stem cells or progenitor cells. In particular, the promoter of the present invention does not drive expression in hematopoietic stem cells and hematopoietic stem / progenitor cells (HSPCs) (see Figure 21).

[0018] As used herein, the term “hematopoietic stem and progenitor cell” or “HSPC” refers to cells identified by the presence of the antigen marker CD34 (CD34+), and is therefore characterized as CD34+ cells and the population of such cells. In certain embodiments, the term “HSPC” refers to cells identified by the presence of the antigen marker CD34 (CD34+) and the absence of the lineage (lin) marker, and is therefore characterized as CD34+ / Lin(-) cells and the population of such cells. Since it is recognized that a population of cells containing CD34+ and / or Lin(-) cells also contains hematopoietic progenitor cells, for the purposes of this application, the term “HSPC” includes hematopoietic stem cells and hematopoietic progenitor cells.

[0019] One skilled in the art is aware of 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 the promoter drives the expression of the fluorescent marker can be detected, for example, by flow cytometry. That is, if the fluorescent marker can be detected in a sufficient amount in the transduced cells, the promoter is said to drive the expression of the transgene in this cell type. However, if no fluorescent marker can be detected or only a very small amount can be detected in the transduced cells, the promoter is said not to drive expression in this cell type. One skilled in the art is further aware that cells can differentiate into other cell types during the transduction procedure. However, one skilled in the art is aware of specific combinations of cell surface markers for determining cell type before and after the transduction procedure. One skilled in the art recognizes that a description of no promoter activity is limited by the sensitivity of promoter-driven transgene product detection and that fluorescent proteins as transgene products, particularly, for example, EGFP with a 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 indicator 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 a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, this refers to a functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, when stimulating or modulating the transcription of a coding sequence in a suitable host cell or other expression system, the promoter sequence is operably linked to the coding sequence. Generally, a promoter transcriptional regulatory sequence operably linked to a transcribed sequence is physically proximal to the transcribed sequence, i.e., is cis-acting.

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

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

[0023] In other words, in certain embodiments, the promoter is the promoter miR223 or a functional fragment thereof. The term “miR223 promoter” refers to the sequence of Sequence ID No. 1 and / or any fragment thereof of at least 200 nucleotides, and / or any sequence or any fragment thereof of at least 200 nucleotides having greater than 95% sequence identity. [ka]

[0024] In this specification, the promoter miR223 drives expression in various myeloid cell types (Figures 11 and 18), but the literature has shown that it does not drive expression in microglia. Surprisingly, we detected miR223 promoter activity in an immortalized microglial cell line (Figure 13).

[0025] The miR223 promoter may 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 may have the same characteristics as the miR223 promoter.

[0026] Therefore, the term “miR223 promoter” also extends to the functional fragment of the miR223 promoter. The functional fragment of the miR223 promoter is a nucleotide sequence containing 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 as shown in SEQ ID NO: 1. The functional fragment of the miR223 promoter is defined as driving expression in the same cell type and at a comparable level as the promoter shown 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, the promoter may include two different nucleotide sequences containing 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, the promoter may include three different nucleotide sequences containing 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, the promoter may include four different nucleotide sequences containing 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 possess the promoter functionality of the miR223 promoter. A promoter is said to possess the functionality of the miR223 promoter if it drives expression at a comparable level in the same cell type and contains at least a certain degree of sequence similarity to the miR223 promoter.

[0029] A promoter is said to have some degree of similarity to the miR223 promoter if it contains 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] Instead, a promoter is said to have a certain degree of similarity to the miR223 promoter if it has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the sequence shown in Sequence ID No. 1.

[0031] Furthermore, if the promoter includes 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, and the contiguous stretch has at least 95% sequence identity with the corresponding fragment of SEQ ID NO: 1, then the promoter can be determined to have a certain degree of similarity to the miR223 promoter.

[0032] In other words, in a particular embodiment, the 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 with SEQ ID NO: 1, and the nucleic acid sequence has miR223 promoter activity.

[0033] The term “sequence identity,” as used herein, is determined by comparing two optimally aligned sequences across a comparison window, where the polynucleotide fragments in the comparison window may contain additions or deletions (e.g., gaps or overhangs) compared to a reference sequence that contains neither additions nor deletions for optimal alignment of the two sequences. The percentage of sequence identity is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues exist in both sequences, obtaining 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. The optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), 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 at Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by inspection.

[0034] In certain embodiments, the promoter is the promoter ITGAM 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 or any fragment thereof of at least 200 nucleotides having greater than 95% sequence identity. [ka]

[0035] In this specification, the promoter ITGAM drives expression in various myeloid cell types (Figures 11 and 18) and microglia (Figure 13).

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

[0037] Therefore, the term “ITGAM promoter” also extends to the functional fragment of the ITGAM promoter. The functional fragment of the ITGAM promoter is a nucleotide sequence containing 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 as shown in SEQ ID NO: 6. The functional fragment of the ITGAM promoter drives expression in the same cell type and at comparable levels as the promoter shown in SEQ ID NO: 6.

[0038] It should be further understood that the present invention encompasses promoters comprising two or more functional fragments of the ITGAM promoter. That is, in certain embodiments, the promoter may include two different nucleotide sequences containing 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, the promoter may include three different nucleotide sequences containing 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, the promoter may include four different nucleotide sequences containing 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 possess the promoter functionality of the ITGAM promoter. A promoter is said to possess the functionality of the ITGAM promoter if it drives expression at a comparable level in the same cell type and contains at least a certain degree of sequence similarity to the ITGAM promoter.

[0040] A promoter is said to have some degree of similarity to the ITGAM promoter if it contains 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] Instead, a promoter is said to have a certain degree of similarity to the ITGAM promoter if it has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the sequence shown in Sequence ID No. 6.

[0042] Furthermore, if the promoter includes 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, and the contiguous stretch has at least 95% sequence identity with the corresponding fragment of SEQ ID NO: 6, then the promoter can be determined to have a certain degree of similarity to the ITGAM promoter.

[0043] In other words, in a particular embodiment, the functional fragment of the 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 with SEQ ID NO: 6, and the nucleic acid sequence has ITGAM promoter activity.

[0044] In certain embodiments, the promoter is promoter AIF1 or a functional fragment thereof. The term “AIF1 promoter” refers to the sequence of Sequence ID No. 5 and / or any fragment thereof of at least 200 nucleotides, and / or any sequence or fragment thereof of at least 200 nucleotides having greater than 95% sequence identity. [ka]

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

[0046] Therefore, the term “AIF1 promoter” also extends to the functional fragment of the AIF1 promoter. The functional fragment of the AIF1 promoter is a nucleotide sequence containing 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 as shown in SEQ ID NO: 5. The functional fragment of the AIF1 promoter drives expression in the same cell type and at comparable levels as the promoter shown 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, the promoter may include two different nucleotide sequences containing 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, the promoter may include three different nucleotide sequences containing 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, the promoter may include four different nucleotide sequences containing 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 possess the promoter functionality of the AIF1 promoter. A promoter is said to possess the functionality of the AIF1 promoter if it drives expression at a comparable level in the same cell type and contains at least a certain degree of sequence similarity to the AIF1 promoter.

[0049] A promoter is said to have some degree of similarity to the AIF1 promoter if it contains 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] Instead, a promoter is said to have a certain degree of similarity to the AIF1 promoter if it has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the sequence shown in Sequence ID No. 5.

[0051] Furthermore, if the promoter includes 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, and the contiguous stretch has at least 95% sequence identity with the corresponding fragment of SEQ ID NO: 5, then the promoter can be determined to have a certain degree of similarity to the AIF1 promoter.

[0052] In other words, in a particular embodiment, the 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 with SEQ ID NO: 5, and 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 Sequence ID No. 2 and / or any fragment thereof of at least 200 nucleotides, and / or any sequence or fragment thereof of at least 200 nucleotides having greater than 95% sequence identity. [ka]

[0054] The P2RY12 promoter may have the sequence of SEQ ID NO: 2. However, those skilled in the art will recognize that the fragment and / or sequence variant of SEQ ID NO: 2 may have the same characteristics as the P2RY12 promoter.

[0055] Therefore, the term “P2RY12 promoter” also extends to functional fragments of the P2RY12 promoter. A functional fragment of the P2RY12 promoter is a nucleotide sequence containing 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 as shown in SEQ ID NO: 1. A functional fragment of the P2RY12 promoter drives expression in the same cell type and at a comparable level as the promoter shown 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, the promoter may include two different nucleotide sequences containing 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, the promoter may include three different nucleotide sequences containing 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, the promoter may include four different nucleotide sequences containing 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 possess the promoter functionality of the P2RY12 promoter. A promoter is said to possess the functionality of the P2RY12 promoter if it drives expression at a comparable level in the same cell type and contains at least a certain degree of sequence similarity to the P2RY12 promoter.

[0058] A promoter is said to have some degree of similarity to the P2RY12 promoter if it contains 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] Instead, a promoter is said to have a certain degree of similarity to the P2RY12 promoter if it has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the sequence shown in SEQ ID NO: 2, SEQ ID NO: 21, or SEQ ID NO: 22.

[0060] Furthermore, if the promoter includes 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, and the contiguous stretch has at least 95% sequence identity with the corresponding fragment of SEQ ID NO: 2, then the promoter can be determined to have a certain degree of similarity to the P2RY12 promoter.

[0061] In other words, in a particular embodiment, the 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 with SEQ ID NO: 1, and the nucleic acid sequence has P2RY12 promoter activity.

[0062] In other words, 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 or any fragment thereof of at least 200 nucleotides having more than 95% sequence identity. [ka]

[0063] The TMEM119 promoter may have the sequence of SEQ ID NO: 3. However, those skilled in the art will recognize that the fragment and / or sequence variant of SEQ ID NO: 3 may have the same characteristics as the TMEM119 promoter.

[0064] Therefore, the term “TMEM119 promoter” also extends to the functional fragment of the TMEM119 promoter. The functional fragment of the TMEM119 promoter is a nucleotide sequence containing 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 as shown in SEQ ID NO: 3. The functional fragment of the TMEM119 promoter drives expression in the same cell type and at a comparable level as the promoter shown in SEQ ID NO: 3. In certain embodiments, the functional fragment of the TMEM119 promoter has the sequence of SEQ ID NO: 23. In certain embodiments, the 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, the promoter may include two different nucleotide sequences containing 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: 3. In certain embodiments, the promoter may include three different nucleotide sequences containing 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: 3. In certain embodiments, the promoter may include four different nucleotide sequences containing 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: 3.

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

[0067] A promoter is said to have some degree of similarity to the TMEM119 promoter if it contains 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] Instead, a promoter is said to have a certain degree of similarity to the TMEM119 promoter if it has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24.

[0069] Furthermore, if the promoter includes 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, and the contiguous stretch has at least 95% sequence identity with the corresponding fragment of SEQ ID NO: 3, then the promoter can be determined to have a certain degree of similarity to the TMEM119 promoter.

[0070] In other words, in a particular embodiment, the 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 with SEQ ID NO: 3, and the nucleic acid sequence has TMEM119 promoter activity.

[0071] In certain embodiments, the promoter is 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 or any fragment thereof of at least 200 nucleotides having greater than 95% sequence identity. [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] Therefore, the term “OLFML3 promoter” also extends to the functional fragment of the OLFML3 promoter. The functional fragment of the OLFML3 promoter is a nucleotide sequence containing 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 as shown in SEQ ID NO: 4. The functional fragment of the OLFML3 promoter drives expression in the same cell type and at a comparable level as the promoter shown in SEQ ID NO: 4. In certain embodiments, the 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, the promoter may include two different nucleotide sequences containing 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: 4. In certain embodiments, the promoter may include three different nucleotide sequences containing 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: 4. In certain embodiments, the promoter may include four different nucleotide sequences containing 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: 4.

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

[0076] A promoter is said to have some degree of similarity to the OLFML3 promoter if it contains 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] Instead, a promoter is said to have a certain degree of similarity to the OLFML3 promoter if it has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25.

[0078] Furthermore, if the promoter includes 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, and the contiguous stretch has at least 95% sequence identity with the corresponding fragment of SEQ ID NO: 4, then the promoter can be determined to have a certain degree of similarity to the OLFML3 promoter.

[0079] In other words, in a particular embodiment, the 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 with SEQ ID NO: 4, and the nucleic acid sequence has OLFML3 promoter activity.

[0080] In certain embodiments, the promoter is a fusion promoter comprising (a) the miR223 promoter, a fragment thereof, or a promoter having miR223 functionality, and (b) a second promoter. In addition to its specific activity in myeloid cells and microglia, the promoter miR223 is attractive for use in cell and gene therapy applications due to its resistance to DNA methylation. This is important because differentiation from stem cells to myeloid cells or microglia-like cells is known to result in large-scale methylation of the promoter sequence, which typically leads to transgene silencing in differentiated cells. Therefore, the promoter miR223 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) TMEM119 promoter, a functional fragment thereof, or a promoter having TMEM119 functionality; (b) P2RY12 promoter, a functional fragment thereof, or a promoter having P2RY12 functionality; (c) OLFML3 promoter, a functional fragment thereof, or a promoter having OLFML3 functionality; (d) ITGAM promoter, a functional fragment thereof, or a promoter having ITGAM functionality; or (e) AIF1 promoter, a functional fragment thereof, or a promoter having AIF1 functionality.

[0082] The terms "miR223 fusion construct" or "miR223 fusion promoter" are, (i) A P2Y12 promoter, or a promoter fragment derived from the P2Y12 promoter, consisting of at least 200 nucleotides of the P2Y12 promoter sequence, fused to the P2Y12 promoter, (ii) A promoter fragment derived from the TMEM119 promoter, consisting of the TMEM119 promoter or at least 200 nucleotides of the TMEM119 promoter sequence, or (iii) 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) 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) The ITGAM promoter, or a promoter fragment derived from the ITGAM promoter, consisting of at least 200 nucleotides of the ITGAM promoter sequence, This refers to a promoter construct consisting of the miR223 promoter.

[0083] In other words, in a particular embodiment, the fusion promoter includes a miR223 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown 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 may include 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 with nucleotide sequence number 26 or 27.

[0085] That is, in a particular embodiment, the fusion promoter includes a miR223 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. It should be understood that the functional fragment is preferably a promoter having the functionality of miR223 and / or TMEM119 as defined above.

[0086] In certain embodiments, a fusion promoter comprising the miR223 promoter and the TMEM119 promoter may include 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 with 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 shown 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 shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. It should be 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 may include 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 with 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 shown 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 shown in SEQ ID NO: 6, or a functional fragment thereof. It should be 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 a miR223 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: 1, or a functional fragment thereof, and an AIF1 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: 5, or a functional fragment thereof. It should be understood that the functional fragment is preferably a promoter having miR223 and / or AIF1 functionality as defined above.

[0091] In a particular embodiment, the present invention provides a promoter that a) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the miR223 promoter, or a functional fragment thereof; or b) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the ITGAM promoter or the sequence shown in SEQ ID NO: 6, or a functional fragment thereof; or c) A TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a miR233 promoter operably linked to a functional fragment thereof, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 1, or a fusion promoter containing a functional fragment thereof. This relates to a vector according to the present invention.

[0092] In a particular embodiment, the present invention provides a promoter that a) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the miR223 promoter, or a functional fragment thereof; or b) A fusion promoter containing a functional fragment thereof, having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the miR233 promoter or the sequence shown in SEQ ID NO: 1. This relates to a vector according to the present invention.

[0093] In a particular embodiment, the present invention provides a promoter that a) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the miR223 promoter, 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 shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24; or a miR233 promoter operably linked to a functional fragment thereof, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: 1, or a fusion promoter containing a functional fragment thereof. This relates to a vector according to the present invention.

[0094] In a particular embodiment, the present invention provides a promoter that a) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the miR223 promoter, 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 with the sequence shown in SEQ ID NO: 23; or a miR233 promoter operably linked to a functional fragment thereof, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 1, or a fusion promoter containing a functional fragment thereof. This relates to a vector according to the present invention.

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

[0096] In certain embodiments, the present invention relates to a vector according to the present invention, which is a fusion promoter comprising (a) a miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in Sequence ID No. 1, operably linked to (b) a TMEM119 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in Sequence ID No. 23.

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

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

[0099] In other words, viral vectors may further include regulatory elements that enable more precise control of the expression of the transgene. The term “transcriptional regulatory element,” as used herein, refers to nucleic acid fragments capable of regulating the expression of one or more genes, preferably the transgene. The transcriptional regulatory element can activate or inhibit the expression of the transgene. Thus, the transcriptional regulatory element, the transgene, and the promoter are operably linked to one another.

[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 transcription activator or repressor. A transcription activator is a protein that activates the expression of a transgene when bound to the transcriptional regulatory element. A transcription repressor is a protein that prevents the expression of a transgene when bound to the transcriptional regulatory element.

[0101] In certain embodiments, transcription activators or repressors can undergo structural changes that determine their binding affinity to transcriptional regulatory elements. For example, if an activator is specifically bound by an inducer molecule, the transcription activator can only bind to transcriptional regulatory elements, thereby activating the expression of a transgene. Conversely, if an activator is specifically bound by an repressor molecule, the transcription repressor can only bind to transcriptional regulatory elements, thereby inactivating the expression of a transgene.

[0102] Those skilled in the art will recognize various systems that can be used to control the expression of a transgene from the promoter of the present invention. In certain embodiments, the present invention includes at least one transcriptional regulatory element comprising a binding site for a transcription activator or repressor, in particular, the transcription activator or repressor is i) Antibiotic-binding domains, particularly tetracycline / doxycycline-binding domains, macrolide-binding domains, or pristinamycin-binding domains; ii) Hormone-binding domains, particularly RU486-binding domains or abscisic acid-binding domains; iii) Steroid-binding domains, in particular ecdysone-binding domains; or iv) Dimerization factor systems, particularly rapamycin-based or rapalog-based dimerization factor systems This invention relates to a viral vector, including the present invention.

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

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

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

[0106] In certain embodiments, the expression of a transgene can be controlled by a dimerization system. A dimerization system is a transcription activator consisting of two separate proteins. The first protein contains a binding site for transcriptional regulatory elements contained in a viral vector, and a drug-binding domain thereon. The second protein contains another drug-binding domain, and an activator or repressor domain, each capable of inducing or repressing the expression of a transgene. Activation or repression by the dimerization system works only in the presence of a dimerization molecule that can be specifically bound by the drug-binding domains of both proteins, thereby bringing the two proteins into close proximity so that the expression of the transgene can be induced or repressed. Well-known examples of dimerization systems include those based on rapamycin or rapalog.

[0107] In a particular embodiment, the present invention relates to a viral vector according to the present invention, wherein the viral vector encodes a riboswitch, and the riboswitch controls the translation of mRNA encoding a therapeutic protein or a combination of therapeutic proteins.

[0108] In place of or in addition to transcriptional regulatory elements, the viral vector according to the present invention can encode a riboswitch that controls the translation of mRNA encoded by the transgene.

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

[0110] In certain embodiments, the present invention provides a therapeutic polypeptide, i) polypeptides that restore cellular function and / or induce cellular responses in cells or tissues; or ii) Polypeptides that enable and / or increase the target specificity of cells This relates to a viral vector according to the present invention.

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

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

[0113] In other words, in certain embodiments, the present invention relates to polypeptides that restore cellular function and / or induce cellular responses in cells, such as 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, 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-alpha R, MSH, G-CSF, GM-CSF, IL-1, IL-6, IL-7, IL-8, IL-31, IL1R, IL31R, IL-10, I 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 L-23, CXCL9 and CXCL-10, PD-1, PD-1L, PD-2 (PDC2), PD-2L, granzyme B, granulosin, CD11b, TIGIT, CD112, CD155, nitric oxide synthase, DNA methyltransferase 3b (DNMT3b), cross-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 polypeptides that restore cellular function and / or induce a cellular response in a cell include at least fragments 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, therapeutic proteins can be proteins that direct target cells to a specific location. For example, a therapeutic protein can be an antigen-binding molecule that directs transduced cells to a specific cell type or tissue. For instance, the expression of a protein that specifically binds to a tumor antigen can direct transduced cells, such as immune cells, to a tumor. In certain embodiments, the antigen-binding molecule may be or contain an antibody. In certain embodiments, the antigen-binding molecule may be or contain a fragment of an antibody. In certain embodiments, the antigen-binding molecule may be a chimeric antigen receptor (CAR).

[0116] In other words, in certain embodiments, the present invention relates to viral vectors according to the present invention in which polypeptides enable and / or increase the target specificity of cells, enabling and / or increasing specificity to tumor antigens, in particular, the tumor antigens being VEGF, VEGF receptor, antagonists to metalloproteinases (e.g., MMP-9), CD40 / CD40L, EGFR, annexin 1, FGFR-1, Her2, St6galnac5, MMP1-28, TIMPS1-4, melanotransferrin, alpha-4-beta-1 integrin, VCAM-1, E-cadherin, alpha-v-beta-3 integrin, alpha-v-beta-5 integrin, alpha-v-beta-6 integrin, alpha-v-beta-8 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 fragments thereof.

[0117] In certain embodiments, the present invention relates to a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene is a) PGRN or a functional fragment thereof; or b) A polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. Regarding viral vectors that code for this.

[0118] In other words, 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 sequences 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. The nucleic acid sequences encoding the said protein are also provided herein. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0119] Progranulin is a precursor protein of granulin. Cleavage of progranulin produces various active 6kDa 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 metalloproteinase are proteases that can cleave progranulin into individual granulin peptides. Progranulin and granulin can be further distinguished by their hypothetical opposing roles in cells. Progranulin is associated with anti-inflammatory activity, while the cleaved granulin peptides have been linked to pro-inflammatory behavior. Mutations in the progranulin (GRN) gene are a major cause of familial frontotemporal dementia. This mutation leads to haploinsufficiency, and therefore to decreased progranulin levels and GRN-related brain degenerative changes that manifest over several years, if not decades. In such cases, progranulin levels can be restored by the viral vector 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 having at least 95% sequence identity with SEQ ID NO: 7, SEQ ID NO: 8, and / or SEQ ID NO: 9, and is a fragment having progranulin activity. A protein is said to have progranulin activity if it can be cleaved into at least one type of 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 certain embodiments, the present invention relates to a viral vector encoding a polypeptide or a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, wherein one or more promoters are a) Myelo-specific promoter or a functional fragment thereof; and / or b) Microglia-specific promoters or functional fragments thereof; and / or c)i) A first promoter which is a bone marrow-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) Second promoter A fusion promoter that includes or will include This includes information about viral vectors.

[0122] In other embodiments, the PGRN or its functional fragment or variant disclosed above may be expressed from a bone marrow-specific promoter or its functional fragment. In other embodiments, the PGRN or its functional fragment or variant disclosed above may be expressed from a microglia-specific promoter or its functional fragment. In other embodiments, the PGRN or its functional fragment or variant disclosed above may be expressed from a fusion promoter, preferably the fusion promoter comprising a bone marrow-specific or microglia-specific promoter or its functional fragment.

[0123] The term "bone marrow-specific promoter," as used herein, refers to any promoter capable of driving expression in myeloid cells. Those skilled in the art will know how to identify whether a promoter can drive expression in myeloid cells. For example, myeloid cells such as the monocytic cell lineage 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 integration of the viral vector into the genome of the myeloid cells, the promoter is determined to be a bone marrow-specific promoter. Bone marrow-specific promoters within the scope of this invention include, but are not limited to, the miR223 promoter, the AIF1 promoter, and the ITGAM promoter.

[0124] In other words, in a particular embodiment, the present invention relates to a viral vector encoding a PGRN or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, wherein the bone marrow-specific promoter is a) miR233 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 1; or b) The ITGAM promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 6; or c) The AIF1 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 5. This concerns viral vectors.

[0125] As used herein, the term “microglia-specific promoter” refers to any promoter capable of driving expression in microglia. Those skilled in the art will know how to identify whether a promoter can drive expression in microglia. For example, a viral vector encoding a fluorescent marker can be transduced into microglia, such as an immortalized microglial cell line, under the control of the promoter in question. If the expression of the fluorescent marker can be detected in the microglia after the integration of the viral vector into the microglial genome, the promoter is determined to be a microglia-specific promoter. Microglia-specific promoters within the scope of this invention include, but are not limited to, the P2RY12 promoter, the TMEM119 promoter, the OLFML3 promoter, the ITGAM promoter, and the AIF1 promoter.

[0126] In other words, in a particular embodiment, the present invention relates to a viral vector encoding a PGRN or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, 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 with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof; or b) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. This concerns viral vectors.

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

[0128] The second promoter may be any promoter known in the art. However, in certain embodiments, the PGRNs disclosed above or their functional fragments or variants may be expressed from a fusion promoter comprising a bone marrow-specific promoter and a microglia-specific promoter. That is, any of the bone marrow-specific promoters disclosed above may be combined with any of the microglia-specific promoters disclosed above, in any order.

[0129] In certain embodiments, the PGRNs disclosed above, or their functional fragments or variants, may be expressed from a fusion promoter comprising miR223, a functional fragment thereof, or a promoter having miR223 functionality, and a microglia-specific promoter.

[0130] In other words, in a particular embodiment, the present invention relates to a viral vector encoding a PGRN or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, wherein the first promoter is a miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 1, or a functional fragment thereof, and the first promoter is i) A TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. ii) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof; iv) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the ITGAM promoter or the sequence shown in SEQ ID NO: 6, or a functional fragment thereof; and / or v) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the AIF1 promoter, or a functional fragment thereof. Regarding a viral vector that is operably linked to it.

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

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

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

[0134] In certain embodiments, the present invention relates to a viral vector encoding a PGRN or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, wherein the promoter is a fusion promoter comprising (a) a miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown 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 with SEQ ID NO: 28.

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

[0136] In certain embodiments, the present invention relates to a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene is a) IL-12 or a functional fragment thereof; or b) A polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 11; and / or a polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 12. Regarding viral vectors that code for this.

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

[0138] Preferably, the viral vector of the present invention encodes both the polypeptide according to SEQ ID NO: 11 and the polypeptide according to SEQ ID NO: 12. In certain embodiments, two IL-12 subunits, alpha and beta, can be linked 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 according to the present invention is a single-chain IL-12 variant. Single-chain IL-12 variants are 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 consists 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 homodimer of p40 are formed after protein synthesis. Therefore, the viral vector of the present invention preferably encodes both the alpha subunit (SEQ ID NO: 11) and the 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 lethal toxicity associated with systemic administration of IL-12 hinders its clinical application in the form of pure cytokine.

[0140] A functional fragment of IL-12 is a fragment of at least 50, at least 100 amino acids, 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 which is a fragment possessing IL-12 activity. Assays for determining whether a protein possesses IL-12 activity are 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 certain embodiments, the present invention relates to a viral vector encoding IL-12 or a functional fragment thereof, including its single-chain variant; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 11 and / or SEQ ID NO: 12, wherein one or more promoters are a) Myelo-specific promoter or a functional fragment thereof; and / or b) Microglia-specific promoters or functional fragments thereof; and / or c)i) A first promoter which is a bone marrow-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) Second promoter A fusion promoter that includes or will include This includes information about viral vectors.

[0142] In other embodiments, IL-12 or its functional fragment or variant disclosed above may be expressed from a bone marrow-specific promoter or its functional fragment. In other embodiments, IL-12 or its functional fragment or variant disclosed above may be expressed from a microglia-specific promoter or its functional fragment. In other embodiments, IL-12 or its functional fragment or variant disclosed above may be expressed from a fusion promoter, preferably the fusion promoter comprising a bone marrow-specific or microglia-specific promoter or its functional fragment.

[0143] In other words, in certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-12 or a functional fragment thereof, or a functional fragment thereof, wherein the bone marrow-specific promoter is a) The miR233 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 1; b) The ITGAM promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 6; c) The AIF1 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 5; or This concerns viral vectors.

[0144] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-12 or a functional fragment thereof, or a functional fragment thereof, wherein a microglia-specific promoter is provided. a) A TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof; or b) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. This concerns viral vectors.

[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 with the sequence shown in SEQ ID NO: 11 and / or SEQ ID NO: 12, wherein the first promoter is a bone marrow-specific promoter and the second promoter is a microglia-specific promoter, or vice versa.

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

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

[0148] That is, in a particular embodiment, 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 with the sequence shown in SEQ ID NO: 11 and / or SEQ ID NO: 12, wherein the first promoter is a miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 1, or a functional fragment thereof, and the first promoter is i) A TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. ii) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof; iv) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the ITGAM promoter or the sequence shown in SEQ ID NO: 6, or a functional fragment thereof; and / or v) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the AIF1 promoter, or a functional fragment thereof. Regarding a viral vector that is operably linked to it.

[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 with the sequence shown in SEQ ID NO: 11 and / or SEQ ID NO: 12, wherein the promoter is the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 11 and / or SEQ ID NO: 12, wherein the first promoter is an ITGAM promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 11 and / or SEQ ID NO: 12, wherein the promoter is a fusion promoter comprising (a) a miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 1, or a functional fragment thereof; and (b) a P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 2, SEQ ID NO: 21, or SEQ ID NO: 22, or a functional fragment thereof. In a particular embodiment, the fusion promoter includes 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 with SEQ ID NO: 26 or SEQ ID NO: 27.

[0152] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-12 or a functional fragment thereof, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising (a) a miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown 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 with SEQ ID NO: 28.

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

[0154] In certain embodiments, the present invention relates to a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene is a) Interferon-gamma (IFN-gamma) or its functional fragments; or b) A polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 10. Regarding viral vectors that code for this.

[0155] In other words, in certain embodiments, the present invention relates to a viral vector encoding interferon-gamma (IFN-gamma). The terms “interferon-gamma,” “IFN-gamma,” or “IFN-γ” refer to the protein sequence of Sequence ID No. 10, and / or any sequence having greater than 95% homology sequence identity. The nucleic acid sequences encoding the said protein are also provided herein. [ka] [ka]

[0156] IFN-gamma is a dimerizable soluble cytokine, 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 crucial cytokine 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 stems partly 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 has developed, 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 is approved by the U.S. Food and Drug Administration for the treatment of chronic granulomatous disease and osteopetrosis. It is currently under study 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 is not yet approved for use in any cancer immunotherapy. However, improved survival has been observed when IFN-gamma is 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 possesses IFN-gamma activity. Assays for determining whether a protein possesses IFN-gamma activity are 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 relates to a viral vector encoding a polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IFN-gamma or a functional fragment thereof, or the sequence shown in SEQ ID NO: 10, wherein one or more promoters are a) Myelo-specific promoter or a functional fragment thereof; and / or b) Microglia-specific promoters or functional fragments thereof; and / or c)i) A first promoter which is a bone marrow-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) Second promoter A fusion promoter that includes or will include This includes information about viral vectors.

[0160] In other embodiments, the IFN-gamma or its functional fragment or variant disclosed above may be expressed from a bone marrow-specific promoter or its functional fragment. In other embodiments, the IFN-gamma or its functional fragment or variant disclosed above may be expressed from a microglia-specific promoter or its functional fragment. In other embodiments, the IFN-gamma or its functional fragment or variant disclosed above may be expressed from a fusion promoter, preferably the fusion promoter comprising a bone marrow-specific or microglia-specific promoter or its functional fragment.

[0161] In other words, in certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IFN-gamma or a functional fragment thereof, or the sequence shown in SEQ ID NO: 10, wherein the bone marrow-specific promoter is a) The miR233 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 1; b) The ITGAM promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 6; c) The AIF1 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 5. This concerns viral vectors.

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

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

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

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

[0166] In other words, in a particular embodiment, 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 with the sequence shown in SEQ ID NO: 10, wherein the first promoter is the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 1, or a functional fragment thereof, and the first promoter is ii) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the TMEM119 promoter, or the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. i) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof; iv) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the ITGAM promoter or the sequence shown in SEQ ID NO: 6, or a functional fragment thereof; and / or v) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the AIF1 promoter, or a functional fragment thereof. Regarding a viral vector that is operably linked to it.

[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 with the sequence shown in SEQ ID NO: 10, wherein the promoter is the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 1, or a functional fragment thereof.

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

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

[0170] In certain embodiments, the present invention relates to a viral vector encoding an IFN-gamma or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 10, wherein the promoter is a fusion promoter comprising (a) a miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown 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 with SEQ ID NO: 28.

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

[0172] In certain embodiments, the present invention relates to a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene is a) GM-CSF or a functional fragment thereof; or b) A polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 13. Regarding viral vectors that code for this.

[0173] In other words, 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 Sequence ID No. 13 and / or any sequence having more than 95% homology sequence identity thereto. The nucleic acid sequences encoding the said protein 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, functioning as a cytokine. Pharmaceutical analogues of naturally occurring GM-CSF are called salglamostim and morglamostim. Unlike granulocyte colony-stimulating factor, which specifically promotes neutrophil proliferation and maturation, GM-CSF affects a wider range of 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 leave the circulation and migrate to tissues, where they mature into macrophages and dendritic cells. Therefore, this is part of the immune / inflammatory cascade, a crucial process in combating infection, where the activation of a small number of macrophages can lead to a rapid increase in their numbers.

[0175] A functional GM-CSF fragment is a fragment having at least 50, at least 100 amino acids, at least 110, at least 120, at least 130, or at least 140 amino acids, having at least 95% sequence identity with SEQ ID NO: 13, and possessing GM-CSF activity. Assays for determining whether a protein possesses GM-CSF activity are 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 certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with GM-CSF or a functional fragment thereof, or the sequence shown in SEQ ID NO: 13, wherein one or more promoters are a) Myelo-specific promoter or a functional fragment thereof; and / or b) Microglia-specific promoters or functional fragments thereof; and / or c)i) A first promoter which is a bone marrow-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) Second promoter A fusion promoter that includes or will include This includes information about viral vectors.

[0177] In other words, in certain embodiments, the GM-CSF or its functional fragment or variant variant disclosed above may be expressed from a bone marrow-specific promoter or its functional fragment. In other embodiments, the GM-CSF or its functional fragment or variant variant disclosed above may be expressed from a microglia-specific promoter or its functional fragment. In other embodiments, the GM-CSF or its functional fragment or variant variant disclosed above may be expressed from a fusion promoter, preferably the fusion promoter comprising a bone marrow-specific or microglia-specific promoter or its functional fragment.

[0178] In other words, in certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with GM-CSF or a functional fragment thereof, or the sequence shown in SEQ ID NO: 13, wherein the bone marrow-specific promoter is a) The miR233 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 1; b) The ITGAM promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 5; b) The AIF1 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 6. This concerns viral vectors.

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

[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 with the sequence shown in SEQ ID NO: 13, wherein the first promoter is a bone marrow-specific promoter and the second promoter is a microglia-specific promoter, or vice versa.

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

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

[0183] In other words, in a particular embodiment, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with GM-CSF or a functional fragment thereof, 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 with the sequence shown in SEQ ID NO: 1, or a functional fragment thereof, and the first promoter is i) A TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. ii) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof; iv) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the ITGAM promoter or the sequence shown in SEQ ID NO: 6, or a functional fragment thereof; and / or v) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the AIF1 promoter, or a functional fragment thereof. Regarding a viral vector that is operably linked to it.

[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 with the sequence shown 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 with the sequence shown in SEQ ID NO: 1, or a functional fragment thereof.

[0185] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with GM-CSF or a functional fragment thereof, 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 with the sequence shown in SEQ ID NO: 6, or a functional fragment thereof.

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

[0187] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with GM-CSF or a functional fragment thereof, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising (a) a miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown 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 with SEQ ID NO: 28.

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

[0189] In certain embodiments, the present invention relates to a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene is a) G-CSF or a functional fragment thereof; or b) A polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 14. Regarding viral vectors that code for this.

[0190] In other words, 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 Sequence ID No. 14 and / or any sequence having greater than 95% homology sequence identity. The nucleic acid sequences encoding the said protein 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 granulocytes and stem cells and release them into the bloodstream. Functionally, it is a cytokine and hormone, a type of colony-stimulating factor, produced by numerous different tissues. Pharmaceutical analogs of naturally occurring 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 myelosuppression 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 G-CSF is used in certain cancer patients to accelerate recovery from chemotherapy-induced neutropenia, reduce mortality, and enable higher-intensity treatment regimens. In a mouse model of Alzheimer's disease, G-CSF has been shown to reduce inflammation, lower amyloid-beta load, and reverse cognitive impairment. Due to its neuroprotective properties, G-CSF is currently being investigated for cerebral ischemia in Phase IIb clinical trials, and several clinical pilot studies have been published for other neurological disorders such as amyotrophic lateral sclerosis (ALS).

[0193] A functional G-CSF fragment is a fragment having at least 50, at least 100 amino acids, at least 120, at least 140, at least 160, or at least 180 amino acids, having at least 95% sequence identity with SEQ ID NO: 14, and possessing G-CSF activity. An assay for determining whether a protein possesses G-CSF activity is, for example, Mickiene et al., Human granulocyte-colony stimulating factor (G-CSF) / stem cell factor (SCF) fusion proteins: design, characterization, and activity; PeerJ. Described in the art by 2020; 8: e9788.

[0194] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with G-CSF or a functional fragment thereof, or the sequence shown in SEQ ID NO: 14, wherein one or more promoters are a) Myelo-specific promoter or a functional fragment thereof; and / or b) Microglia-specific promoters or functional fragments thereof; and / or c)i) A first promoter which is a bone marrow-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) Second promoter A fusion promoter that includes or will include This includes information about viral vectors.

[0195] In other words, in certain embodiments, the G-CSF or its functional fragment or variant variant disclosed above may be expressed from a bone marrow-specific promoter or its functional fragment. In other embodiments, the G-CSF or its functional fragment or variant variant disclosed above may be expressed from a microglia-specific promoter or its functional fragment. In other embodiments, the G-CSF or its functional fragment or variant variant disclosed above may be expressed from a fusion promoter, preferably the fusion promoter comprising a bone marrow-specific or microglia-specific promoter or its functional fragment.

[0196] In other words, in certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with G-CSF or a functional fragment thereof, or the sequence shown in SEQ ID NO: 14, wherein the bone marrow-specific promoter is a) The miR233 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 1; c) The ITGAM promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 6; or b) The AIF1 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 5. This concerns viral vectors.

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

[0198] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with G-CSF or a functional fragment thereof, or a functional fragment thereof, wherein the first promoter is a bone marrow-specific promoter and the second promoter is a microglia-specific promoter, or vice versa.

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

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

[0201] In other words, in a particular embodiment, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with G-CSF or a functional fragment thereof, 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 with the sequence shown in SEQ ID NO: 1, or a functional fragment thereof, and the first promoter is i) A TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. ii) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof; iv) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the ITGAM promoter or the sequence shown in SEQ ID NO: 6, or a functional fragment thereof; and / or v) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the AIF1 promoter, or a functional fragment thereof. Regarding a viral vector that is operably linked to it.

[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 with the sequence shown 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 with the sequence shown in SEQ ID NO: 1, or a functional fragment thereof.

[0203] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with G-CSF or a functional fragment thereof, 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 with the sequence shown in SEQ ID NO: 6, or a functional fragment thereof.

[0204] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with G-CSF or a functional fragment thereof, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising (a) a miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 1, or a functional fragment thereof; and (b) a P2RY12 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with 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 shown in SEQ ID NO: 14, or a functional fragment thereof, wherein 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 shown 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 shown in SEQ ID NO: 3, SEQ ID NO: 23 or SEQ ID NO: 24, or a functional fragment thereof, and is a fusion promoter. 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 shown in SEQ ID NO: 14, or a functional fragment thereof, wherein 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 shown 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 shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof, and is a fusion promoter. 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 certain embodiments, the invention is a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene is 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 shown 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 shown 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 shown in SEQ ID NO: 15 encoding, and relates to a viral vector.

[0208] That is, in certain embodiments, the invention relates to a viral vector encoding a GM-CSF - INF-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 INF-gamma as defined above, or any functional fragment or variant thereof. GM-CSF and INF-gamma, and functional fragments or variants thereof, can be expressed as separate polypeptides from the viral vector of the invention. In certain embodiments, GM-CSF and INF-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:

Chemical formula

Chemical formula

[0210] 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 sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence shown in SEQ ID NO: 15, or a first polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence shown 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 shown in SEQ ID NO: 13, wherein one or more promoters are a) Myelo-specific promoter or a functional fragment thereof; and / or b) Microglia-specific promoters or functional fragments thereof; and / or c)i) A first promoter which is a bone marrow-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) Second promoter A fusion promoter that includes or will include This includes information about viral vectors.

[0211] In other embodiments, the GM-CSF-INF-gamma co-expression construct or a functional fragment or variant thereof disclosed above may be expressed from a bone marrow-specific promoter or a functional fragment thereof. In other embodiments, the GM-CSF-INF-gamma co-expression construct or a functional fragment or variant thereof disclosed above may be expressed from a microglia-specific promoter or a functional fragment thereof. In other embodiments, the GM-CSF-INF-gamma co-expression construct or a functional fragment or variant thereof disclosed above may be expressed from a fusion promoter, preferably the fusion promoter comprising a bone marrow-specific or microglia-specific promoter or a functional fragment thereof.

[0212] In other words, in a particular embodiment, 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 shown in SEQ ID NO: 15, or a first polypeptide or a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence shown in SEQ ID NO: 10, and a second polypeptide or a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence shown in SEQ ID NO: 13, wherein the bone marrow-specific promoter is a) The miR233 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 1; b) The ITGAM promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 6; c) AIF1 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 5, This concerns viral vectors.

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

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

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

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

[0217] In other words, in a particular embodiment, the present invention relates to a GM-CSF-INF-gamma coexpression construct or a functional fragment thereof; or a viral vector encoding a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence shown in SEQ ID NO: 15, or a first polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence shown 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 shown in SEQ ID NO: 13, wherein the first promoter is a miR233 promoter, or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: 1, i) A TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. ii) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof; iv) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the ITGAM promoter or the sequence shown in SEQ ID NO: 6, or a functional fragment thereof; and / or v) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the AIF1 promoter, or a functional fragment thereof. Relating to a viral vector operably linked thereto.

[0218] In certain embodiments, the 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 shown in SEQ ID NO: 15, or a first polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown 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 shown 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 shown in SEQ ID NO: 1, or a functional fragment thereof.

[0219] In certain embodiments, the 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 shown in SEQ ID NO: 15, or a first polypeptide having at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown 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 shown in SEQ ID NO: 13, or a functional fragment thereof, wherein the first promoter is the ITGAM promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 6, or a functional fragment thereof.

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

[0221] In certain embodiments, the present invention relates to a GM-CSF-INF-gamma co-expression construct or a functional fragment thereof; or a nucleic acid sequence encoding at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 15; or a first polypeptide or a functional fragment thereof encoding at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 10; and a second polypeptide or a functional fragment thereof encoding at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 13. The present invention relates to a viral vector in which 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 with the sequence shown 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 with the sequence shown 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 with SEQ ID NO: 28.

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

[0223] In certain embodiments, the present invention relates to a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene is a) G-CSF and IFN-gamma, or functional fragments thereof; or b) A first polypeptide, or a functional fragment thereof, having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 10, and a second polypeptide, or a functional fragment thereof, having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 14; or c) Polypeptides having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 16 Regarding viral vectors that code for this.

[0224] In other words, in certain embodiments, the present invention relates to a viral vector encoding a G-CSF-INF-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 INF-gamma as defined above, or any functional fragment or variant thereof. G-CSF and INF-gamma, as well as 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 INF-gamma can be expressed as a fusion protein.

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

[0226] An exemplary G-CSF-INF-gamma coexpression construct may include the following nucleic acid sequences: [ka]

[0227] In certain embodiments, the present invention relates to a viral vector encoding a G-CSF-INF-gamma coexpression construct or a functional fragment thereof; or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 16, or a functional fragment thereof; or a first polypeptide or a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 10, and a second polypeptide or a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 14, wherein one or more promoters are a) myelo-specific promoter or a functional fragment thereof; and / or b) Microglia-specific promoters or functional fragments thereof; and / or c)i) A first promoter which is a bone marrow-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) Second promoter A fusion promoter that includes or will include This includes information about viral vectors.

[0228] In other embodiments, the G-CSF-INF-gamma co-expression construct or its functional fragment or variant variant disclosed above may be expressed from a bone marrow-specific promoter or its functional fragment. In other embodiments, the G-CSF-INF-gamma co-expression construct or its functional fragment or variant variant disclosed above may be expressed from a microglia-specific promoter or its functional fragment. In other embodiments, the G-CSF-INF-gamma co-expression construct or its functional fragment or variant variant disclosed above may be expressed from a fusion promoter, preferably the fusion promoter comprising a bone marrow-specific or microglia-specific promoter or its functional fragment.

[0229] In other words, in a particular embodiment, the present invention relates to a viral vector encoding a G-CSF-INF-gamma coexpression construct or a functional fragment thereof; or a nucleic acid sequence or a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 16; or a first polypeptide or a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 10; and a second polypeptide or a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 14, wherein the bone marrow-specific promoter is a) The miR233 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 1; b) The ITGAM promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 6; or c) The AIF1 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 5. This concerns viral vectors.

[0230] In certain embodiments, the present invention relates to a viral vector encoding a G-CSF-INF-gamma coexpression construct or a functional fragment thereof; or a nucleic acid sequence or a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 16; or a first polypeptide or a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 10; and a second polypeptide or a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 14, wherein the microglia-specific promoter is a) A TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof; b) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. This concerns viral vectors.

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

[0232] The second promoter may be any promoter known in the art. However, in certain embodiments, the G-CSF-INF-gamma coexpression constructs or functional fragments or variants thereof disclosed above may be expressed from a fusion promoter comprising a bone marrow-specific promoter and a microglia-specific promoter. That is, any of the bone marrow-specific promoters disclosed above may be combined with any of the microglia-specific promoters disclosed above, in any order.

[0233] In certain embodiments, the G-CSF-INF-gamma coexpression constructs disclosed above, or their functional fragments or mutant variants, may be expressed from a fusion promoter comprising miR223, its functional fragment, or a promoter having miR223 functionality, and a microglia-specific promoter.

[0234] In other words, in a particular 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 a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 16, or a first polypeptide or a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 10, and a second polypeptide or a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 14, wherein the first promoter is a miR233 promoter, or a promoter or a functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 1, i) A TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. ii) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof; iv) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the ITGAM promoter or the sequence shown in SEQ ID NO: 6, or a functional fragment thereof; and / or v) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the AIF1 promoter, or a functional fragment thereof. Regarding a viral vector that is operably linked to it.

[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 a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 16; or a first polypeptide or a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 10; and a second polypeptide or a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 14, wherein the promoter is a miR233 promoter; or a promoter or a functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 1.

[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 a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 16; or a first polypeptide or a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 10; and a second polypeptide or a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 14, wherein the first promoter is an ITGAM promoter; or a promoter or a functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 6.

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

[0238] In certain embodiments, the present invention encodes a G-CSF-INF-gamma co-expression construct or a functional fragment thereof; or a nucleic acid sequence or a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 16; or a first polypeptide or a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 10; and a second polypeptide or a functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 14. 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 with the sequence shown 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 with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. In a particular embodiment, 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 with SEQ ID NO: 28.

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

[0240] In certain embodiments, the present invention relates to a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene is a) IL-2 or a functional fragment thereof; or b) A polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 17. Regarding viral vectors that code for this.

[0241] In other words, 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 Sequence ID No. 17 and / or any sequence having more than 95% homology sequence identity thereto. The nucleic acid sequences encoding the said protein 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 white blood cells (leukocytes, often lymphocytes) that are responsible for immunity. IL-2 is part of the body's natural response to microbial infection, and its role is to distinguish between foreign ("non-self") and "self." IL-2 mediates its effects by binding to IL-2 receptors expressed by lymphocytes. The main sources of IL-2 are activated CD4+ T cells and activated CD8+ T cells.

[0243] Aldesleukin is a form of recombinant interleukin-2. It is manufactured using recombinant DNA technology, marketed as a protein therapy, and trademarked as Proleukin. It is approved by the Food and Drug Administration (FDA) and in several European countries for the treatment of cancer (malignant melanoma, renal cell carcinoma) at high intermittent doses and has been used extensively at continuous doses.

[0244] A functional fragment of IL-2 is a fragment having at least 50, at least 100 amino acids, at least 110, at least 120, at least 130, or at least 140 amino acids, having at least 95% sequence identity with SEQ ID NO: 17, and possessing IL-2 activity. Assays for determining whether a protein has IL-2 activity are 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 certain embodiments, the present invention relates to a viral vector encoding a polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-2 or a functional fragment thereof, or the sequence shown in SEQ ID NO: 17, wherein one or more promoters are a) Myelo-specific promoter or a functional fragment thereof; and / or b) Microglia-specific promoters or functional fragments thereof; and / or c)i) A first promoter which is a bone marrow-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) Second promoter A fusion promoter that includes or will include This includes information about viral vectors.

[0246] In other embodiments, IL-2 or a functional fragment or variant disclosed above may be expressed from a bone marrow-specific promoter or a functional fragment thereof. In other embodiments, IL-2 or a functional fragment or variant disclosed above may be expressed from a microglia-specific promoter or a functional fragment thereof. In other embodiments, IL-2 or a functional fragment or variant disclosed above may be expressed from a fusion promoter, preferably the fusion promoter comprising a bone marrow-specific or microglia-specific promoter or a functional fragment thereof.

[0247] In other words, in certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-2 or a functional fragment thereof, or the sequence shown in SEQ ID NO: 17, wherein the bone marrow-specific promoter is a) The miR233 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 1; b) The ITGAM promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 6; or c) The AIF1 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 5. This concerns viral vectors.

[0248] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-2 or a functional fragment thereof, or the sequence shown in SEQ ID NO: 17, wherein a microglia-specific promoter is provided. a) A TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof; b) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. This concerns viral vectors.

[0249] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-2 or a functional fragment thereof, or a functional fragment thereof, wherein the first promoter is a bone marrow-specific promoter and the second promoter is a microglia-specific promoter, or vice versa.

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

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

[0252] In other words, in a particular embodiment, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-2 or a functional fragment thereof, 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 with the sequence shown in SEQ ID NO: 1, or a functional fragment thereof, and the first promoter is i) A TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. ii) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof; iv) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the ITGAM promoter or the sequence shown in SEQ ID NO: 6, or a functional fragment thereof; and / or v) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the AIF1 promoter, or a functional fragment thereof. Regarding a viral vector that is operably linked to it.

[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 with the sequence shown in SEQ ID NO: 17, wherein the promoter is the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 1, or a functional fragment thereof.

[0254] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-2 or a functional fragment thereof, 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 with the sequence shown in SEQ ID NO: 6, or a functional fragment thereof.

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

[0256] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-2 or a functional fragment thereof, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising (a) a miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown 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 with SEQ ID NO: 28.

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

[0258] In certain embodiments, the present invention relates to a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene is a) IL-15 or a functional fragment thereof; or b) A polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 18. Regarding viral vectors that code for this.

[0259] In other words, 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 Sequence ID No. 18 and / or any sequence having more than 95% homology sequence identity thereto. The nucleic acid sequences encoding the said protein are also provided herein. [ka]

[0260] Interleukin-15 (IL-15) is a cytokine that has structural similarities to interleukin-2 (IL-2). Like IL-2, IL-15 binds to a complex composed of the IL-2 / IL-15 receptor beta chain (CD122) and common gamma chain (gamma-C, CD132), and signals through this complex. IL-15 is secreted by mononuclear phagocytes (and some other cells) after viral infection. This cytokine induces the proliferation of natural killer cells, i.e., 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. Survival signals that maintain memory T cells in the absence of antigens are provided by IL-15. 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. Similarly, in humans with celiac disease, IL-15 suppresses apoptosis in T lymphocytes by inducing Bcl-2 and / or Bcl-xL.

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

[0262] A functional fragment of IL-15 is a fragment having at least 50, at least 100 amino acids, at least 110, at least 120, at least 130, or at least 140 amino acids, having at least 95% sequence identity with SEQ ID NO: 18, and possessing IL-15 activity. Assays for determining whether a protein possesses IL-15 activity are 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 certain embodiments, the present invention relates to a viral vector encoding a polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-15 or a functional fragment thereof, or the sequence shown in SEQ ID NO: 18, wherein one or more promoters are a) Myelo-specific promoter or a functional fragment thereof; and / or b) Microglia-specific promoters or functional fragments thereof; and / or c)i) A first promoter which is a bone marrow-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) Second promoter A fusion promoter that includes or will include This includes information about viral vectors.

[0264] In other words, in certain embodiments, IL-15 or its functional fragment or variant variant disclosed above may be expressed from a bone marrow-specific promoter or its functional fragment. In other embodiments, IL-15 or its functional fragment or variant variant disclosed above may be expressed from a microglia-specific promoter or its functional fragment. In other embodiments, IL-15 or its functional fragment or variant variant disclosed above may be expressed from a fusion promoter, preferably the fusion promoter comprising a bone marrow-specific or microglia-specific promoter or its functional fragment.

[0265] In other words, in certain embodiments, the present invention relates to a viral vector encoding a polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-15 or a functional fragment thereof, or the sequence shown in SEQ ID NO: 18, wherein the bone marrow-specific promoter is a) The miR233 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 1; b) The ITGAM promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 6; or c) The AIF1 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 5. This concerns viral vectors.

[0266] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-15 or a functional fragment thereof, or the sequence shown in SEQ ID NO: 18, wherein a microglia-specific promoter is provided. a) A TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof; b) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. This concerns viral vectors.

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

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

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

[0270] In other words, in a particular embodiment, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-15 or a functional fragment thereof, 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 with the sequence shown in SEQ ID NO: 1, or a functional fragment thereof, and the first promoter is i) A TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. ii) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof; iv) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the ITGAM promoter or the sequence shown in SEQ ID NO: 6, or a functional fragment thereof; and / or v) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the AIF1 promoter, or a functional fragment thereof. Regarding a viral vector that is operably linked to it.

[0271] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-15 or a functional fragment thereof, 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 with the sequence shown in SEQ ID NO: 1, or a functional fragment thereof.

[0272] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-15 or a functional fragment thereof, 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 with the sequence shown in SEQ ID NO: 6, or a functional fragment thereof.

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

[0274] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-15 or a functional fragment thereof, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising (a) a miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown 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 with SEQ ID NO: 28.

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

[0276] In certain embodiments, the present invention relates to a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene is a) IL-21 or a functional fragment thereof; or b) A polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 19. Regarding viral vectors that code for this.

[0277] In other words, 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 Sequence ID No. 19 and / or any sequence having sequence identity with more than 95% homology thereto. The nucleic acid sequences encoding the said protein are also provided herein. [ka]

[0278] Interleukin-21 (IL-21) is a cytokine with potent regulatory effects in immune system cells, 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, showing that it enriches a population of central memory type CTLs with a distinctive CD28+CD127hiCD45RO+ phenotype and IL-2 production capacity. Tumor-reactive antigen-specific CTLs generated by priming in the presence of IL-21 resulted in a stable "helper-independent" phenotype. IL-21 is also described as having antitumor effects through a continuous and enhanced CD8+ cellular response to achieve persistent tumor immunity.

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

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

[0282] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-21 or a functional fragment thereof, or the sequence shown in SEQ ID NO: 19, wherein one or more promoters are a) Myelo-specific promoter or a functional fragment thereof; and / or b) Microglia-specific promoters or functional fragments thereof; and / or c)i) A first promoter which is a bone marrow-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) Second promoter A fusion promoter that includes or will include This includes information about viral vectors.

[0283] In other embodiments, IL-21 or its functional fragment or variant may be expressed from a bone marrow-specific promoter or its functional fragment. In other embodiments, IL-21 or its functional fragment or variant may be expressed from a microglia-specific promoter or its functional fragment. In other embodiments, IL-21 or its functional fragment or variant may be expressed from a fusion promoter, preferably the fusion promoter comprising a bone marrow-specific or microglia-specific promoter or its functional fragment.

[0284] In other words, in certain embodiments, the present invention relates to a viral vector encoding a polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-21 or a functional fragment thereof, or the sequence shown in SEQ ID NO: 19, wherein the bone marrow-specific promoter is a) The miR233 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 1; b) The ITGAM promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 6; or c) The AIF1 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 5. This concerns viral vectors.

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

[0286] a) A TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof; b) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. This concerns viral vectors.

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

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

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

[0290] In other words, in a particular embodiment, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-21 or a functional fragment thereof, 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 with the sequence shown in SEQ ID NO: 1, or a functional fragment thereof, and the first promoter is i) A TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. ii) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof; iv) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the ITGAM promoter or the sequence shown in SEQ ID NO: 6, or a functional fragment thereof; and / or v) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the AIF1 promoter, or a functional fragment thereof. Regarding a viral vector that is operably linked to it.

[0291] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-21 or a functional fragment thereof, 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 with the sequence shown in SEQ ID NO: 1, or a functional fragment thereof.

[0292] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-21 or a functional fragment thereof, 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 with the sequence shown in SEQ ID NO: 6, or a functional fragment thereof.

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

[0294] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IL-21 or a functional fragment thereof, or a functional fragment thereof, wherein the promoter is a fusion promoter comprising (a) a miR233 promoter or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown 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 with SEQ ID NO: 28.

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

[0296] In certain embodiments, the present invention relates to a viral vector comprising a transgene under the control of one or more promoters, wherein the transgene is a) IFN-alpha or its functional fragments; or b) A polypeptide or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 20. Regarding viral vectors that code for this.

[0297] In other words, 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 Sequence ID No. 20 and / or any sequence having more than 95% homology sequence identity. The nucleic acid sequences encoding the said protein are also provided herein. [ka]

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

[0299] When administered orally, IFNα2 is broken down by digestive enzymes and loses its activity. Therefore, IFNα2 is primarily administered by subcutaneous or intramuscular injection. Once in the bloodstream, IFNα2 is rapidly eliminated by the kidneys. Due to the short lifespan of IFNα2 in the body, several injections per week are required. Pegylated interferon alpha-2a and pegylated interferon 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) demonstrated efficacy in treating patients diagnosed with certain viral infections (such as chronic viral hepatitis B and C) or certain types of cancer (melanoma, renal cell carcinoma, and various hematological malignancies).

[0301] A functional IFN-alpha fragment is a fragment of at least 50, at least 100 amino acids, 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: 20 and possesses IFN-alpha activity. An assay for determining whether a protein possesses IFN-alpha activity is, for example, Moll et al., "The differential activity of interferon-α subtypes is consistent among distinct target genes." and cell types; described in the art by Cytokine. 2011 Jan; 53(1): 52-59.

[0302] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IFN-alpha or a functional fragment thereof, or the sequence shown in SEQ ID NO: 20, wherein one or more promoters are a) Myelo-specific promoter or a functional fragment thereof; and / or b) Microglia-specific promoters or functional fragments thereof; and / or c)i) A first promoter which is a bone marrow-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) Second promoter A fusion promoter that includes or will include This includes information about viral vectors.

[0303] In other words, in certain embodiments, the IFN-alpha or its functional fragment or variant variant disclosed above may be expressed from a bone marrow-specific promoter or its functional fragment. In other embodiments, the IFN-alpha or its functional fragment or variant variant disclosed above may be expressed from a microglia-specific promoter or its functional fragment. In other embodiments, the IFN-alpha or its functional fragment or variant variant disclosed above may be expressed from a fusion promoter, preferably the fusion promoter comprising a bone marrow-specific or microglia-specific promoter or its functional fragment.

[0304] In other words, in certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IFN-alpha or a functional fragment thereof, or the sequence shown in SEQ ID NO: 20, wherein the bone marrow-specific promoter is a) The miR233 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 1; b) The ITGAM promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 6; or c) The AIF1 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 5. This concerns viral vectors.

[0305] In certain embodiments, the present invention relates to a viral vector encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with IFN-alpha or a functional fragment thereof, or the sequence shown in SEQ ID NO: 20, wherein a microglia-specific promoter is provided. a) A TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof; b) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. This concerns viral vectors.

[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 with the sequence shown in SEQ ID NO: 20, wherein the first promoter is a bone marrow-specific promoter and the second promoter is a microglia-specific promoter, or vice versa.

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

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

[0309] In other words, in a particular embodiment, the present invention relates to a viral vector encoding an IFN-alpha or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 20, wherein the first promoter is the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 1, or a functional fragment thereof, and the first promoter is i) A TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. ii) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof; iv) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the ITGAM promoter or the sequence shown in SEQ ID NO: 6, or a functional fragment thereof; and / or v) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the AIF1 promoter, or a functional fragment thereof. Regarding a viral vector that is operably linked to it.

[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 with the sequence shown in SEQ ID NO: 20, wherein the promoter is the miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 1, or a functional fragment thereof.

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

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

[0313] In certain embodiments, the present invention relates to a viral vector encoding an IFN-alpha or a functional fragment thereof; or a polypeptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 20, wherein the promoter is a fusion promoter comprising (a) a miR233 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown 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 with SEQ ID NO: 28.

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

[0315] In certain embodiments, the present invention relates to a viral vector according to the present invention, wherein one or more promoters are a) Myelo-specific promoter or a functional fragment thereof; and / or b) Microglia-specific promoters or functional fragments thereof; and / or c)i) A first promoter which is a bone marrow-specific promoter or a microglia-specific promoter or a functional fragment thereof; and ii) Second promoter A fusion promoter that includes or will include This includes information about viral vectors.

[0316] In other words, any of the transgenes or functional fragments or variants disclosed above can be operably ligated to one or more promoters. In certain embodiments, the transgenes or functional fragments or variants disclosed above can be operably ligated to a bone marrow-specific promoter or a functional fragment thereof. In certain embodiments, the transgenes or functional fragments or variants disclosed above can be operably ligated to a microglia-specific promoter or a functional fragment thereof. In certain embodiments, the transgenes or functional fragments or variants disclosed above can be operably ligated to a fusion promoter comprising a bone marrow-specific or microglia-specific promoter or a functional fragment thereof and a second promoter. In certain embodiments, the present invention is a viral vector according to the present invention, wherein the bone marrow-specific promoter is a) miR233 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 1; or b) The ITGAM promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 6; or c) The AIF1 promoter or a functional fragment thereof; or a promoter or functional fragment thereof having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 5. This concerns viral vectors.

[0317] The term "bone marrow-specific promoter," as used herein, refers to any promoter capable of driving expression in myeloid cells. Those skilled in the art will know how to identify whether a promoter can drive expression in myeloid cells. For example, myeloid cells such as the monocytic cell lineage 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 integration of the viral vector into the genome of the myeloid cells, the promoter is determined to be a bone marrow-specific promoter. Bone marrow-specific promoters within the scope of this invention include, but are not limited to, the miR223 promoter, the AIF1 promoter, and the ITGAM promoter.

[0318] In other words, in a particular embodiment, the present invention relates to a viral vector according to the present invention, wherein a microglia-specific promoter is a) A TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof; or b) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof. This concerns viral vectors.

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

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

[0321] In other words, the fusion promoter may preferably include a bone marrow-specific promoter and a microglia-specific promoter. In certain embodiments, the microglia-specific promoter is fused to the 5' end of the bone marrow-specific promoter. In certain embodiments, the microglia-specific promoter is fused to the 3' end of the bone marrow-specific promoter.

[0322] In certain embodiments, the present invention relates to a viral vector according to the present invention, wherein the first promoter is a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the miR233 promoter or the sequence shown in SEQ ID NO: 1, or a functional fragment thereof, and the first promoter is i) A TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. ii) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof; iv) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the ITGAM promoter or the sequence shown in SEQ ID NO: 6, or a functional fragment thereof; and / or v) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the AIF1 promoter, or a functional fragment thereof. Regarding a viral vector that is operably linked to it.

[0323] In a particular embodiment, the present invention relates to a viral vector according to 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 with the sequence shown 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 with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof.

[0324] In certain embodiments, the present invention relates to a viral vector comprising at least one transcriptional regulatory element, wherein the at least one transcriptional regulatory element is arranged to inhibit or activate the transcriptional activity of a promoter.

[0325] In certain embodiments, the present invention relates to a transcriptional regulatory element comprising at least one transcriptional regulatory element comprising a binding site for a transcriptional activator or repressor, in particular, the transcriptional activator or repressor comprising i) an antibiotic binding domain, in particular a tetracycline / doxycycline binding domain, a macrolide binding domain, or a pristinamycin binding domain; ii) Hormone-binding domains, particularly RU486-binding domains or abscisic acid-binding domains; iii) Steroid-binding domains, particularly ecdysone-binding domains; iv) Dimerization factor systems, particularly rapamycin-based or rapalog-based dimerization factor systems This invention relates to a viral vector, including the present invention.

[0326] In a particular embodiment, the present invention relates to a viral vector according to the present invention, wherein the viral vector encodes a riboswitch, and the riboswitch controls the translation of mRNA encoding a therapeutic protein or a combination of the therapeutic proteins.

[0327] In other words, a viral vector encoding any one of the transgenes disclosed above, or any functional fragment or variant thereof, may include a regulatory element that enables control of the expression of the transgene. Preferably, the regulatory element is one of the regulatory elements disclosed elsewhere in this specification.

[0328] In certain embodiments, the present invention is a) Retroviral vectors, in particular lentiviral vectors, more specifically lentiviral SIN vectors; or b) Foam virus vector; or c) Viral vectors selected from the group consisting of adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, parvovirus vectors, coronavirus vectors, and alpha-retrovirus vectors. This relates to a viral vector according to the present invention.

[0329] The viral vector according to the present invention may be any type of viral vector that enables the delivery of a transgene to a mammalian cell, or preferably a human cell.

[0330] In certain embodiments, the viral vector is a retroviral vector. As used herein, the term “retrovirus” refers to a virus comprising a viral outer envelope glycoprotein shell, including but not limited to a membrane-fusion-active vesicular stomatitis virus (VSV) glycoprotein (VSVG) that encapsulates viral RNA, as well as viral proteins necessary for the reverse transcription of its genomic RNA into a linear double-stranded DNA copy and subsequent covalent integration of its genomic DNA into the host genome.

[0331] Retroviruses are a common tool for gene delivery (Miller, 2000, Nature. 357: 455-460). When a virus is integrated into the host genome, it is called a “provirus.” Proviruses act as templates for RNA polymerase II, directing the expression of RNA molecules that encode necessary structural proteins and enzymes that help explain the production of new viral particles. Examples of retroviruses include, but are not limited to, Moloney's mouse leukemia virus (M-MuLV), Moloney's mouse sarcoma virus (MoMSV), Harvey's mouse sarcoma virus (HaMuSV), mouse mammary cancer virus (MuMTV), gibbon leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, friend mouse leukemia virus, mouse 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. An example lentivirus is HIV (Human Immunodeficiency Virus). This includes, but is not limited to, HIV types 1 and 2; Visna-Maedi virus (VMV); Capricorn encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Monkey immunodeficiency virus (SIV). In one embodiment, an HIV-based vector skeleton (i.e., an HIV cis-acting sequence element) is preferred.

[0333] The term “vector” is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The nucleic acid to be transferred is generally ligated into, i.e., inserted into, the vector nucleic acid molecule. A vector may contain sequences that direct autonomous replication in a cell, or sequences 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-deficient retroviruses and lentiviruses.

[0334] Within the scope of this invention, viral vectors are used for transduction of target cells. The term "transduction" refers to the process of physically bringing a viral vector into contact with a target cell, subsequently introducing viral nucleic acid into the target cell, and in the case of retroviruses, reverse transcribing it into DNA and incorporating it into the target cell's genome, with the goal of creating a state that enables this.

[0335] The term "lentiviral vector" can be used to refer to a lentiviral-infected particle consisting of a biological membrane studded with viral envelope glycoproteins that encapsulates a lentiviral capsid structure formed by lentiviral proteins, or a simple biological membrane without a viral envelope protein shell that has membrane fusion capabilities. The capsid structure encapsulates lentiviral RNA, as well as lentiviral proteins necessary for reverse transcription and stable integration into the genome of target cells.

[0336] Lentiviral vectors enable 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 along with in vitro transduction, while AAV-infected particles can be used for the delivery of DNA to non-dividing cells by in vivo injection.

[0337] Preferably, the viral vector according to the present invention is a self-inactivating lentiviral vector. A "self-inactivating" (SIN) vector is a viral or lentiviral vector in which the replication-deficient vector, for example, 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. Consequently, the vector can infect and then integrate into the host genome only once, and cannot be transmitted further. This is because the right (3')LTR U3 region is blocked during viral replication, preventing the left (5')LTR The U3 region is used as a template, and therefore, viral transcripts cannot be produced without the U3 enhancer promoter. If viral transcripts are not produced, processing and packaging into virions are impossible, and thus the life cycle of the virus ends.

[0338] In certain embodiments, the viral vector may be a foamy viral vector. The term “foamy viral vector,” as used herein, refers to a viral vector that utilizes a portion 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 a particular embodiment, the viral vector is selected from the group consisting of adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, parvovirus vectors, coronavirus vectors, and alpha-retrovirus vectors.

[0340] As used herein, the term “adenovirus vector” refers to a viral vector or plasmid containing structural and functional genetic elements or portions thereof, primarily derived from adenoviruses.

[0341] As used herein, the term “adenovirus” refers to members of the family Adenoviridae. Adenoviridae 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 adenovirus vectors are known to those skilled in the art (e.g., 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), (See S164-S171).

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

[0344] As used herein, the term "alpha-retroviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements or portions thereof, primarily derived from alpha-retroviruses. As used herein, the term "alpha-retrovirus" refers to any virus of the genus Alpharetrovirus. Methods for obtaining alpha-retroviral vectors are known to those skilled in the art (see, for example, Garoff, Henrik, and Kejun Li, 1998, Gene Therapy. 61–69).

[0345] In certain embodiments, the viral vector may be an adeno-associated virus (AAV) vector. Currently, there are two classes of recombinant AAVs (rAAVs) in use: single-stranded AAVs (ssAAVs) and self-complementary AAVs (scAAVs). ssAAVs are packaged as either a sense (positive strand) or antisense (negative strand) genome.

[0346] In other words, in certain embodiments, the viral vector is a DNA-based viral vector. In such embodiments, the viral DNA can be directly incorporated into the genome of a target cell without reverse transcription of the viral DNA.

[0347] In certain embodiments, the present invention is a) a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the miR223 promoter, or a functional fragment thereof; and b) Microglia-specific promoters, or their functional fragments. The present invention relates to a fusion promoter comprising a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the miR223 promoter or the sequence shown in Sequence ID No. 1, or a functional fragment thereof, which is operably linked to the microglia-specific promoter or the functional fragment thereof.

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

[0349] Therefore, in certain embodiments, the present invention provides a microglia-specific promoter, a) A TMEM119 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof; b) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown 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 with the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 25, or a functional fragment thereof, d) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the ITGAM promoter or the sequence shown in SEQ ID NO: 6, or a functional fragment thereof; or e) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the AIF1 promoter, or a functional fragment thereof. This relates to a fusion promoter according to the present invention.

[0350] In certain embodiments, the fusion promoter includes the miR223 promoter and the P2RY12 promoter. That is, in certain embodiments, the present invention is a) a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the miR223 promoter, or a functional fragment thereof; and b) A P2RY12 promoter, or a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 2, SEQ ID NO: 21, or SEQ ID NO: 22, or a functional fragment thereof. Regarding fusion promoters, including those mentioned.

[0351] In certain embodiments, the miR223-P2RY12 fusion promoter according to the present invention includes a nucleotide sequence shown in SEQ ID NO: 26 or SEQ ID NO: 27. In certain embodiments, the miR223-P2RY12 fusion promoter according to the present invention includes 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 with the nucleotide sequence shown in SEQ ID NO: 26 or SEQ ID NO: 27.

[0352] In certain embodiments, the fusion promoter includes the miR223 promoter and the TMEM119 promoter. That is, in certain embodiments, the present invention is a) a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the miR223 promoter, 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 with the sequence shown in SEQ ID NO: 3, SEQ ID NO: 23, or SEQ ID NO: 24, or a functional fragment thereof. Regarding fusion promoters, including those mentioned.

[0353] In certain embodiments, the miR223-TMEM119 fusion promoter according to the present invention includes the nucleotide sequence shown in SEQ ID NO: 28. In certain embodiments, the miR223-TMEM119 fusion promoter according to the present invention includes 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 with the nucleotide sequence shown in SEQ ID NO: 28.

[0354] In certain embodiments, the fusion promoter includes the miR223 promoter and the OLFML3 promoter. That is, in certain embodiments, the present invention is a) a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the miR223 promoter, 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 with the sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9, or a functional fragment thereof. Regarding fusion promoters, including those mentioned.

[0355] In certain embodiments, the miR223-OLFML3 fusion promoter according to the present invention includes the nucleotide sequence shown in SEQ ID NO: 29. In certain embodiments, the miR223-OLFML3 fusion promoter according to the present invention includes 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 with the nucleotide sequence shown in SEQ ID NO: 29.

[0356] In certain embodiments, the fusion promoter includes the miR223 promoter and the AIF1 promoter. That is, in certain embodiments, the present invention is a) a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the miR223 promoter, or a functional fragment thereof; and b) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the AIF1 promoter or the sequence shown in SEQ ID NO: 5, or a functional fragment thereof. Regarding fusion promoters, including those mentioned.

[0357] In certain embodiments, the fusion promoter includes the miR223 promoter and the ITGAM promoter. That is, in certain embodiments, the present invention is a) a promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the miR223 promoter, or a functional fragment thereof; and b) A promoter having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the ITGAM promoter or the sequence shown in SEQ ID NO: 6, or a functional fragment thereof. Regarding fusion promoters, including those mentioned.

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

[0359] In certain embodiments, the present invention relates to a transcriptional regulatory element comprising at least one transcriptional regulatory element comprising a binding site for a transcriptional activator or repressor, in particular, the transcriptional activator or repressor comprising i) an antibiotic binding domain, in particular a tetracycline / doxycycline binding domain, a macrolide binding domain, or a pristinamycin binding domain; ii) Hormone-binding domains, particularly RU486-binding domains or abscisic acid-binding domains; iii) Steroid-binding domains, particularly ecdysone-binding domains; iv) Dimerization factor systems, particularly rapamycin-based or rapalog-based dimerization factor systems The present invention relates to a fusion promoter, including the present invention.

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

[0361] In other words, the fusion promoter of the present invention may include a regulatory element that enables more precise control of the expression of the transgene. Preferably, the regulatory element is one of the regulatory elements disclosed elsewhere in this specification.

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

[0363] In certain embodiments, the present invention provides a fusion promoter, a) Contains the sequence shown in Sequence ID No. 28: or b) A sequence having 90%, 91%, 92%, 93%, or 95% sequence identity with the sequence shown in Sequence ID No. 28, The present invention relates to a fusion promoter that drives expression in microglia and / or myeloid cells.

[0364] In certain embodiments, the present invention relates to a host cell containing a viral vector according to the present invention.

[0365] In other words, the present invention further relates to a host cell containing a viral vector according to the present invention. In certain embodiments, the host cell may be a cell used for the production of the viral vector according to 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., a HSC) or its progenitor cell (e.g., a macrophage) containing viral nucleic acid, regardless of its virus-producing capacity, that is infected with an infectious viral particle.

[0366] When a host cell 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 producing cell," the host cell is also said to contain the viral vector according to the present invention. Therefore, the viral vector does not necessarily have to be in a circular form contained within the host cell.

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

[0368] In certain embodiments, the host cell may be a hematopoietic stem cell. That is, in certain embodiments, the present invention relates to a hematopoietic stem cell transduced with one of the viral vectors disclosed herein.

[0369] The term "hematopoietic stem cell," or "HSC," or "HSPC," is synonymous with the term "hematopoietic stem cell," relating to any cell population obtained by (but not limited to) bone marrow aspiration, apheresis after stem cell recruitment, or from umbilical cord blood, and / or relating to any cell population enriched with CD34-positive or CD133-positive cells by any method (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” means that the population contains a greater number and / or a higher percentage of CD34-positive cells than those found in the cell population prior to the enrichment step. Various methods for CD34-positive cell enrichment are known to those skilled in the art (e.g., Baldwin, K. et. al., 2015, Stem cells, 33(5), 1532-1542; Wojciechowski, Joel C et al., 2008, British journal of haematology 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 a CD34-positive myeloid-enriched population. In a more preferred embodiment, the host cells are hematopoietic stem and progenitor cells in a CD34-positive myeloid-enriched population. In the most preferred embodiment, the host cells are hematopoietic stem cells in a CD34-positive myeloid-enriched population.

[0372] In other embodiments, the host cell may be a myeloid cell. That is, in certain embodiments, the present invention relates to granulocytes (neutrophils, eosinophils, and basophils), monocytes, macrophages, Kupffer cells, or mast cells transduced with one 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 will recognize the methods for enriching and / or identifying the cell types disclosed above, as well as methods for transducing them with a viral vector.

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

[0375] In other words, 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, the pharmaceutical composition comprises a viral vector according to the present invention. In such embodiments, the pharmaceutical composition is preferably used for transduction of target cells, such as hematopoietic stem cells, in vivo. Alternatively, the pharmaceutical composition can be administered directly to the target in which it is needed, so that the viral vector contained in the pharmaceutical composition transduces 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 other embodiments, the pharmaceutical composition comprises a host cell containing a viral vector according to the present invention. Such a host cell can be obtained by transducing the host cell with any one of the vectors according to the present invention. The pharmaceutical composition containing the transduced host cell can be administered to a target in need. Those skilled in the art will know how to formulate the transduced host cell in a pharmaceutical composition.

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

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

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

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

[0382] The viral vector and / or host cell according to the present invention may, if necessary, be formulated in a pharmaceutical composition together with one or more further therapeutic agents currently used to prevent or treat the disorder in question.

[0383] The vectors of the present invention can be administered parenterally, preferably intravascularly (including intravenously) and intrathecally to a subject. When administered parenterally, the vector is preferably given in an injection-suitable pharmaceutical vehicle such as a sterile aqueous solution or dispersion. After administration, the subject is monitored to detect changes in gene expression. The dose 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 the administration of the target gene in the vectors of the present invention. The dose of vector delivered using the method of the present invention varies depending on the desired response by the host and the vector used.

[0384] Within the scope of the present invention, it is assumed that the viral vector, host cells, or pharmaceutical composition according to the present invention will be administered into the bloodstream or cerebrospinal fluid (liquor cerebrospinalis) (or brain tissue) of a subject. As used herein, “introducing host cells into the bloodstream of a subject” includes, without limitation, introducing such cells into one of the veins or arteries of the subject by injection. Such administration may be, for example, one, multiple, and / or one or more times over a long period. A single injection is preferred, but in some cases, repeated injections over time (e.g., four times a year, twice a year, or once a year) may be necessary. Such administration may also preferably be 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 to 0.1 M, preferably 0.05 M phosphate buffer or 0.8% saline. Furthermore, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include 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 buffering media. Parenteral vehicles include sodium chloride solutions, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate and fixative oils. Intravenous vehicles include fluid and nutrient supplements, electrolyte supplements such as Ringer's dextrose, and supplements based on Ringer's dextrose. Fluids commonly used for IV administration can be found, for example, in Remington: The Science and Practice of Pharmacy, 20th Ed., p. 808, Lippincott Williams & Wilkins (2000). For example, preservatives and other additives such as antibacterial agents, antioxidants, chelating agents, and inert gases may be present.

[0385] In this specification, it is preferred that the viral vectors, host cells, or pharmaceutical compositions according to the present invention be administered into the bloodstream of the target. However, the viral vectors, host cells, or pharmaceutical compositions according to the present invention may also be administered directly to the target tissue. That is, in certain embodiments, the viral vectors, host cells, or pharmaceutical compositions according to the present invention may be injected directly into the brain. Alternatively, the viral vectors, host cells, or pharmaceutical compositions according to the present invention may be administered by direct CNS injection, injection into CSF, intrathecal injection, and / or intravascular administration.

[0386] Alternatively, the viral vector, host cells, or pharmaceutical composition according to the present invention can be administered directly into the tumor.

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

[0388] In other words, the viral vectors, host cells, or pharmaceutical compositions according to the present invention can be used to treat the subject requiring treatment. The term “treatment,” as used herein, includes preventative (e.g., prophylactic), curative, or elective treatments, and “to treat,” as used herein, also includes preventative, curative, and elective treatments. The term “subject,” as used herein, refers to animals, preferably mammals, and more preferably humans.

[0389] In certain embodiments, the present invention relates to a viral vector, a host cell, or a pharmaceutical composition according to the present invention for use in the treatment of diseases or disorders that have their origin or signs in the brain or are based on the brain.

[0390] Targeting brain cells for therapeutic purposes is challenging due to the selective permeability of the blood-brain barrier. Within this invention, the inventors target brain diseases or disorders through cell and gene therapy. Therefore, hematopoietic stem cells or populations of cells containing hematopoietic stem cells can be transformed with 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, in particular, can cross the blood-brain barrier during transient leakage of the blood-brain barrier following treatment-related radiation or chemotherapy, for example, busulfan administration. Once inside the brain, hematopoietic stem cells can differentiate into macrophages exhibiting 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 vector of the present invention has been demonstrated to be active in both macrophage and microglia cells, and is therefore particularly suitable for targeting the brain.

[0391] While it is preferable to administer hematopoietic stem cells containing the viral vector according to the present invention into the bloodstream of the target, the viral vector, host cells, or pharmaceutical composition according to the present invention can also be administered directly into the brain (intracranial).

[0392] In particular embodiments, the present invention relates to viral vectors, host cells, or pharmaceutical compositions according to the present invention for use in the prevention and / or treatment of PGRN-related diseases or disorders, wherein the viral vector encodes a 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 according to the present invention can be used to restore progranulin levels in the brain of subjects suffering from PGRN-related diseases or disorders. For this reason, it is preferable that the transgene encoded in the viral vector is a polynucleotide encoding a polypeptide that has at least 95%, 96%, 97%, 98%, or 99% sequence identity with the PGRN gene or the sequence shown in PGRN functionality and SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

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

[0395] In other words, PGRN-related diseases can be neurodegenerative diseases or disorders. 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, host cell, or pharmaceutical composition for use according to the present invention, wherein the neurodegenerative disease or disorder is a frontotemporal degenerative disease or disorder. In certain embodiments, the present invention relates to a viral vector, host cell, or 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 certain embodiments, the present invention relates to a viral vector, host cell, or 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 certain embodiments, the present invention relates in particular to viral vectors, host cells, or pharmaceutical compositions according to the present invention for use in the treatment of cancer, lymphoma, and / or sarcoma, 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] In other words, the viral vector of the present invention can be used in the treatment of cancer. Hereinafter, the promoter of the present invention has been demonstrated to be active in various myeloid cells and microglia. Therefore, the viral vector according to the present invention, or host cells containing the viral vector according to 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 a viral vector according to the present invention can be administered to a patient with cancer. The hematopoietic stem cells can differentiate into myeloid cells, migrate to the tumor site, and induce an immune response against the tumor. The myeloid cells may contain a transgene encoding one of the cytokines disclosed herein to increase the immune response against the tumor. Alternatively, or in addition to this, the transgene may encode an antigen-binding protein that directs the myeloid cells toward the tumor and induces a more pronounced immune response against the tumor.

[0401] In certain embodiments, the present invention relates to cancer, lymphoma and / or sarcoma, and brain tumors. The present invention relates to a viral vector, host cell, or pharmaceutical composition for use in the form of brain metastasis.

[0402] In other words, the viral vector or host cells according to 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 vector according to the present invention can migrate to the brain and differentiate into macrophages exhibiting 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, thereby initiating an immune response against tumors in the brain.

[0403] In certain embodiments, the present invention relates to a viral vector, host cell, or pharmaceutical composition for use according to the present invention, wherein the brain tumor is selected from the group consisting of glioblastoma, glioma, gangliocyte, astrocytoma, oligodendroglioma, PNET (primitive neuroectodermal tumor), medulloblastoma, CNS lymphoma, meningioma, retinoblastoma, and neuroblastoma.

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

[0405] As used herein, the term "cancer" refers to a disease characterized by dysregulated cell proliferation and / or growth. This term includes benign and malignant cancerous diseases, such as tumors, and may refer to invasive or non-invasive cancers. This term encompasses all types of cancer, including carcinoma, sarcoma, lymphoma, leukemia, germ cell tumors, and blastoma.

[0406] In certain embodiments, the present invention relates to viral vectors, host cells, or pharmaceutical compositions for use according to the present invention, wherein a viral vector, host cell, or pharmaceutical composition is administered in conjunction with a treatment that reduces the integrity of the blood-brain barrier, and in particular, the treatment that reduces the integrity of the blood-brain barrier is bone marrow conditioning, CNS conditioning, and / or blood-brain barrier conditioning.

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

[0408] Alternatively, the AAV-based viral vector according to the present invention, or a pharmaceutical composition containing the AAV-based viral vector according to the present invention, can be directly applied to brain compartments for in vivo infection of cells requiring it.

[0409] To more efficiently replace microglia in the brain with the transduced cells of the present invention, it is preferable to deplete endogenous microglia before administration of the transduced cells. Various treatment regimens that reduce the integrity of the blood-brain barrier have been reported to cause microglial depletion. For example, Capotondo et al. demonstrated that brain pre-treatment is helpful in the success of microglial rearrangement after hematopoietic stem cell transplantation (Proc Natl Academia). Sci US A. 2012 Sep 11; 109(37): 15018-15023).

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

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

[0412] In certain embodiments, the present invention relates to viral vectors, host cells, or pharmaceutical compositions for use according to the present invention, wherein blood-brain barrier pretreatment includes radiotherapy or targeted radiotherapy.

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

[0414] In other words, the viral vector, host cells, or pharmaceutical composition according to the present invention can be administered to the target in need 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days after treatment that reduces the integrity of the blood-brain barrier.

[0415] While 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, it is important to understand that viral vectors 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 cancers in any organ or tissue that are reachable by myeloid cells such as macrophages or monocytes.

[0416] In certain embodiments, the present invention relates to a viral vector, a host cell, or a pharmaceutical composition according to the present invention for use in the treatment of autoimmune diseases.

[0417] In other words, the viral vector, host cell, or pharmaceutical composition according to 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 resulting from an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive response to autoantigens. 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 mumps, 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, spondyloarthritis, 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 antagonists, IL-2, IL-4, IL-10, TGF beta, FOXP3, T-bet, GATA-3, CD36 family (CD36-L1, CD36-L2) binding CD1b, CD1c, CD1D, and MHC-associated protein 1 (MR1) for T cell receptor recognition.

[0420] In certain embodiments, the present invention relates to a viral vector, a host cell, or a pharmaceutical composition according to the present invention for use in the treatment of autoinflammatory diseases.

[0421] It should be understood that the term “autoinflammatory disease” as used herein encompasses 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, anti-synthetase syndrome, scleritis, macrophage activation syndrome, Behcet's syndrome, PAPA syndrome, Blau syndrome, gout, adult and juvenile Still's disease, cryopyrinopathy, Mackle-Wells syndrome, familial cold-induced autoinflammatory syndrome, neonatal-onset multiorgan inflammatory disease, familial Mediterranean fever, chronic infantile neurocutaneous arthritis syndrome, systemic juvenile idiopathic arthritis, hyper-IgD syndrome, Schnitzler syndrome, and TNF receptor-associated periodic syndromes (TRAPS).

[0422] Transgenes that can be used to treat autoinflammatory diseases include IL-1 receptor antagonists and IL-1 beta.

[0423] In certain embodiments, the present invention relates to a viral vector, a host cell, or a pharmaceutical composition according to the present invention for use in the treatment of allergic diseases.

[0424] As used herein, the term "allergic disease" refers to any symptom, tissue damage, or loss of tissue function caused by an allergy, and includes, but is 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 any part of IgE, including Fc, Fab, including the variable and hypervariable regions of Fab; or genes encoding proteins, including antibodies and other receptor-binding proteins, against any receptor of cells implied to mediate allergic reactions, including mast cells, eosinophils, B cells, and T cells. Furthermore, soluble and potentially neutralizing binding proteins and peptides or antibodies should be induced by genes against any cytokine, including IL-1, IL-4, IL-33, and any other cytokine, including any form of interleukin and chemokine associated with allergic diseases.

[0426] In certain embodiments, the present invention relates to viral vectors, host cells, or pharmaceutical compositions according to the present invention for use in hematopoiesis and solid organ transplantation.

[0427] In other words, the viral vectors, host cells, or pharmaceutical compositions according to the present invention can be administered to the target in need before hematopoiesis or solid organ transplantation. Transgenes that can be used in hematopoiesis and solid organ transplantation include IL-1, IL-1R antagonists, IL-2, IL-4, IL-10, TGF beta, FOXP3, T-bet, GATA-3, CD36 family (CD36-L1, CD36-L2) binding CD1b, CD1c, CD1D, and MHC-related protein 1 (MR1) for T cell receptor recognition.

[0428] In certain embodiments, the present invention relates to a method for treating a brain-related disease or disorder in which the brain is the origin or manifestation of the brain in a subject of interest, a) A modification step comprising the step of genetically modifying a population of hematopoietic stem cells and / or CD34-enriched myeloid cells, wherein the population of hematopoietic stem cells and / or CD34-enriched myeloid cells is brought into contact with the viral vector of the present invention; or a modification step comprising the step of genetically modifying a population of myeloid cells and / or enriched myeloid cells, wherein the population of myeloid cells and / or enriched myeloid cells is brought into contact with the viral vector of the present invention, b) The step of intravenously administering the genetically modified cells obtained from step (a) to the target in need, c) A step of treating a brain-related disease or disorder in the subject of need, which has its origin or signs in the brain. Regarding methods including

[0429] In other words, 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 according to the present invention can migrate to the brain of a person suffering from a brain-based disease or disorder and replace microglia.

[0430] Therefore, cells such as hematopoietic stem cells or myeloid cells can be transduced ex vivo using the viral vector according to the present invention. The transduced population of cells can then be administered to a target in need. In a particular embodiment, transduced hematopoietic stem cells are administered to a target in need. This embodiment may be advantageous because stem cells have a higher potential to cross the blood-brain barrier than other cell types. However, the host cells may be myeloid cells such as monocytes and / or macrophages. However, monocytes and / or macrophages are preferably used in targets with a compromised blood-brain barrier.

[0431] In certain embodiments, the present invention relates to a method according to the present invention, wherein a population of hematopoietic stem cells and / or enriched CD34-positive myeloid cells, or a population of myeloid cells and / or enriched myeloid cells, is obtained from a subject or exogenous donor in need.

[0432] In other words, in certain embodiments, the method of the present invention involves the use of autologous cells. Those skilled in the art will recognize methods for enriching a particular cell type from the blood of a subject. Consequently, a particular type of blood cell can be enriched from the blood of a subject in need, transduced using a viral vector according to the present invention, and administered back to the subject in need. Autologous cells have the advantage of reducing the risk of immunogenic reactions.

[0433] In other embodiments, the cells administered to the target in need may originate from an exogenous donor. Those skilled in the art will know methods for identifying a suitable donor or for manipulating the cells and / or target in a way that reduces the risk of immunogenic reactions.

[0434] In certain embodiments, the present invention relates to a method for treating a brain-related disease or disorder in which the brain is the origin or manifestation of the brain in a subject of interest, a) A step of mobilizing hematopoietic stem cells in the target population, b) Following the recruitment of hematopoietic stem cells in step (a), the step of intravenously administering the viral vector according to the present invention to a subject requiring it, c) A step of treating a brain-related disease or disorder in the subject of need, which has its origin or signs in the brain. Regarding methods including

[0435] In other words, the viral vector of the present invention, or a pharmaceutical composition containing the viral vector according to the present invention, can also be administered directly to the subject in need. Preferably, the subject is pretreated with an agent that induces the mobilization of hematopoietic stem cells in the subject so that the mobilized hematopoietic stem cells can be infected with the viral vector according to the present invention in vivo. Agents commonly used to stimulate the mobilization of hematopoietic stem cells from bone marrow are G-CSF and plerixafor. However, other agents that stimulate the mobilization of hematopoietic stem cells 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. Microglia-like cells can express one or more transgenes required for the prevention and / or treatment of brain-based diseases or disorders. For example, microglia-like cells can express PGRN when used for the prevention and / or treatment of one of the PGRN-related diseases or disorders disclosed herein. Alternatively, microglia-like cells can express one of the cytokines disclosed herein when used in the treatment of brain tumors.

[0437] In certain embodiments, the present invention relates to a method according to the present invention in which the recruitment of hematopoietic stem cells in a target subject comprises the administration of G-CSF and / or plerixafor. Plerixafor (INN and USAN, trade name Mozobil) is an immunostimulant used to recruit hematopoietic stem cells into the bloodstream in cancer patients.

[0438] In certain embodiments, the present invention relates to a method according to the present invention wherein a disease or disorder having its origin or signs in the brain or based on the brain is a PGRN-related disease or disorder, and in particular a PGRN-related disease or disorder is a neurodegenerative disease or disorder, and in particular a frontotemporal degenerative disease or neurodegenerative disorder, and in particular a frontotemporal 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, and in particular the viral vector encodes a PGRN or a functional fragment thereof.

[0439] In other words, viral vectors encoding progranulin or a polypeptide having PGRN functionality and having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown 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-related neurodegenerative diseases disclosed herein.

[0440] In certain embodiments, the present invention relates to a method according to the present invention in which a disease or disorder having its origin or signs in the brain or based on the brain is a brain tumor, and in particular the brain tumor is selected from the group consisting of glioma, glioblastoma, ganglioblastoma, astrocytoma, oligodendroglioma, PNET (primitive neuroectodermal tumor), medulloblastoma, CNS lymphoma and neuroblastoma, or 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 malignancy including any form of leukemia and lymphoma, and in particular the viral vector codes for 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 comprising an additional step of transiently reducing the integrity of the blood-brain barrier, and in particular, a method of the present invention wherein the step of reducing the integrity of the blood-brain barrier comprises bone marrow conditioning, CNS conditioning and / or blood-brain barrier conditioning.

[0442] In certain embodiments, the present invention relates to a method according to the present invention in which a treatment to reduce the integrity of the blood-brain barrier is performed before the administration of genetically modified cells to the target in need, and in particular, the time interval between the treatment to reduce the integrity of the blood-brain barrier and the administration of genetically modified cells is performed after the treatment to reduce the integrity of the blood-brain barrier.

[0443] Therapies to impair the integrity of the blood-brain barrier may be any one of the therapies disclosed herein. In certain embodiments, therapies to impair the integrity of the blood-brain barrier may be administered 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days prior to the administration of the viral vector, host cells, or pharmaceutical composition according to the present invention.

[0444] As described above, the viral vector, host cell, or pharmaceutical composition according to the present invention can also be used to treat cancer in other parts of the body. That is, in certain embodiments, the present invention is a method for treating cancer in a target area of ​​interest, a) A step of mobilizing hematopoietic stem cells in the target population, b) Following the recruitment of hematopoietic stem cells in step (a), the step of intravenously administering the viral vector according to the present invention to a subject requiring it, c) Steps to treat cancer in the target population Regarding methods including

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

[0446] In certain embodiments, the present invention relates to a method for expressing a transgene in the brain and / or CNS of a target, a) A modification step comprising the step of genetically modifying a population of hematopoietic stem cells and / or CD34-enriched myeloid cells, wherein the population of hematopoietic stem cells and / or CD34-enriched myeloid cells is brought into contact with a viral vector according to the present invention; or a modification step comprising the step of genetically modifying a population of myeloid cells and / or enriched myeloid cells, wherein the population of myeloid cells and / or enriched myeloid cells is brought into contact with a viral vector according to the present invention, b) The step of administering the genetically modified cells obtained from step (a) intravenously or intrathecally to the target subject as needed, c) The step of expressing the transgene encoded by the viral vector in the target brain and / or CNS. Regarding methods including

[0447] In other words, this method can be used to express a transgene in the brain or central nervous system of the desired target. To do this, a population of cells can be transduced ex vivo by a viral vector according to the present invention. In certain embodiments, the population of cells may be a population of hematopoietic stem cells or a population of enriched CD34-positive myeloid cells. Preferably, the population of enriched CD34-positive myeloid cells includes hematopoietic stem cells and / or hematopoietic progenitor cells. In certain embodiments, the population of cells may be an enriched population of myeloid cells. Myeloid cells may be any of the myeloid cells disclosed herein. In certain embodiments, myeloid cells may be macrophages.

[0448] The term “population of cells” is used to indicate multiple cells. For example, a population of hematopoietic stem cells refers to multiple stem cells. A population of hematopoietic stem cells may consist exclusively of hematopoietic stem cells. However, as used herein, “population of hematopoietic stem cells” is understood to preferably be a population of cells that include hematopoietic stem cells. That is, “population of hematopoietic stem cells” may include other cell types, in particular CD34-positive cell types. Those skilled in the art will recognize 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. A population of enriched CD34-positive bone marrow cells may 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] A myeloid cell-enriched population 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 by flow cytometry to enrich a specific type(s) of myeloid cells.

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

[0451] In certain embodiments, the present invention relates to a method according to the present invention in which a population of hematopoietic stem cells and / or enriched CD34-positive myeloid cells, or a population of myeloid cells and / or enriched myeloid cells, is obtained from a subject or an exogenous donor.

[0452] In other words, the cell population can include autologous or allogeneic cells, as described above.

[0453] In certain embodiments, the present invention relates to a method for expressing a transgene in the brain and / or CNS of a target, a) A step of mobilizing hematopoietic stem cells in the subject, b) Following the recruitment of hematopoietic stem cells in step (a), the step of intravenously administering the viral vector according to the present invention to a subject requiring it, c) The step of expressing the transgene encoded by the viral vector in the target brain and / or CNS. Regarding methods including

[0454] In other words, in certain embodiments, the transgene can be delivered to a target that requires it in vivo. That is, preferably, the viral vector according to the present invention can be directly administered to the target after the target has received stem cell mobilization therapy. Therefore, in certain embodiments, the present invention relates to a method according to the present invention in which the mobilization of hematopoietic stem cells in the target includes the administration of G-CSF or plerixafor.

[0455] In certain embodiments, the present invention relates to a method according to the present invention, wherein the method includes an additional step of reducing the integrity of the blood-brain barrier, and in particular, the step of temporarily reducing the integrity of the blood-brain barrier includes bone marrow conditioning, CNS conditioning and / or blood-brain barrier conditioning.

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

[0457] In certain embodiments, the present invention relates to a method according to the present invention in which a treatment to reduce the integrity of the blood-brain barrier is performed before the administration of genetically modified cells to the target in need, and in particular, the time interval between the treatment to reduce the integrity of the blood-brain barrier and the administration of genetically modified cells is performed after the treatment to reduce the integrity of the blood-brain barrier.

[0458] In other words, a treatment to reduce 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 before administration of the viral vector according to the present invention or a pharmaceutical composition containing the viral vector according to the present invention.

[0459] In certain embodiments, the present invention relates to a method for treating a brain-related disease or disorder in which the brain is the origin or manifestation of the brain in a subject of interest, a) The step of administering the viral vector according to the present invention to the brain or intrathecal cavity of the target requiring it, b) Steps to treat a brain-related disease or disorder in the subject of need, which has its origin or signs in the brain. Regarding methods including

[0460] In other words, the viral vector according to the present invention or a pharmaceutical composition containing the viral vector according to the present invention can be directly administered into the brain or spinal canal (intrathecal cavity). In such embodiments, the viral vector is preferably an AAV-based viral vector. Therefore, in certain embodiments, the present invention relates to a viral vector according to the present invention in which the viral vector is an AAV-based viral vector.

[0461] It should be understood that this method may be used to treat or prevent any brain-based disease or disorder disclosed herein, in particular neurodegenerative diseases and disorders, as well as cancer. The term “intrathecal cavity” as used herein means administration into or within the fluid-filled space between the thin layers of tissue covering the brain and spinal cord.

[0462] The present invention provides novel retroviral vectors for use in the treatment of diseases or disorders originating in or based on the brain, particularly PGRN-related 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, ganglioblastoma, astrocytoma, oligodendroglioma, PNET (primitive neuroectodermal), medulloblastoma, CNS lymphoma, and neuroblastoma or any other CNS tumor, and further, retroviral vectors 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 malignancies, including any form of leukemia and lymphoma.

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

[0464] The vector according to the present invention includes safety features of a myelo- / microglia-specific promoter for phagocytic cell-specific expression, preferably a miR223 gene promoter, or a fusion promoter construct containing a miR223 promoter, in order to drive transgene expression, particularly the expression of PGRN cDNA.

[0465] Therefore, in specific embodiments, the present invention relates to the introduction of a PGRN coding expression cassette containing a bone marrow / microglia-specific promoter, but not limited to, the miR223 promoter, into HSCs using a lentiviral autoinactivated (SIN) gene therapy vector.

[0466] In another specific embodiment, the present invention relates to the introduction of a PGRN coding expression cassette comprising a bone marrow / 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 coding expression cassette comprising a bone marrow / microglia-specific (specific) promoter, but not limited to, a miR223 fusion promoter, and more particularly, a fusion promoter in which the miR223 promoter or a functional portion thereof is fused with the whole or a functional portion of a promoter selected from the group consisting of the TMEM119 promoter, P2RY12 promoter, OLFML3 promoter, AIF1 promoter, and ITGAM promoter.

[0468] In one embodiment, the present invention relates to the use of a TMEM119 promoter construct, a P2RY12 promoter construct, an OLFML3 promoter construct, or a fusion construct comprising miR223 fused to TMEM119, miR223 fused to P2RY12, or miR223 fused to the OLFML3 promoter, for driving PGRN expression in HSC-derived monocyte / macrophages, dendritic cells, and microglia-like cells or microglia after macrophage migration to the brain.

[0469] Following the transduction of HSCs, the patient is administered the ex vivo-treated HSCs. In specific embodiments, the ex vivo-treated HSCs are administered intravenously.

[0470] For HSC transplantation, the patient's bone marrow is pre-treated with a suitable pre-conditioning compound or treatment, particularly with busulfan, threosulfan, radiotherapy, or a biological agent capable of depleting endogenous brain microglia, but preferably with busulfan. This procedure allows HSC-derived transgenic monocyte / macrophages or dendritic cells to enter the brain and achieve a substantial level of chimeric phenomena in the brain of HSC-derived monocyte / macrophages, microglia-like macrophages, dendritic cells, and / or microglial cells, thereby enabling the delivery of a sufficient amount of PGRN or other transgenes to the brain.

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

[0472] The retroviral vectors according to the present invention can also be used to target bone marrow-derived macrophages and microglia involved in brain tumors and metastases. The specific vector architecture according to the present invention, which includes a bone marrow / microglia-specific promoter, is the basis for successful protein expression 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 retroviral vector constructs described herein for the treatment of patients suffering from brain tumors, especially brain tumors selected from the group consisting of glioblastoma, glioma, ganglioblastoma, 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 the retroviral vector construct described herein for the treatment of patients suffering from brain metastases originating from any form of breast cancer, lung cancer, colon cancer, testicular cancer, kidney cancer and melanoma or any other solid tumor, as well as any hematological malignancy including any form 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 a combination of therapeutic polypeptides, in the control of a fusion promoter, a bone marrow / microglia-specific promoter, or a combination of bone marrow-specific and microglia-specific promoters, in particular, under the control of a fusion promoter, which drives the expression of a therapeutic polypeptide or a combination of therapeutic polypeptides in HSC-derived myeloid cells, HSC-derived blood monocytes / macrophages, dendritic cells, and brain microglia or microglia-like cells after macrophage migration to the brain.

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

[0477] 3. Bone marrow / microglia-specific promoters, (a) AIF1 promoter or ITGAM promoter; or (b) A fusion promoter comprising a promoter or promoter fragment having promoter functionality of the miR223 promoter and a promoter or promoter fragment having promoter functionality of a promoter selected from the group consisting of the TMEM119 promoter, the P2RY12 promoter, and the OLFML3 promoter. A retroviral vector according to Embodiment 1, which is a promoter or promoter fragment having promoter functionality selected from a promoter.

[0478] 4. The retroviral vector according to Embodiment 3, wherein the promoter or promoter fragment having miR233 promoter functionality has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 1, or is a fragment thereof at least 200 nucleotides in length, and the 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%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 2, or is a fragment thereof at least 200 nucleotides in length, and the promoter or fragment still has 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%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 3, or is a fragment thereof at least 200 nucleotides in length, and the promoter or fragment still has promoter functionality of the TMEM119 promoter.

[0481] 7. A retroviral vector according to 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%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 4, or is a fragment thereof at least 200 nucleotides in length, and the promoter or fragment still has 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%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 5, or is a fragment thereof at least 200 nucleotides in length, and the promoter or fragment still has promoter functionality of the AIF1 promoter.

[0483] 9. A retroviral vector according to 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%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 6, or is a fragment thereof at least 200 nucleotides in length, and the promoter or fragment still has promoter functionality of the ITGAM promoter.

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

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

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

[0487] 13. The retroviral vector according to Embodiment 12, wherein the partial sequence of the PGRN polypeptide is at least 40 amino acids long.

[0488] 14. Therapeutic polypeptides include FasL / Fas, Trail / TRAIL-R, lymphotoxin beta, decoy receptors 1-3, TNF-alpha, TNF-alpha-R, 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, granulysine, nitric oxide synthase, and DNA methyltransferase. Insulatorase 3b (DNMT3b), cross-domain protein 1A (JMJD1A), histone deacetylase 3 (HDAC3) and HDAC9, CSF1 receptor (CSF1R) or CSD1R-ligand IL-34, all chemokines, chemokine receptors, VEGF, VEGF-receptors, antagonists to metalloproteinases (e.g., MMP-9), tumor-specific ligands and receptors such as CD40 / CD40L, EGFR, Annexin 1, FGFR- A retroviral vector according to any one embodiment of Embodiments 1 to 10, selected from the group consisting of 1, Her2, St6galnac5, MMP1-28 and their counterparts TIMPS1-4 (tissue metalloproteinase inhibitors), melanotransferrin, alpha-4-beta-1 integrin and its ligand endothelial cell VCAM-1, E-cadherin, alpha-v-beta-3 integrin, alpha-v-beta-5 integrin, alpha-v-beta-6 integrin, alpha-v-beta-8 integrin, single nucleotide variant neoantigen, INDEL frameshift neoantigen, splice variant antigen, fusion protein neoantigen, endogenous retroelement antigen, tumor-specific antigen, in particular tumor-specific antigen 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), soluble and membrane-bound.

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

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

[0491] 17. The retroviral vector according to Embodiment 15, wherein the therapeutic polypeptide has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence shown in SEQ ID NO: 10, or is a fragment thereof of at least 50 amino acids in length, and the polypeptide still provides IFN-gamma functionality.

[0492] 18. A retroviral vector according to any one embodiment of Embodiments 1 to 17, which is a lentiviral vector, particularly a lentiviral SIN vector.

[0493] 19. A retroviral vector according to any one embodiment of Embodiments 1 to 17, which is a foamy viral vector.

[0494] 20. A viral vector according to any one embodiment of Embodiments 1 to 17, which is an adenovirus vector and / or a herpesvirus vector, or an alpha-retrovirus vector.

[0495] 21. A retroviral vector according to any one embodiment of Embodiments 1 to 20 for use in therapeutic purposes.

[0496] 22. A retroviral vector according to any one embodiment of Embodiments 1 to 20, for use in the treatment of diseases or disorders of which originate in the brain or are based on the brain or nervous system.

[0497] 23. A retroviral vector according to any one embodiment of Embodiments 1 to 13 and 18 to 232 for use in the treatment of PGRN-related diseases or disorders.

[0498] 24. The retroviral vector according to Embodiment 23, wherein the PGRN-related disease or disorder is a neurodegenerative disease or disorder.

[0499] 25. The retroviral vector according to Embodiment 24, wherein the neurodegenerative disease or disorder is a frontotemporal degenerative disease or disorder.

[0500] 26. The retroviral vector according to Embodiment 25, wherein the frontotemporal degenerative disease or disorder is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease.

[0501] 27. A retroviral vector according to any one embodiment of embodiments 1 to 10 and 14 to 22 for use in the treatment of brain tumors.

[0502] 28. A retroviral vector according to Embodiment 27 for use in the treatment of brain tumors selected from the group consisting of glioblastoma, glioma, ganglioblastoma, astrocytoma, oligodendroglioma, PNET (primitive neuroectodermal), medulloblastoma, CNS lymphoma, and neuroblastoma or any other CNS tumor.

[0503] 29. The retroviral vector according to Embodiment 27 for use in the treatment of brain metastases originating from any form of breast cancer, lung cancer, colon cancer, testicular cancer, kidney cancer and melanoma or any other solid tumor, as well as any hematological malignancy including any form of leukemia and lymphoma.

[0504] 30. A method for treating a disease or disorder having its origin in or based on the brain, comprising ex vivo genetic modification of a population of hematopoietic stem cells and / or enriched CD34-positive myeloid cells of a patient suffering from such disease or disorder by retroviral transduction with a retroviral vector described in any one embodiment of Embodiments 1 to 22, and administration of the modified cells to the patient.

[0505] 31. The method according to Embodiment 30, wherein the retroviral vector is the vector described in any one embodiment of Embodiments 1 to 13 and 18 to 22, and the patient suffers from a PGRN-related disease or disorder.

[0506] 32. The method according to Embodiment 31, wherein the PGRN-related disease or disorder is a neurodegenerative disease or disorder.

[0507] 33. The method according to Embodiment 32, wherein the neurodegenerative disease or disorder is a frontotemporal degenerative disease or disorder.

[0508] 34. The method according to Embodiment 33, wherein the frontotemporal degenerative disease or disorder is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease.

[0509] 35. The method according to Embodiment 30, wherein the retroviral vector is the vector described in any one embodiment of Embodiments 1 to 10 and 14 to 21, and the patient suffers from a brain tumor.

[0510] 36. The method according to Embodiment 35, wherein the brain tumor is selected from the group consisting of glioma, glioblastoma, ganglioblastoma, astrocytoma, oligodendroglioma, PNET (primitive neuroectodermal), medulloblastoma, CNS lymphoma, and neuroblastoma or any other CNS tumor.

[0511] 37. The method according to embodiment 35, wherein the patient has brain metastases originating from any form of breast cancer, lung cancer, colon cancer, testicular cancer, kidney cancer and melanoma or any other solid tumor, as well as any hematological malignancy, including any form of leukemia and lymphoma.

[0512] 38....

Claims

1. A pharmaceutical composition for use in a method for treating a brain-based disease or disorder, wherein the composition comprises a viral vector, the viral vector comprises a nucleic acid molecule encoding a therapeutic polypeptide under the control of a promoter or promoter fragment, the promoter or promoter fragment drives the expression of the therapeutic polypeptide in myeloid cells and microglia, and the promoter or promoter fragment is inactive in progenitor cells and / or stem cells.

2. The pharmaceutical composition for use according to Claim 1, wherein the promoter is selected from the group consisting of the miR223 promoter, the ITGAM promoter, the AIF1 promoter, the TMEM119 promoter, the P2RY12 promoter, and the OLFML3 promoter.

3. The pharmaceutical composition for use according to claim 1, wherein the promoter is a fusion promoter comprising a miR223 promoter operably linked to a second promoter which is a microglia-specific promoter.

4. The pharmaceutical composition for use according to any one of claims 1 to 3, wherein the therapeutic polypeptide is selected from the group consisting of progranulin (PGRN), interleukin-12 (IL-12), interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF), or functional fragments thereof.

5. The pharmaceutical composition for use according to claim 4, wherein the therapeutic polypeptide is progranulin (PGRN) or a functional fragment thereof.

6. The pharmaceutical composition for use according to claim 4, wherein the therapeutic polypeptide is a cytokine selected from the group consisting of IL-12, IFN-gamma, GM-CSF, and G-CSF.

7. The pharmaceutical composition for use according to any one of claims 1 to 3, wherein the nucleic acid molecule encodes both GM-CSF and IFN-gamma.

8. The pharmaceutical composition for use according to claim 7, wherein the nucleic acid molecule encodes both GM-CSF and IFN-gamma as a fusion protein.

9. The pharmaceutical composition for use according to any one of claims 1 to 8, wherein the viral vector is a retroviral vector.

10. The pharmaceutical composition for use according to claim 9, wherein the retroviral vector is a lentiviral vector.

11. The pharmaceutical composition for use according to claim 10, wherein the lentiviral vector is a self-inactivating (SIN) lentiviral vector.

12. The pharmaceutical composition for use according to any one of claims 1 to 11, wherein the viral vector further comprises at least one transcriptional regulatory element arranged to inhibit or activate the promoter.

13. The pharmaceutical composition for use according to claim 12, wherein the transcriptional regulatory element comprises a tetracycline / doxycycline binding domain.

14. The pharmaceutical composition for use according to any one of claims 1 to 13, wherein the brain-based disease or disorder is a neurodegenerative disease.

15. The pharmaceutical composition for use according to claim 14, wherein the neurodegenerative disease is frontotemporal dementia caused by a mutation in the PGRN gene.

16. The pharmaceutical composition for use according to any one of claims 1 to 13, wherein the brain-based disease or disorder is cancer.

17. The pharmaceutical composition for use according to claim 16, wherein the cancer is a brain tumor.

18. The pharmaceutical composition for use according to claim 17, wherein the brain tumor is a glioblastoma.

19. The pharmaceutical composition for use according to claim 16, wherein the cancer is a brain metastasis originating from a cancer selected from the group consisting of breast cancer, lung cancer, and colon cancer.

20. A pharmaceutical composition for use according to any one of claims 1 to 19, wherein the use comprises (a) ex vivo genetically modifying hematopoietic stem cells (HSCs) using the viral vector to produce genetically modified HSCs, and (b) administering the genetically modified HSCs to a subject in need thereof.

21. The pharmaceutical composition for use according to claim 20, wherein the hematopoietic stem cells are derived from a CD34-positive enriched cell population.

22. The pharmaceutical composition for use according to claim 20 or 21, wherein the use further comprises administering a treatment that reduces the integrity of the blood-brain barrier prior to the administration of the genetically modified HSC.

23. The pharmaceutical composition for use according to claim 22, wherein the treatment for reducing the integrity of the blood-brain barrier is a bone marrow conditioning treatment selected from the group consisting of busulfan administration, treosulfan administration, radiotherapy, and targeted radiotherapy.

24. The pharmaceutical composition for use according to claim 22 or 23, wherein the genetically modified HSC is administered half a day to 15 days after the treatment that reduces the integrity of the blood-brain barrier.

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