Artificial expression construct for regulating gene expression in non-neuronal cells of the central nervous system

Abstract

An artificial expression construct for regulating gene expression in a target type of central nervous system cell will be described. The artificial expression constructs of the present disclosure can be used for the expression of synthetic genes or the regulation of gene expression in non-neuronal cells such as oligodendrocytes, microglia, astrocytes, and endothelial cells.

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 351,631, filed on June 13, 2022, the content of which is hereby incorporated by reference in its entirety as if fully set forth herein.

[0002] Statement regarding research and development funded by the federal government This invention was made with government support under grant numbers MH114126 and MH120095 awarded by the National Institutes of Health (NIH). The U.S. government has certain rights in this invention.

[0003] Reference to the sequence listing The Sequence Listing accompanying this application is provided in XML format rather than as a hard copy, and this Sequence Listing is hereby incorporated by reference in this specification. The name of the XML file containing this Sequence Listing is 2WU3521.xml. This XML file is 271 KB in size, was created on June 13, 2023, and was electronically submitted via the Patent Center.

[0004]

Background Art

[0005] ​To fully understand brain biology, it is necessary to distinguish and define various types of cells, investigate them in detail, and identify artificial expression constructs that can label and perturb these cells. In mice, the use of driver strains expressing recombinase has successfully labeled cell populations sharing marker gene expression. However, the generation, maintenance, and use of such strains capable of labeling specific types of cells with high specificity are costly, often require the mating of transgenic animals among three species, and the desired experimental animals can be obtained only at low frequencies. Furthermore, since these tools require germline transgenic animals, they cannot be applied to humans.

Summary of the Invention

Means for Solving the Problems

[0006] The present disclosure provides artificial expression constructs that induce gene expression in a target central nervous system cell population. Examples of the target central nervous system cell population include non-neuronal cells in the brain such as glial cells present in the brain parenchyma, and these non-neuronal cells can be further classified into oligodendrocytes, microglia, astrocytes, and oligodendrocyte progenitor cells, as well as pericytes, endothelial cells, and ependymal cells present in other cell compartments.

[0007] In certain embodiments of the artificial expression constructs of the present disclosure, the following enhancers are utilized to induce gene expression in a target central nervous system cell population. The combinations of the target cell populations and enhancers used in the present disclosure are shown below in the order of target cell population / enhancer. Oligodendrocytes / eHGT_409h, eHGT_410h, eHGT_361h, eHGT_395m, eHGT_401h, eHGT_391h, eHGT_403h, 3xCore_eHGT_641m, 3xcore2_eHGT_400m, 3xcore2_eHGT_401h, 3xcore2_eHGT_409h, 3xcore2_eHGT 410h, and eHGT_363h; Microglia / eHGT_331h, eHGT_332h, eHGT_333h, eHGT_335h, eHGT_334h, eHGT_360h, eHGT_562h, eHGT_563h, eHGT_564h, eHGT_565h, eHGT_566m, eHGT_567m, eHGT_568m, eHGT_569m, and eHGT_734m; Astrocyte / eHGT_377m, eHGT_389m, eHGT_380h, eHGT_374h, 3xcore1_eHGT_387m, eHGT_385h, eHGT_386h, eHGT_378m, eHGT_642m, 3xcore2_eHGT_390h, 3xcore2_eHGT_380h, 3xcore2_eHGT_374m, 3xcore2_eHGT_375m, and eHGT_358h; Layered astrocytes of the first layer / eHGT_312h, eHGT_313h, eHGT_315h, and eHGT_316h; and Endothelial cells / eHGT_603m.

[0008] In certain embodiments, these artificial enhancer factors are concatamerized enhancer cores. Examples of such enhancer cores or concatamerized enhancer cores include the cores of eHGT_374m, eHGT_375m, eHGT_380h, eHGT_387m, eHGT_390h, eHGT_400m, eHGT_401h, eHGT_409h, eHGT_410h, and / or eHGT_641m, or concatamerized cores thereof. By using such artificial enhancer factors, a transgene can be rapidly expressed to obtain high expression, compared to using the full length of the original (natural) enhancer alone.

[0009] Certain embodiments of the enhancer core include Core_eHGT_641m, core2_eHGT_400m, core2_eHGT_401h, core2_eHGT_409h, core2_eHGT_410h, core1_eHGT_387m, core2_eHGT_390h, core2_eHGT_380h, core2_eHGT_374m, and core2_eHGT_375m. In certain embodiments, these enhancer cores are concatenated and have 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the core sequence. In certain embodiments, concatamers containing 3 copies of the selected enhancer core include 3xCore_eHGT_641m, 3xcore2_eHGT_400m, 3xcore2_eHGT_401h, 3xcore2_eHGT_409h, 3xcore2_eHGT_410h, 3xcore1_eHGT_387m, 3xcore2_eHGT_390h, 3xcore2_eHGT_380h, 3xcore2_eHGT_374m, and 3xcore2_eHGT_375m.

[0010] Certain embodiments provide an artificial expression construct comprising the features of the vectors described herein, and these vectors include CN3062, CN3063, CN2979, CN2498, CN2677, CN2505, CN2499, CN4004, CN3575, CN4254, CN4255, CN4256, CN2806, CN2807, CN2808, CN2809, CN2812, CN2813, CN2678, CN2679, CN2680, CN2681, CN2833, CN2834, CN2835, CN2836, CN2627, CN2675, CN2344, CN3060, CN3061, CN3098, CN2493, CN3568, CN2495, CN2496, CN2827, CN2846, CN3572, CN4253, CN4257, CN2410, CN2345, CN2346, CN4268, CN4522, and CN4523.

Brief Description of the Drawings

[0011] Part of the drawings submitted in this application is considered easier to understand in color. The applicants consider the color versions of these drawings to be part of the original application and reserve the right to submit color images of the drawings in later procedures.

[0012] [Figs. 1A - 1D] Fluorescent images of sagittal sections of mouse brains transduced with intravenously delivered CN3060 virus are shown. This virus is packaged in a PHP.eB capsid. The black parts indicate the expression of SYFP. Each image is a montage image. (Figure 1A) shows the whole brain, (Figure 1B) shows the visual cortex (VISp), (Figure 1C) shows the deep cerebellar nuclei (DCN), and (Figure 1D) shows the cerebellar cortex (Bergmann glia).

[0013] [Figs. 2A - 2D] (Figure 2A) shows fluorescent images of sagittal sections of mouse brains transduced with intravenously delivered CN2499 virus. The SYFP2 transgene is induced by the enhancer eHGT_403h. This virus is packaged in a PHP.eB capsid. The images are montage images. The brain was co-stained with propidium iodide (PI) and 4’,6-diamidino-2-phenylindole (DAPI). Note that a large number of reporters labeling oligodendrocytes are concentrated in midbrain and hindbrain structures, as well as other white matter tracts. (Figures 2B - 2D) show fluorescent images of tissues other than the brain of mice transduced with intravenously delivered CN2499 virus. The SYFP transgene (black) is induced by the enhancer eHGT_403h. This virus is packaged in a PHP.eB capsid. Each image is a montage image. (Figure 2B) shows the lumbar spinal cord, (Figure 2C) shows the liver, and (Figure 2D) shows the heart. Strong expression of oligodendrocytes is seen in the spinal cord, while little expression of oligodendrocytes is observed in the heart and liver.

[0014] [Figs. 3A - 3B]Fluorescent images of sagittal sections of mouse brains transduced with intravenously delivered CN3061 virus are shown. The SYFP2 transgene (black) was induced by the enhancer eHGT_389m. This virus was packaged in the PHP.eB capsid. Each image is a montage. (Fig. 3A) shows the whole brain, and (Fig. 3B) shows the visual cortex (VISp - the dashed square in Fig. 4A). Note that reporter expression is seen in scattered cells showing the morphology of astrocytes.

[0015] [Figs. 4A - 4B] Fluorescent images of sagittal sections of mouse brains transduced with intravenously delivered CN3098 virus are shown. The SYFP2 transgene (black) was induced by the enhancer eHGT_380h. This virus was packaged in the PHP.eB capsid. Each image is a montage. (Fig. 4A) shows the whole brain, and (Fig. 5B) shows the visual cortex (VISp - the dashed square in Fig. 4A). Note that reporter expression is seen in scattered cells showing the morphology of astrocytes.

[0016] [Figs. 5A - 5B] Fluorescent images of sagittal sections of mouse brains transduced with intravenously delivered CN2498 virus are shown. The SYFP2 transgene (black) was induced by the enhancer eHGT_395m. This virus was packaged in the PHP.eB capsid. Each image is a montage. (Fig. 5A) shows the whole brain, and (Fig. 5B) shows the visual cortex (VISp - the dashed square in Fig. 5A). Note that reporter expression is seen in cells showing the morphology of oligodendrocytes and is concentrated in the white matter tracts.

[0017] [Figs. 6A - 6B]Fluorescent images of sagittal sections of mouse brains transduced with intravenously delivered CN2677 virus are shown. The SYFP2 transgene (black) was induced by the enhancer eHGT_401h. This virus was packaged in the PHP.eB capsid. Each image is a montage. (Figure 6A) shows the whole brain, and (Figure 6B) shows the visual cortex (VISp - the dashed square in Figure 6A). Note that reporter expression was seen in cells showing the morphology of oligodendrocytes and was concentrated in white matter tracts.

[0018] [Figs. 7A - 7B] Widespread astrocyte - specific labeling in the mouse brain by CN3572 is shown. (Figure 7A) shows an epifluorescence microscopy image of native SYFP2 expression in a sagittal section of a mouse brain 24 days after injection of 2.5×1011 viral genome copies of the AAV vector CN3572 (rAAV - 3xcore2_eHGT_390h - minBglobin - SYFP2 - WPRE3 - BGHpA) of serotype PHP.eB into the retro - orbital venous plexus. Abbreviations: CTX, cortex; STR, striatum; HPF, hippocampus; TH, thalamus; CB, cerebellum; BS, brainstem. Scale bar = 1 mm. (Figure 7B) shows an enlarged view of the area framed in Figure 7A, showing more detailed cell labeling of astrocytes in the CTX region. Scale bar = 200 microns.

[0019] [Figs. 8A - 8B] Widespread oligodendrocyte - specific labeling in the mouse brain by CN3575 is shown. (Figure 8A) shows an epifluorescence microscopy image of native SYFP2 expression in a sagittal section of a mouse brain 27 days after injection of 5.0×1011 viral genome copies of the AAV vector CN3575 (rAAV - 3xcore2_eHGT_400m - minBglobin - SYFP2 - WPRE3 - BGHpA) of serotype PHP.eB into the retro - orbital venous plexus. Scale bar = 1 mm. (Figure 8B) shows an enlarged view of the area framed in Figure 8A, showing more detailed cell labeling of astrocytes in the CTX region. Scale bar = 250 microns.

[0020] [Fig. 9]As arrays that support the present disclosure, eHGT_409h (SEQ ID NO: 1); eHGT_410h (SEQ ID NO: 2); eHGT_361h (SEQ ID NO: 3); eHGT_395m (SEQ ID NO: 4); eHGT_401h (SEQ ID NO: 5); eHGT_391h (SEQ ID NO: 6); eHGT_403h (SEQ ID NO: 7); eHGT_641m (SEQ ID NO: 166); Core_eHGT_641m (SEQ ID NO: 8); 3xCore_eHGT_641m (SEQ ID NO: 9); eHGT_400m (SEQ ID NO: 167); core2_eHGT_400m (SEQ ID NO: 10); 3xcore2_eHGT_400m (SEQ ID NO: 11); core2_eHGT_401h (SEQ ID NO: 12); 3xcore2_eHGT_401h (SEQ ID NO: 13); core2_eHGT_409h (SEQ ID NO: 14); 3xcore2_eHGT_409h (SEQ ID NO: 15); core2_eHGT_410h (SEQ ID NO: 16); 3xcore2_eHGT_410h (SEQ ID NO: 17); eHGT_331h (SEQ ID NO: 18); eHGT_332h (SEQ ID NO: 19); eHGT_333h (SEQ ID NO: 20); eHGT_335h (SEQ ID NO: 21); eHGT_334h (SEQ ID NO: 22); eHGT_360h (SEQ ID NO: 23); eHGT_562h (SEQ ID NO: 24); eHGT_563h (SEQ ID NO: 25); eHGT_564h (SEQ ID NO: 26); eHGT_565h (SEQ ID NO: 27); eHGT_566m (SEQ ID NO: 28); eHGT_567m (SEQ ID NO: 29); eHGT_568m (SEQ ID NO: 30); eHGT_569m (SEQ ID NO: 31); eHGT_734m (SEQ ID NO: 32); eHGT_312h (SEQ ID NO: 33); eHGT_313h (SEQ ID NO: 34); eHGT_377m (SEQ ID NO: 35); eHGT_389m (SEQ ID NO: 36); eHGT_380h (SEQ ID NO: 37); eHGT_374h (SEQ ID NO: 38); eHGT_387m (SEQ ID NO: 168); core1_eHGT_387m (SEQ ID NO: 39); 3xcore1_eHGT_387m (SEQ ID NO: 40); eHGT_385h (SEQ ID NO: 41); eHGT_386h (SEQ ID NO: 42); eHGT_378m (SEQ ID NO: 43); eHGT_642m (SEQ ID NO: 44); eHGT_390h (SEQ ID NO: 169); core2_eHGT_390h (SEQ ID NO: 47);3xcore2_eHGT_390h (SEQ ID NO: 48); core2_eHGT_380h (SEQ ID NO: 51); 3xcore2_eHGT_380h (SEQ ID NO: 52); eHGT_374m (SEQ ID NO: 170); core2_eHGT_374m (SEQ ID NO: 53); 3xcore2_eHGT_374m (SEQ ID NO: 54); eHGT_603m (SEQ ID NO: 55); eHGT_315h (SEQ ID NO: 155); eHGT_316h (SEQ ID NO: 156); eHGT_358h (SEQ ID NO: 157); eHGT_363h (SEQ ID NO: 158); eHGT_375m (SEQ ID NO: 171); core2_eHGT_375m (SEQ ID NO: 159); 3xcore2_eHGT_375m (SEQ ID NO: 160); β-globin minimal promoter (pBGmin / minBGlobin / minBGprom) (SEQ ID NO: 56); minCMV promoter (SEQ ID NO: 57); mutant minCMV promoter (removal of SacI restriction enzyme site) (SEQ ID NO: 58); minRho promoter (SEQ ID NO: 59); minRho* promoter (SEQ ID NO: 60); Hsp68 minimal promoter (proHsp68) (SEQ ID NO: 61); SYFP2 (SEQ ID NO: 62); EGFP (SEQ ID NO: 63); optimized Flp recombinase (FlpO) (SEQ ID NO: 64); improved Cre recombinase (iCre) (SEQ ID NO: 65); SP10 insulator (SP10ins) (SEQ ID NO: 66); 3xSP10ins (SEQ ID NO: 67); 3XFLAG (SEQ ID NO: 68); 10aa (SEQ ID NO: 69); H2B (SEQ ID NO: 70); WPRE3 (SEQ ID NO: 71); WPRE (SEQ ID NO: 72); BGHpA (SEQ ID NO: 73); hGHpA (SEQ ID NO: 74); P2A (SEQ ID NO: 75); T2A (SEQ ID NO: 76); E2A (SEQ ID NO: 77); F2A (SEQ ID NO: 78); exemplary plasmid backbone 1 - left ITR (SEQ ID NO: 79); exemplary plasmid backbone 1 - right ITR (SEQ ID NO: 80); exemplary plasmid backbone 2 - left ITR (SEQ ID NO: 81); exemplary plasmid backbone 2 - right ITR (SEQ ID NO: 82); PHP.eB capsid (SEQ ID NO: 83); AAV9 VP1 capsid protein (SEQ ID NO: 84); tet-transactivator version 2 (tTA2) (SEQ ID NO: 85); GTPase HRas [Homo sapiens] (SEQ ID NO: 86);Substance P [Homo sapiens] at positions 58 to 68 of Protachykinin-1 (SEQ ID NO: 87); Oxytocin [Homo sapiens] at positions 20 to 28 of Oxytocin-Neurophysin 1 (SEQ ID NO: 88); GCaMP6m (SEQ ID NO: 89); GCaMP6s (SEQ ID NO: 90); GCaMP6f (SEQ ID NO: 91); CN3062 (SEQ ID NO: 92); CN3063 (SEQ ID NO: 93); CN2979 (SEQ ID NO: 94); CN2498 (SEQ ID NO: 95); CN2677 (SEQ ID NO: 96); CN2505 (SEQ ID NO: 97); CN2499 (SEQ ID NO: 98); CN4004 (SEQ ID NO: 99); CN3575 (SEQ ID NO: 100); CN4254 (SEQ ID NO: 101); CN4255 (SEQ ID NO: 102); CN4256 (SEQ ID NO: 103); CN2806 (SEQ ID NO: 104); CN2807 (SEQ ID NO: 105); CN2808 (SEQ ID NO: 106); CN2809 (SEQ ID NO: 107); CN2812 (SEQ ID NO: 108); CN2813 (SEQ ID NO: 109); CN2678 (SEQ ID NO: 110); CN2679 (SEQ ID NO: 111); CN2680 (SEQ ID NO: 112); CN2681 (SEQ ID NO: 113); CN2833 (SEQ ID NO: 114); CN2834 (SEQ ID NO: 115); CN2835 (SEQ ID NO: 116); CN2836 (SEQ ID NO: 117); CN2627 (SEQ ID NO: 118); CN2675 (SEQ ID NO: 119); CN2344 (SEQ ID NO: 120); CN3060 (SEQ ID NO: 121); CN3061 (SEQ ID NO: 122); CN3098 (SEQ ID NO: 123); CN2493 (SEQ ID NO: 124); CN3568 (SEQ ID NO: 125); CN2495 (SEQ ID NO: 126); CN2496 (SEQ ID NO: 127); CN2827 (SEQ ID NO: 128); CN2846 (SEQ ID NO: 129); CN3572 (SEQ ID NO: 131); CN4253 (SEQ ID NO: 133); CN4257 (SEQ ID NO: 134); CN2410 (SEQ ID NO: 135); CN2345 (SEQ ID NO: 161); CN2346 (SEQ ID NO: 162); CN4522 (SEQ ID NO: 163); CN4523 (SEQ ID NO: 164); and CN4268 (SEQ ID NO: 165).;

Mode for Carrying Out the Invention

[0021] To fully understand the biology of the brain, it is necessary to distinguish and define various types of cells, investigate them in detail, and identify artificial expression constructs that can label and perturb these cells (Tasic, Curr. Opin. Neurobiol. 50, 242-249 (2018); Zeng & Sanes, Nat. Rev. Neurosci. 18, 530-546 (2017)). In mice, the use of driver strains that express recombinase has been successful in labeling cell populations that share marker gene expression (Daigle et al., Cell 174, 465-480.e22 (2018); Taniguchi, et al., Neuron 71, 995-1013 (2011); Gong et al., J. Neurosci. 27, 9817-9823 (2007)). However, the generation, maintenance, and use of such strains that can label specific types of cells with high specificity are costly, often require the mating of transgenic animals among three species, and the desired experimental animals can be obtained only at low frequencies. Furthermore, since these tools require germline transgenic animals, they cannot be applied to humans.

[0022] The present disclosure provides artificial expression constructs that induce gene expression in a target central nervous system cell population. Examples of the target central nervous system cell population include non-neuronal cells in the brain such as glial cells present in the brain parenchyma, and these non-neuronal cells can be further classified into oligodendrocytes, microglia, astrocytes, and oligodendrocyte progenitor cells, as well as pericytes, endothelial cells, and ependymal cells present in other cell compartments.

[0023] In certain embodiments of the artificial expression constructs of the present disclosure, the following enhancers are utilized to induce gene expression in a target central nervous system cell population. The combinations of target cell populations and enhancers used in the present disclosure are shown below in the order of target cell population / enhancer. Oligodendrocytes / eHGT_409h, eHGT_410h, eHGT_361h, eHGT_395m, eHGT_401h, eHGT_391h, eHGT_403h, 3xCore_eHGT_641m, 3xcore2_eHGT_400m, 3xcore2_eHGT_401h, 3xcore2_eHGT_409h, 3xcore2_eHGT 410h, and eHGT_363h; Microglia / eHGT_331h, eHGT_332h, eHGT_333h, eHGT_335h, eHGT_334h, eHGT_360h, eHGT_562h, eHGT_563h, eHGT_564h, eHGT_565h, eHGT_566m, eHGT_567m, eHGT_568m, eHGT_569m, and eHGT_734m; Astrocytes / eHGT_377m, eHGT_389m, eHGT_380h, eHGT_374h, 3xcore1_eHGT_387m, eHGT_385h, eHGT_386h, eHGT_378m, eHGT_642m, 3xcore2_eHGT_390h, 3xcore2_eHGT_380h, 3xcore2_eHGT_374m, 3xcore2_eHGT_375m, and eHGT_358h; Layer 1 Interlaminar Astrocytes / eHGT_312h, eHGT_313h, eHGT_315h, and eHGT_316h; and Endothelial Cells / eHGT_603m.

[0024] In certain embodiments, these artificial enhancer factors are concatemerized enhancer cores. Examples of such enhancer cores or concatemerized enhancer cores include the cores of eHGT_374m, eHGT_375m, eHGT_380h, eHGT_387m, eHGT_390h, eHGT_400m, eHGT_401h, eHGT_409h, eHGT_410h, and / or eHGT_641m, or concatemerized versions of these cores. By using such artificial enhancer factors, a transgene can be rapidly expressed to obtain high expression levels, more so than when using the full length of the original (natural) enhancer alone.

[0025] Certain embodiments of the enhancer core include Core_eHGT_641m, core2_eHGT_400m, core2_eHGT_401h, core2_eHGT_409h, core2_eHGT_410h, core1_eHGT_387m, core2_eHGT_390h, core2_eHGT_380h, core2_eHGT_374m, and core2_eHGT_375m. In certain embodiments, these enhancer cores are concatemerized and have 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the core sequence. In certain embodiments, examples of concatemers containing 3 copies of the selected enhancer core include 3xCore_eHGT_641m, 3xcore2_eHGT_400m, 3xcore2_eHGT_401h, 3xcore2_eHGT_409h, 3xcore2_eHGT_410h, 3xcore1_eHGT_387m, 3xcore2_eHGT_390h, 3xcore2_eHGT_380h, 3xcore2_eHGT_374m, and 3xcore2_eHGT_375m.

[0026] Certain embodiments provide artificial expression constructs that include the features of the vectors described herein, and such vectors include CN3062, CN3063, CN2979, CN2498, CN2677, CN2505, CN2499, CN4004, CN3575, CN4254, CN4255, CN4256, CN2806, CN2807, CN2808, CN2809, CN2812, CN2813, CN2678, CN2679, CN2680, CN2681, CN2833, CN2834, CN2835, CN2836, CN2627, CN2675, CN2344, CN3060, CN3061, CN3098, CN2493, CN3568, CN2495, CN2496, CN2827, CN2846, CN3572, CN4253, CN4257, CN2410, CN2345, CN2346, CN4268, CN4522, and CN4523.

[0027] Various aspects of the present disclosure will be described in more detail below, along with additional options. The various aspects of the present disclosure will be described below in the following items, namely, (i) artificial expression constructs and vectors for targeted expression of genes in target types of cells; (ii) compositions for administration; (iii) cell lines containing the artificial expression constructs; (iv) transgenic animals; (v) methods of use; (vi) kits and commercial packages; (vii) exemplary embodiments; and (viii) conclusions. These headings are presented for the purpose of systematic description only and do not limit the scope or interpretation of the present disclosure.

[0028] (i) Artificial expression constructs and vectors for targeted expression of genes in target types of cells The artificial expression constructs disclosed herein include (i) an enhancer sequence that induces targeted expression of a coding sequence in target types of central nervous system cells, (ii) the coding sequence to be expressed, and (iii) a promoter. The artificial expression constructs of the present disclosure may further include other regulatory factors, if necessary or beneficial.

[0029] In certain embodiments, an "enhancer" or "enhancer element" is a cis-acting sequence that increases the amount of transcription associated with a promoter and can function in either the forward or reverse direction with respect to the promoter and the transcribed coding sequence, and can be located either upstream or downstream with respect to the promoter or the transcribed coding sequence. In the art, various methods or techniques are known for measuring the function of enhancer element sequences. Specific examples of enhancer sequences used in the artificial expression constructs disclosed herein include eHGT_409h, eHGT_410h, eHGT_361h, eHGT_395m, eHGT_401h, eHGT_391h, eHGT_403h, 3xCore_eHGT_641m, 3xcore2_eHGT_400m, 3xcore2_eHGT_401h, 3xcore2_eHGT_409h, 3xcore2_eHGT 410h, eHGT_363h, eHGT_331h, eHGT_332h, eHGT_333h, eHGT_335h, eHGT_334h, eHGT_360h, eHGT_562h, eHGT_563h, eHGT_564h, eHGT_565h, eHGT_566m, eHGT_567m, eHGT_568m, eHGT_569m, eHGT_734m, eHGT_312h, eHGT_313h, eHGT_315h, eHGT_316h, eHGT_377m, eHGT_389m, eHGT_380h, eHGT_374h, 3xcore1_eHGT_387m, eHGT_385h, eHGT_386h, eHGT_378m, eHGT_642m, 3xcore2_eHGT_390h, 3xcore2_eHGT_380h, 3xcore2_eHGT_374m, 3xcore2_eHGT_375m, eHGT_358h, and eHGT_603m.

[0030] In certain embodiments, the enhancer used for the targeted type of central nervous system cell is an enhancer that is utilized only in the targeted type of central nervous system cell or an enhancer that is predominantly utilized in the targeted type of central nervous system cell. The enhancer used in the targeted type of central nervous system cell is an enhancer that enhances gene expression in the targeted central nervous system. In certain embodiments, the enhancer used in the targeted type of central nervous system cell enhances gene expression in the targeted central nervous system but does not substantially induce gene expression in other types of cells that are not the target, and thus is also a target central nervous system enhancer having cell-type specific transcriptional activity.

[0031] When a heterologous coding sequence operably linked to the enhancer disclosed herein is expressed in the targeted type of cell, this administered heterologous coding sequence is expressed in the intended type of cell.

[0032] When a heterologous coding sequence is selectively expressed in a selected cell, the heterologous coding sequence administered in the intended type of cell is expressed, but is not substantially expressed in other types of cells. This will be described in more detail below. In certain embodiments, not being substantially expressed in other types of cells means that the expression in the reference cell is less than 50% compared to the target type of cell; the expression in the reference cell is less than 40% compared to the target type of cell; the expression in the reference cell is less than 30% compared to the target type of cell; the expression in the reference cell is less than 20% compared to the target type of cell; or the expression in the reference cell is less than 10% compared to the target type of cell. In certain embodiments, "reference cell" refers to a non-target cell. The non-target cell may be present within the same anatomical structure as the target cell and / or may project to a common anatomical region. In certain embodiments, the reference cell is present within an anatomical structure adjacent to the anatomical structure containing the target type of cell. In certain embodiments, the reference cell is a non-target cell having a gene expression profile different from that of the target cell.

[0033] In certain embodiments, the transcript of the coding sequence may be low-expressed in non-selected types of cells, for example, expressed at less than 1% or at an expression level of 1%, 2%, 3%, 5%, 10%, 15% or 20% of the expression level of the transcript in the selected cell. In certain embodiments, the target type of central nervous system cell is the only type of cell that can express the correct combination of various transcription factors that can bind to the enhancers disclosed herein and induce gene expression. Thus, in certain embodiments, expression occurs only in the target type of cell.

[0034] In certain embodiments, the target cell types (e.g., non-neuronal cells) can be identified based on the transcriptional profiles described in Tasic et al., Nature 563, 72-78 (2018) and Hodge et al., Nature 573, 61-68 (2019). For reference, various cell types and their prominent features are described below.

[0035] Sub - classification of non - neuronal cells: · Astrocytes: Glial cells derived from the neuroectoderm, which express the Aqp4 marker and often also the GFAP marker, but do not express the neuronal marker SNAP25. Astrocytes may have a characteristic star-like morphology and are involved in supporting the metabolism of other cells in the brain. In mice and humans, multiple types of astrocyte morphologies are observed. · Oligodendrocytes: Glial cells derived from the neuroectoderm that express the Sox10 marker. This classification includes oligodendrocyte progenitor cells (OPCs). Oligodendrocytes are a subcategory mainly responsible for myelinating neurons. · VLMC: Vascular leptomeningeal cells (VLMC) are part of the meninges surrounding the outer layer of the cortex and express Lum and Col1a1 as marker genes. · Pericytes: Vascular-related cells that express Kcnj8 and Abcc9 as marker genes. Pericytes surround endothelial cells, are important for regulating blood flow in capillaries, and are involved in blood-brain barrier permeability. · Smooth muscle cells (SMC): Specialized smooth muscle cells, which are vascular-related cells that express the Acta2 marker gene. Smooth muscle cells cover arterioles in the brain and are involved in blood-brain barrier permeability. · Endothelial cells: Cells lining the blood vessels in the brain. Endothelial cells express the Tek marker and the PDGF-B marker. · Microglia: An immune cell derived from hematopoietic cells, a macrophage localized in brain tissue, a perivascular macrophage (PVM) that may migrate from the blood and be associated with brain tissue, and may be seen as a byproduct of a brain incision procedure. Microglia are known to express Cx3cr1, Tmem119, and PTPRC (CD45).

[0036] In certain embodiments, the coding sequence is a heterologous coding sequence encoding an effector factor. An effector factor is a sequence that is expressed to obtain an intended effect, and the intended effect is actually achieved by this effector factor. Examples of effector factors include reporter genes / proteins and functional genes / proteins.

[0037] Exemplary reporter genes / proteins include those expressed by Addgene ID No. 83894 (pAAV-hDlx-Flex-dTomato-Fishell_7), ID No. 83895 (pAAV-hDlx-Flex-GFP-Fishell_6), ID No. 83896 (pAAV-hDlx-GiDREADD-dTomato-Fishell-5), ID No. 83898 (pAAV-mDlx-ChR2-mCherry-Fishell-3), ID No. 83899 (pAAV-mDlx-GCaMP6f-Fishell-2), ID No. 83900 (pAAV-mDlx-GFP-Fishell-1), and ID No. 89897 (pcDNA3-FLAG-mTET2(N500)). Exemplary reporter genes include, in particular, an expressible fluorescent protein or an expressible biotin; blue fluorescent proteins (e.g., eBFP, eBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire); cyan fluorescent proteins (e.g., eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan, mTurquoise); green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green (mAzamigreen), CopGFP, AceGFP, avGFP, ZsGreen1, Oregon Green TM (Thermo Fisher Scientific)); luciferase; orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato, dTomato); red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRuby, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred, Texas Red TM(Thermo Fisher Scientific); far-red fluorescent proteins (e.g., mPlum and mNeptune); yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, SYFP2, Venus, YPet, PhiYFP, ZsYellow1); or reporter genes encoding tandemly linked complexes.

[0038] GFP is composed of 238 amino acids (26.9 kDa) and was first isolated from jellyfish of the genus Aequorea victoria / Aequorea aequorea / Aequorea forskalea, which emit green fluorescence when exposed to blue light. GFP isolated from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor excitation peak at 475 nm. Its emission peak is at 509 nm, in the low-wavelength region of green light in the visible spectrum. GFP obtained from Renilla reniformis has one major excitation peak at 498 nm. Because it can be used for a wide range of applications and further improvements are desired by researchers, various GFP mutants have been created. The first major improvement was a single point mutation (S65T) reported by Roger Tsien in Nature in 1995. This mutation dramatically improved the spectral properties of GFP, increasing fluorescence and photostability and shifting the major excitation peak to 488 nm while maintaining the emission peak at 509 nm. By adding a point mutation (F64L) that improves the folding efficiency at 37°C to GFP, enhanced GFP (EGFP) was obtained. EGFP has an extinction coefficient (denoted as ε) of 55,000 L / mol·cm, which is also known as the optical cross-sectional area per molecule of 9.13×10 -21 m 2 and is also known as the optical cross-sectional area per molecule. In addition, Superfolder GFP was reported in 2006 as a series of GFP mutants that can fold rapidly and mature even when fused to peptides with insufficient folding.

[0039] "Yellow Fluorescent Protein" (YFP) is a genetic variant of the green fluorescent protein derived from Aequorea victoria. Its excitation peak is at 514 nm and its emission peak is at 527 nm.

[0040] Exemplary functional molecules include ion transporters, cell transport proteins, enzymes, transcription factors, neurotransmitters, calcium reporters, channelrhodopsins, guide RNAs, nucleases, microRNAs, or designer receptors exclusively activated by designer drugs (DREADDs), each having functionality.

[0041] An ion transporter is an integral membrane protein responsible for transporting ions across the cell membrane. Ion transporters are found in most cell types and are important for regulating cell excitability and homeostasis. Ion transporters are involved in numerous cellular processes such as action potentials, synaptic transmission, hormone secretion, and muscle contraction. Many biological processes important for living cells involve the transport of cations such as calcium ions (Ca 2+ ), potassium ions (K + ), and sodium ions (Na + ) through ion channels. In certain embodiments, examples of ion transporters include voltage-gated sodium channels (e.g., SCN1A), potassium channels (e.g., KCNQ2), and calcium channels (e.g., CACNA1C).

[0042] Exemplary enzymes, transcription factors, receptors, membrane proteins, cell transport proteins, signal transduction molecules and neurotransmitters include enzymes such as lactase, lipase, helicase, α-glucosidase, aromatic L-amino acid decarboxylase (AADC), amylase; transcription factors such as SP1, AP-1, heat shock factor protein 1, C / EBP (CCAAT / enhancer binding protein), Oct-1; receptors such as transforming growth factor receptor β1, platelet-derived growth factor receptor, epidermal growth factor receptor, vascular endothelial growth factor receptor, interleukin 8 receptor α; membrane proteins and cell transport proteins such as clathrin, dynamin, caveolin, Rab4A, Rab-11A; signal transduction molecules such as nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), platelet-derived growth factor (PDGF), transforming growth factor β (TGFβ), epidermal growth factor (EGF), GTPase, HRas; and neurotransmitters such as cocaine- and amphetamine-regulated transcript, substance P, oxytocin, somatostatin.

[0043] In certain embodiments, examples of functional molecules include reporters that indicate the function and state of cells, such as calcium reporters. Intracellular calcium concentration is an important predictor of many cellular activities, such as neuronal activation, muscle cell contraction, and signal transduction by second messengers. As a sensitive and simple technique for monitoring intracellular calcium concentration, there is a method using a calcium indicator encoded by a gene (GECI). Among GECIs, a calcium sensor named GCaMP that uses green fluorescent protein (GFP) is highly efficient and widely used. GCaMP is formed by fusing M13 and calmodulin protein to the N-terminus and C-terminus of circularly permuted GFP. Some types of GCaMP exhibit characteristic fluorescence emission spectra (Zhao et al., Science, 2011, 333(6051): 1888-1891). Exemplary GECIs that exhibit green fluorescence include GCaMP3, GCaMP5G, GCaMP6s, GCaMP6m, GCaMP6f, jGCaMP7s, jGCaMP7c, jGCaMP7b, jGCaMP7f, jGCaMP8s, jGCaMP8m, and jGCaMP8f. Furthermore, GECIs that exhibit red fluorescence include jRGECO1a and jRGECO1b. AAV products containing GECIs are commercially available.For example, from Vigene Biosciences, AAV products such as AAV8-CAG-GCaMP3 (Catalog No. BS4-CX3AAV8), AAV8-Syn-FLEX-GCaMP6s-WPRE (Catalog No. BS1-NXSAAV8), AAV8-Syn-FLEX-GCaMP6s-WPRE (Catalog No. BS1-NXSAAV8), AAV9-CAG-FLEX-GCaMP6m-WPRE (Catalog No. BS2-CXMAAV9), AAV9-Syn-FLEX-jGCaMP7s-WPRE (Catalog No. BS12-NXSAAV9), AAV9-CAG-FLEX-jGCaMP7f-WPRE (Catalog No. BS12-CXFAAV9), AAV9-Syn-FLEX-jGCaMP7b-WPRE (Catalog No. BS12-NXBAAV9), AAV9-Syn-FLEX-jGCaMP7c-WPRE (Catalog No. BS12-NXCAAV9), AAV9-Syn-FLEX-NES-jRGECO1a-WPRE (Catalog No. BS8-NXAAAV9), AAV8-Syn-FLEX-NES-jRCaMP1b-WPRE (Catalog No. BS7-NXBAAV8) are provided.

[0044] In certain embodiments, calcium reporters include NTnC, a calcium indicator (GECI) encoded by a gene; a chimera in which myosin light chain kinase, GFP, and calmodulin are fused; TN-XXL, a calcium indicator; a BRET-based auto-luminescent calcium indicator; and / or OeNL(Ca2+)-18μ, a calcium indicator protein.

[0045] In certain embodiments, functional molecules include modulators of neuronal activity-like channelrhodopsins (e.g., channelrhodopsin-1, channelrhodopsin-2, and variants thereof). Channelrhodopsins are a subfamily of rhodopsin (retinylidene protein) that function as light-gated ion channels. In addition to channelrhodopsin-1 (ChR1) and channelrhodopsin-2 (ChR2), several channelrhodopsin variants have been developed. For example, Lin et al. (Biophys J, 2009, 96(5): 1803-14) describe the generation of chimeras of the transmembrane domains of ChR1 and ChR2 using site-directed mutagenesis. Zhang et al. (Nat Neurosci, 2008, 11(6): 631-3) describe VChR1, a channelrhodopsin variant that is shifted to red light. VChR1 has low photosensitivity and reduced membrane trafficking and expression. Other known channelrhodopsin variants include the ChR2 variants described in Nagel, et al., Proc Natl Acad Sci USA, 2003, 100(24): 13940-5, ChR2 / H134R (Nagel, G., et al., Curr Biol, 2005, 15(24): 2279-84), and ChD / ChEF / ChIEF (Lin, J. Y., et al., Biophys J, 2009, 96(5): 1803-14), all of which are activated by blue light (470 nm) but not sensitive to orange / red light. Other variants are described in Lin, Experimental Physiology, 2010, 96.1: 19-25; Knopfel et al., The Journal of Neuroscience, 2010, 30(45): 14998-15004; and Mardinly et al., Nat Neurosci. 2018, 21(6):881-893.

[0046] In certain embodiments, functional molecules include DNA and RNA editing tools such as CRISPR / Cas (e.g., guide RNAs and nucleases such as Cas, Cas9, cpf1). Further, functional molecules include recombinant Cpf1 as described in US Patent Publication No. 2018 / 0030425, US Patent Publication No. 2016 / 0208243, WO / 2017 / 184768, and Zetsche et al. (2015) Cell 163: 759-771; single-stranded gRNAs (see, e.g., Jinek et al. (2012) Science 337:816-821; Jinek et al. (2013) eLife 2:e00471; Segal (2013) eLife 2:e00563), editases, guide RNA molecules, microRNAs, or homologous recombination donor cassettes.

[0047] In certain embodiments, functional molecules include localization cassettes. In certain embodiments, localization cassettes are used to localize molecules (e.g., vectors, proteins, sensors) to specific compartments below the cellular unit, such as the cell body, axons, and dendrites of neurons. In certain embodiments, localization cassettes include cell body tags (e.g., cell body (EE-RR)) for localization to the cell body; axonal tags (e.g., those derived from GAP43) or synaptophysin (sy) for localization to axons; hydrophobic tails for localization to the cell membrane; and hydrophobic or alkyl chains for localization to the endoplasmic reticulum. In certain embodiments, localization cassettes are fused to sensor molecules such as GECIs. In certain embodiments, fusion proteins of localization cassettes and GECIs include cell body-jGCaMP8s, axon-jRGECO1a, syGCaMP5G, and cell body-jGCaMP7s.

[0048] In certain embodiments, the functional molecule includes a tag cassette. Examples of tag cassettes include His tag (HHHHHH; SEQ ID NO: 136), Flag tag (DYKDDDDK; SEQ ID NO: 137), Xpress tag (DLYDDDDK; SEQ ID NO: 138), Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO: 139), calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 140), polyglutamic acid tag, HA tag (YPYDVPDYA; SEQ ID NO: 141), Myc tag (EQKLISEEDL; SEQ ID NO: 142), Strep tag (meaning the original STREP® tag) (WRHPQFGG; SEQ ID NO: 143), STREP tag II (WSHPQFEK; SEQ ID NO: 144; (Institut fur Bioanalytik (IBA), Germany); see, for example, U.S. Patent Publication No. 7,981,632), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 145), Softag 3 (TQDPSRVG; SEQ ID NO: 146), and V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 147). In certain embodiments, the tag cassette includes a fusion tag cassette such as 3XFLAG. In certain embodiments, 3XFLAG includes the sequence shown in SEQ ID NO: 68.

[0049] The sequences of the aforementioned functional molecules are publicly available. For example, lactase (e.g., GenBank: EAX11622.1), lipase (e.g., GenBank: AAA60129.1), helicase (e.g., GenBank: AMD82207.1), amylase (e.g., GenBank: AAA51724.1), α-glucosidase (e.g., GenBank: ABI53718.1), transcription factor SP1 (e.g., UniProtKB / Swiss-Prot: P08047.3), transcription factor AP-1 (e.g., NP_002219.1), heat shock factor protein 1 (e.g., UniProtKB / Swiss-Prot: Q00613.1), CCAAT / enhancer-binding protein (C / EBP) β isoform a (e.g., NP_005185.2), Oct-1 (e.g., UniProtKB / Swiss-Prot: P14859.2), TGFβ (e.g., GenBank: CAF02096.2), glial cell line-derived neurotrophic factor (GDNF) (e.g., NP_001177397.1), platelet-derived growth factor receptor (e.g., GenBank: AAA60049.1), epidermal growth factor receptor (e.g., GenBank: CAA25240.1), vascular endothelial growth factor receptor (e.g., GenBank: AAC16449.2), interleukin 8 receptor α (e.g., GenBank: AAB59436.1), caveolin (e.g., GenBank: CAA79476.1), dynamin (e.g., GenBank: AAA88025.1), clathrin heavy chain 1 isoform 1 (e.g., NP_004850.1), clathrin heavy chain 2 isoform 1 (e.g., NP_009029.3), clathrin light chain A isoform a (e.g., NP_001824.1), clathrin light chain B isoform a (e.g., NP_001825.1), ras-related protein Rab-4A isoform 1 (e.g., NP_004569.2), ras-related protein Rab-11A (e.g., UniProtKB / Swiss-Prot: P62491.3), platelet-derived growth factor (e.g., GenBank: AAA60552.1), transforming growth factor β3 (e.g., GenBank: AAA61161.1), nerve growth factor (e.g., GenBank: CAA37703.1), EGF (e.g., GenBank: CAA34902.2) Cocaine- and Amphetamine-Regulated Transcript (A chain) (e.g., PDB:1HY9_A), protachykinin-1 (e.g., UniProtKB-P20366), oxytocin neurophysin 1 (e.g., UniProtKB-P01178), somatostatin (e.g., GenBank:AAH32625.1), genetically encoded green calcium indicator NTnC (A chain) [synthetic construct] (e.g., PDB:5MWC_A), calcium indicator TN-XXL [synthetic construct] (e.g., GenBank:ACF93133.1), BRET-based auto-luminescent calcium indicator [synthetic construct] (e.g., GenBank:ADF42668.1), calcium indicator protein OeNL(Ca2+)-18μ [synthetic construct] (e.g., GenBank:BBB18812.1), myosin light chain kinase, green fluorescent protein, calmodulin chimera (A chain) [synthetic construct] (e.g., PDB:3EKJ_A), channelopsin 1 (e.g., UniProtKB-F8UVI5), channelopsin 1 (e.g., GenBank:AER58217.1), channelrhodopsin 2 (e.g., UniProtKB-B4Y105), channelrhodopsin 2 [synthetic construct] (e.g., GenBank:ABO64386.1), CRISPR-associated protein (Cas) (e.g., GenBank:AKG27598.1), Cas9 [synthetic construct] (e.g., GenBank:AST09977.1), CRISPR-associated endonuclease Cpf1 (e.g., UniProtKB / Swiss-Prot:U2UMQ6.1), ribonuclease 4 or ribonuclease L (e.g., UniProtKB / Swiss-Prot:Q05823.2), deoxyribonuclease IIβ (e.g., GenBank:AAF76893.1), sodium channel protein type 1 subunit α (e.g., UniProtKB-P35498), member 2 of the voltage-gated potassium channel subfamily KQT (e.g., UniProtKB-O43526) and voltage-dependent L-type calcium channel subunit α-1C (e.g., UniProtKB-Q13936) may be mentioned.

[0050] Additional effector factors include Cre, iCre, dgCre, FlpO and tTA2. iCre refers to Cre with codon optimization. dgCre is a GFP / Cre recombinase fusion gene enhanced by fusing the first 159 amino acids of the dihydrofolate reductase gene (DHFR or folA) of the Escherichia coli K12 strain chromosome to the N-terminus, has a G67S mutation, and has a destabilizing domain mutation R12Y / Y100I by recombination. FlpO is a codon-optimized form of FLPe, and protein expression and FRT recombination efficiency in mouse cells are significantly improved. The FLP / FRT system, like the Cre / LoxP system, is widely used in gene expression (the generation of conditional knockout mice using the FLP / FRT system is also widely carried out). tTA2 refers to the tetracycline transactivator.

[0051] Exemplary expressible factors include expression products that do not contain effector factors, such as non-functional proteins or defective proteins. In certain embodiments, by utilizing such expressible factors, a method for testing the effects of the corresponding functional molecules can be implemented. In certain embodiments, the expressible factors have lost their functionality or have defects due to recombinant mutations that inactivate their functions. In these embodiments, the non-expressible factors are as structurally similar as possible to the corresponding functional molecules.

[0052] Exemplary self-cleaving peptides include 2A peptides that produce two proteins from one mRNA. The 2A sequence is a short sequence (e.g., 20 amino acids in length) and is often used in constructs with size limitations. Specific examples include P2A, T2A, E2A, and F2A. In certain embodiments, the artificial expression construct includes an internal ribosome entry site (IRES) sequence. The IRES can initiate translation by ribosomes from a second internal site on the mRNA molecule and can produce two proteins from one mRNA.

[0053] The artificial expression construct may encode nuclear translocation proteins such as histone H1, histone H2A, histone H2B, histone H3, histone H4, HPhA and H2B* which are histone-like proteins.

[0054] The coding sequences encoding the molecules described herein (e.g., RNA or protein) can be obtained from publicly available databases or publications. The coding sequences may further include various sequence polymorphisms, mutations and / or sequence variants, and such changes do not affect the function of the encoded molecule. "Encoding" refers to the property that a nucleic acid sequence such as a vector, plasmid, gene, cDNA, mRNA, etc. functions as a template for the synthesis of other molecules such as proteins.

[0055] The term "gene" may include not only the coding sequence but also regulatory regions such as promoters, enhancers, insulators and / or post-transcriptional regulatory factors (e.g., terminal regions). Further, the term may include any introns spliced from the mRNA transcript and other DNA sequences, as well as variants resulting from alternative splice sites. These sequences may further include degenerate codons of sequences or reference sequences that may be introduced to confer codon selectivity in a particular type of organism or cell.

[0056] Examples of promoters include common promoters, tissue-specific promoters, cell-specific promoters, and / or cytoplasm-specific promoters. Examples of promoters also include strong promoters, weak promoters, constitutive expression promoters, and / or inducible promoters. Inducible promoters induce expression in response to specific conditions, signals, or cellular events. For example, the promoter may be an inducible promoter that requires a specific ligand, small molecule, transcription factor, or hormone protein for induction of transcription from the promoter. Specific examples of promoters include minBglobin (also called minBGprom), CMV, minCMV, minCMV* (minCMV* is minCMV with the SacI restriction site removed), minRho, minRho* (minRho* is minRho with the SacI restriction site removed), the SV40 early promoter, the Hsp68 minimal promoter (proHSP68), and the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter. A minimal promoter by itself does not have the activity to induce gene expression alone, but can be activated to induce gene expression when linked to neighboring enhancer factors.

[0057] In certain embodiments, the expression construct is provided by being incorporated into a vector. A "vector" refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule, such as an expression construct. The nucleic acid to be transferred is usually ligated to the nucleic acid molecule of the vector and, for example, inserted into the nucleic acid molecule of the vector. The vector may contain a sequence that induces self-replication of the cell and may contain a sequence that allows integration into the DNA of the host cell. Examples of useful vectors include plasmids (e.g., DNA plasmids and RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.

[0058] The term "viral vector" is widely used to refer to a nucleic acid molecule containing viral-derived components that facilitate the transfer and expression of non-native nucleic acid molecules in cells. "Adeno-associated virus vector" refers to a viral vector or plasmid containing structural and functional gene factors or a part thereof mainly derived from AAV. "Retroviral vector" refers to a viral vector or plasmid containing structural and functional gene factors or a part thereof mainly derived from retroviruses. "Lentiviral vector" refers to a viral vector or plasmid containing structural and functional gene factors or a part thereof mainly derived from lentiviruses and the like. "Hybrid vector" refers to a vector containing structural and functional gene factors derived from two or more viruses.

[0059] "Adenovirus vector" refers to a construct containing an adenovirus sequence that is sufficient to (a) promote the packaging of an artificial expression construct and (b) express a coding sequence cloned in the sense or antisense direction. Recombinant adenovirus vectors include genetically modified forms of adenoviruses. Since the genetic constitution of adenoviruses is a linear double-stranded DNA virus of 36 kb, most of the adenovirus DNA can be replaced with a foreign sequence of up to 7 kb. Unlike retroviruses, adenovirus DNA can replicate using episomes without causing genotoxicity, and thus is not integrated into the chromosome even when host cells are infected with adenoviruses. Furthermore, adenoviruses are structurally stable, and genome rearrangement is not detected after extensive amplification.

[0060] Adenoviruses are particularly suitable for use as gene transfer vectors because they have a medium-sized genome, ease of manipulation, high titer, a wide range of target cells, and high infectivity. At both ends of the adenovirus genome are inverted terminal repeats (ITRs) that are 100-200 base pairs in length, and the ITRs are cis-acting factors necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the adenovirus genome contain various transcription units that are divided by the initiation of viral DNA replication. The E1 region (E1A and E1B) encodes proteins that are responsible for regulating the transcription of the adenovirus genome and some cellular genes. Expression of the E2 region (E2A and E2B) synthesizes proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and the suppression (shut-off) of host cell protein biosynthesis. Late gene products, which include most of the adenovirus capsid proteins, are expressed only after significant processing of a single primary transcript induced by the major late promoter (MLP). The MLP is particularly efficient in the late stage of infection, and all mRNAs induced by this promoter have a 5'-leader (TPL) sequence that is divided into three parts and is preferentially selected by the mRNA for translation.

[0061] The characteristics of adenovirus vectors other than the requirement that the adenovirus vector be replication-deficient or at least conditionally defective are not considered to be as important in ensuring the implementation of the specific embodiments disclosed herein. The adenovirus can be of any of the known 42 serotypes or subgroups A to F. Adenovirus type 5 is a human adenovirus for which much biochemical and genetic information is known and has been historically used in most constructs using adenovirus as a vector. Therefore, in certain embodiments, to obtain a conditionally replication-deficient adenovirus vector for use in specific embodiments, an adenovirus of serotype 5 of subgroup C is preferably used as the starting material.

[0062] As described herein, a normal vector is replication-defective and lacks the E1 region of adenovirus. Therefore, it is most convenient to introduce a polynucleotide encoding a gene of interest at a position where the coding sequence of the E1 region has been removed. However, the insertion position of the construct within the adenovirus sequence is not so important. The polynucleotide encoding the gene of interest may be inserted into the deleted E3 region of the E3 replacement vector, or may be inserted into the E4 region. The deletion of the E4 region is complemented by a helper cell line or a helper virus.

[0063] Adeno-associated virus (AAV) is a parvovirus that was found as a contaminant in stocks of adenovirus. AAV is a ubiquitous virus (85% of the US population has anti-AAV antibodies) and does not cause disease. Furthermore, since the replication of AAV depends on the presence of a helper virus (e.g., adenovirus), AAV is also classified as a dependovirus. Various serotypes have been isolated, among which AAV-2 has been the most extensively characterized. AAV has single-stranded linear DNA, which is encapsulated by the capsid proteins VP1, VP2, and VP3 to form an icosahedral virion with a diameter of 20 - 24 nm.

[0064] The length of AAV DNA is 4.7 kilobases. AAV DNA contains two open reading frames and is flanked by two ITRs. There are mainly two types of genes in the AAV genome, namely rep and cap. The rep gene encodes proteins responsible for AAV virus replication, and the cap gene encodes capsid proteins VP1-3. Each ITR forms a T-shaped hairpin structure. These terminal repeat sequences are the only cis components of AAV required for chromosomal integration. Therefore, AAV can be used as a vector that can remove all viral coding sequences and replace them with a gene cassette for delivery. Three AAV virus promoters have been identified and named p5, p19, and p40 respectively according to their positions on the map. Transcription from p5 and p19 results in the production of rep proteins, and transcription from p40 produces capsid proteins.

[0065] AAV has an excellent safety profile and can be expressed in target cell populations by modifying the capsid and genome, making it outstanding for use in the present disclosure. scAAV refers to self-complementary AAV. pAAV refers to plasmid adeno-associated virus. rAAV refers to recombinant adeno-associated virus.

[0066] Other viral vectors may also be used. For example, vectors derived from viruses such as vaccinia virus, poliovirus, and herpes virus may be used. These vectors provide beneficial features for various mammalian cells.

[0067] Retroviruses are often used as gene delivery tools. A "retrovirus" is an RNA virus whose genomic RNA is reverse transcribed to produce a double-stranded linear DNA copy, which is integrated into the host genome via covalent bonds. The retrovirus integrated into the host genome is called a "provirus". The provirus functions as a template for RNA polymerase II and induces the expression of RNA molecules encoding the structural proteins and enzymes required for the production of new virus particles.

[0068] Examples of retroviruses suitable for use in certain embodiments include Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), Rous sarcoma virus (RSV), and lentivirus.

[0069] "Lentivirus" refers to the complex retrovirus group (or genus of complex retroviruses). Examples of lentiviruses include HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna-maedi virus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In certain embodiments, an HIV-based vector backbone (i.e., the cis-acting sequence elements of HIV) can be used.

[0070] When using vectors, in some types, safety can be improved by replacing the U3 region of the 5'LTR, which induces the transcription of the viral genome in the production of virus particles, with a heterologous promoter. Examples of heterologous promoters that can be used for this purpose include, for example, the Simian virus 40 (SV40) (e.g., early or late) promoter, the Cytomegalovirus (CMV) (e.g., immediate early) promoter, the Moloney murine leukemia virus (MoMLV) promoter, the Rous sarcoma virus (RSV) promoter, and the Herpes simplex virus (HSV) (thymidine kinase) promoter. Normal promoters can induce high-level transcription independently of Tat. Replacement of the U3 region with a heterologous promoter results in the deletion of the complete U3 sequence from the virus production system, thus reducing the possibility of recombination that produces replicable viruses. In certain embodiments, the heterologous promoter has the additional advantage of being able to control the way the viral genome is transcribed. For example, the heterologous promoter may be an inducible promoter such that the whole or a part of the viral genome is transcribed only in the presence of an inducer. Examples of inducers include one or more compounds or physiological conditions, such as the culture temperature or culture pH of the host cell.

[0071] In certain embodiments, the viral vector contains the TAR factor. "TAR" refers to the "trans-activation response" genetic factor present in the R region of the lentiviral LTR. This factor interacts with the lentiviral trans-activator (tat) genetic factor to enhance viral replication. However, this genetic factor is not required in embodiments where the U3 region of the 5'LTR is replaced with a heterologous promoter.

[0072] The "R region" refers to the region within the retroviral LTR from the start point of the cap site (i.e., the transcription start point) to just before the start point of the poly(A) strand. The R region is also defined as the region sandwiched between the U3 region and the U5 region. The R region plays a role in moving nascent DNA from one end of the genome to the other during reverse transcription.

[0073] In certain embodiments, the expression of heterologous sequences in viral vectors can be increased by incorporating post-transcriptional regulatory elements and efficient polyadenylation sites into the viral vectors, and in this case, a transcription termination signal may further be incorporated into the viral vectors. The expression of heterologous nucleic acids can be increased by various post-transcriptional regulatory elements. Examples of post-transcriptional regulatory elements include the post-transcriptional regulatory element of woodchuck hepatitis virus (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the post-transcriptional regulatory element of hepatitis B virus (HPRE) (Smith et al., Nucleic Acids Res. 26(21):4818-4827, 1998); and other post-transcriptional regulatory elements (Liu et al., 1995, Genes Dev., 9:1766). In certain embodiments, the vector contains a post-transcriptional regulatory element such as WPRE or HPRE. In certain embodiments, the vector lacks or does not contain a post-transcriptional regulatory element such as WPRE or HPRE.

[0074] Expression of a heterologous gene can be increased by a factor that can induce efficient transcription termination and polyadenylation of a heterologous nucleic acid transcript. Transcription termination signals are typically found downstream of polyadenylation signals. In certain embodiments, the vector includes a polyadenylation signal at the 3' end of a polynucleotide encoding a molecule (e.g., a protein) to be expressed. A "poly(A) site" or "poly(A) sequence" refers to a DNA sequence that induces both transcription termination and polyadenylation of a nascent RNA transcript transcribed by RNA polymerase II. The polyadenylation sequence can improve the stability of the mRNA by adding a poly(A) tail to the 3' end of the coding sequence, thereby contributing to an improvement in translation efficiency. In certain embodiments, BGHpA, hGHpA, or SV40pA may be utilized. In certain embodiments, a preferred embodiment of the expression construct includes a terminator factor. The terminator factor can increase the amount of transcription and minimize transcription from the construct to another plasmid sequence by read-through.

[0075] In certain embodiments, the viral vector further comprises one or more insulator elements. The insulator element may protect sequences expressed from the viral vector, such as effector elements or expressible elements, from integration site effects. Integration site effects occur via cis-acting elements within genomic DNA and mean that the transferred sequence may or may not be expressed (i.e., position effect; see, for example, Burgess-Beusse et al., PNAS., USA, 99:16433, 2002; and Zhan et al., Hum. Genet., 109:471, 2001). In certain embodiments, the viral transfer vector comprises one or more insulator elements in the 3' LTR, and when the provirus integrates into the host genome, this insulator is integrated into both the 5' LTR and the 3' LTR during replication of the 3' LTR. Insulators suitable for use in certain embodiments include the chicken β-globin insulator (see Chung et al., Cell 74:505, 1993; Chung et al., PNAS USA 94:575, 1997; and Bell et al., Cell 98:387, 1999), the SP10 insulator (Abhyankar et al., JBC 282:36143, 2007), or other small CTCF recognition sequences that function as enhancer-blocking insulators (Liu et al., Nature Biotechnology, 33:198, 2015).

[0076] In addition to those described above, various types of suitable expression vectors are also known to those skilled in the art. Examples of these known expression vectors include commercially available expression vectors designed for general recombinant operations, such as plasmids containing one or more reporter genes and regulatory factors required for expressing the reporter genes in cells. For example, numerous vectors are commercially available from Invitrogen, Stratagene, Clontech, etc., and are described in various accompanying guidebooks. In certain embodiments, suitable expression vectors include any plasmid, cosmid, or phage construct capable of expressing the encoded gene in mammalian cells, such as pUC plasmid systems and Bluescript plasmid systems.

[0077] Certain embodiments of the vectors disclosed herein include those described in the following table.

Table 1

[0078] Sub-component sequences within large vector sequences can be readily identified by those skilled in the art based on the description of the present disclosure (see Figure 9). The nucleotides between the identifiable sub-components described in the above table are restriction enzyme recognition sites used for the assembly (cloning) of constructs and, in some cases, additional nucleotides with no identifiable function. These segments of the complete vector sequence can be regulated using various cloning techniques and / or various vectors. Usually, short 6-base palindromic sequences represent artifacts during vector construction that are not important for the function of the vector.

[0079] In certain embodiments, a vector (e.g., AAV) having a capsid capable of crossing the blood-brain barrier (BBB) is selected. In certain embodiments, the vector is engineered to contain a capsid that crosses the blood-brain barrier. Examples of AAVs having viral capsids that cross the blood-brain barrier include AAV9 (Gombash et al., Front Mol Neurosci. 2014; 7:81), AAVrh.10 (Yang, et al., Mol Ther. 2014; 22(7): 1299-1309), AAV1R6, AAV1R7 (Albright et al., Mol Ther. 2018; 26(2): 510), rAAVrh.8 (Yang et al., supra), AAV-BR1 (Marchio et al., EMBO Mol Med. 2016; 8(6): 592), AAV-PHP.S (Chan et al., Nat Neurosci. 2017; 20(8): 1172), AAV-PHP.B (Deverman et al., Nat Biotechnol. 2016; 34(2): 204), AAV-PPS (Chen et al., Nat Med. 2009; 15: 1215), and PHP.eB. In certain embodiments, the capsid of PHP.eB differs from AAV9 in that the amino acid residues 586 and later, S-AQ-A (SEQ ID NO: 148), are changed to S-DGTLAVPFK-A (SEQ ID NO: 149) when compared to AAV9 as a reference. In certain embodiments, PHP.eb refers to the sequence of SEQ ID NO: 83.

[0080] AAV9 is a natural AAV serotype that, unlike many other natural serotypes, can cross the blood-brain barrier (BBB) via intravenous injection. Since AAV9 can transduce a broad area of the central nervous system (CNS), treatment can be performed with minimal invasion (Naso et al., BioDrugs. 2017; 31(4): 317). Such cases have been reported in relation to, for example, clinical trials for the treatment of spinal muscular atrophy (SMA) syndrome by AveXis (AVXS-101, NCT03505099) and clinical trials for the treatment of CLN3 gene-related neuronal ceroid lipofuscinosis (NCT03770572).

[0081] AAVrh.10 is an AAV first isolated from cynomolgus monkeys and has a weakly positive human serum reactivity compared to other common serotypes used for gene delivery purposes (Selot et al., Front Pharmacol. 2017; 8: 441) and has been evaluated in several clinical trials (LYS-SAF302, LYSOGENE and NCT03612869).

[0082] AAV1R6 and AAV1R7 are two variants isolated from a library of chimeric AAV vectors (substituting the capsid domain of AAVrh.10 with the capsid domain of AAV1) that retain the ability to cross the BBB and transduce the central nervous system, but transduction into liver endothelium and vascular endothelium is significantly suppressed.

[0083] rAAVrh.8 is also an AAV isolated from cynomolgus monkeys and shows extensive transduction into glial cells and neurons in clinically relevant areas by peripheral administration, with suppressed tropism for peripheral tissues compared to other vectors.

[0084] AAV-BR1 is an AAV2 variant that presents the NRGTEWD epitope (SEQ ID NO: 151) and was isolated during in vivo screening of a random AAV display peptide library. AAV-BR1 highly expresses the transgene in the brain, shows high specificity, and has minimal off-target affinity (including affinity for the liver) (Korbelin et al., EMBO Mol Med. 2016; 8(6): 609).

[0085] AAV-PHP.S (Addgene, Watertown, Massachusetts) is a variant of AAV9 generated by the CREATE method, encodes the 7-mer sequence QAVRTSL (SEQ ID NO: 152), transduces neurons in the enteric nervous system, and strongly transduces peripheral sensory afferent nerves projecting to the spinal cord and brainstem.

[0086] AAV-PHP.B (Addgene, Watertown, Massachusetts) is a variant of AAV9 generated by the CREATE method and encodes the 7-mer sequence TLAVPFK (SEQ ID NO: 153). AAV-PHP.B transfers genes to the entire central nervous system with higher efficiency than AAV9 and transduces most astrocytes and neurons in multiple central nervous system regions.

[0087] AAV-PPS is an AAV2 variant produced by inserting the DSPAHPS epitope (SEQ ID NO: 154) into the AAV2 capsid and shows dramatically improved brain tropism compared to AAV2.

[0088] For further information on capsids that cross the blood-brain barrier, see Chan et al., Nat. Neurosci. 2017 Aug: 20(8): 1172-1179.

[0089] (ii) Compositions for administration The artificial expression constructs and vectors of the present disclosure (referred to herein as bioactive components) can be formulated using carriers suitable for administration to cells, tissue sections, animals (e.g., mice and non-human primates), or humans. The bioactive components contained in the compositions described herein can be prepared as the neutral form, as the free base, or as a pharmaceutically acceptable salt.

[0090] Examples of pharmaceutically acceptable salts include acid addition salts (formed from the free amino groups of proteins), which can be formed using inorganic acids such as hydrochloric acid and phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, and mandelic acid. Salts formed from free carboxyl groups can be derived from inorganic bases such as sodium, potassium, ammonium, calcium, and iron hydroxide, or organic bases such as isopropylamine, trimethylamine, histidine, and procaine.

[0091] Examples of carriers for bioactive components include solvents, dispersion media, vehicles, coating agents, diluents, isotonic agents, absorption delaying agents, buffers, solutions, suspensions, colloids, etc. The use of such carriers for bioactive components is well known in the art. Conventional media or agents can be used in combination with the compositions described herein, except when they are incompatible with the bioactive components of the present disclosure.

[0092] "Pharmaceutically acceptable carrier" refers to a carrier that does not cause allergic or similar adverse reactions when administered to humans, or in certain embodiments, when administered intravenously (e.g., into the retro-orbital venous plexus).

[0093] In certain embodiments, the compositions of the present disclosure can be formulated for intravenous, intracerebral, intraocular, intravitreal, parenteral, subcutaneous, intracerebroventricular, intramuscular, intrathecal, intraspinal, intraperitoneal, oral, or nasal inhalation administration, or for direct injection or direct administration to one or more cells, tissues, or organs.

[0094] The compositions of the present disclosure may include liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, and / or nanoparticles.

[0095] The formation and use of liposomes are widely known to those skilled in the art. Liposomes have been developed to improve stability in serum and half-life in the blood (see, for example, U.S. Patent No. 5,741,516). Furthermore, various methods of using liposomes and liposome-like preparations as drug carrier candidates have been reported (see, for example, U.S. Patent No. 5,567,434; U.S. Patent No. 5,552,157; U.S. Patent No. 5,565,213; U.S. Patent No. 5,738,868; and U.S. Patent No. 5,795,587).

[0096] The present disclosure also provides a pharmaceutically acceptable nanocapsule formulation of the bioactive ingredient of the present disclosure. Generally, nanocapsule formulations can encapsulate compounds in a stable and reproducible manner (Quintanar-Guerrero et al., Drug Dev Ind Pharm 24(12):1113-1128, 1998; Quintanar-Guerrero et al., Pharm Res. 15(7):1056-1062, 1998; Quintanar-Guerrero et al., J. Microencapsul. 15(1):107-119, 1998; Douglas et al., Crit Rev Ther Drug Carrier Syst 3(3):233-261, 1987). To avoid side effects due to the large amount of polymer taken up by cells, such ultrafine particles can be designed using polymers that can degrade in vivo. The use of biodegradable polyalkylcyanoacrylate nanoparticles that meet such requirements is also contemplated in the present disclosure. Such fine particles can be easily prepared, for which, for example, Couvreur et al., J Pharm Sci 69(2):199-202, 1980; Couvreur et al., Crit Rev Ther Drug Carrier Syst. 5(1)1-20, 1988; zur Muhlen et al., Eur J Pharm Biopharm, 45(2):149-155, 1998; Zambaux et al., J Control Release 50(1-3):31-40, 1998; and U.S. Patent No. 5,145,684 are described.

[0097] Injectable compositions include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions (U.S. Patent No. 5,466,468). Injectable compositions for delivery by injection are in the form of sterile fluids that can be delivered using a syringe. In certain embodiments, the injectable composition is usually stable during the manufacturing process and storage and may contain one or more preservative compounds to prevent contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or a dispersion medium, and this solvent or dispersion medium includes, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof, and / or vegetable oils. To maintain appropriate fluidity, for example, coating agents such as lecithin can be used, or in the case of dispersions, the particle size can be maintained at the required size, and / or surfactants can be used. To prevent the action of microorganisms, various antibacterial and / or antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. can be used. In various embodiments, the injectable composition includes, for example, isotonic agents such as saccharides and sodium chloride. Sustained absorption of the injectable composition can be achieved, for example, by incorporating an agent that delays absorption, such as aluminum monostearate or gelatin, into the injectable composition. If necessary, a suitable buffer can be added to the injectable composition, and the diluted liquid is initially made isotonic using sufficient physiological saline or glucose.

[0098] The dispersion may be prepared using glycerol, liquid polyethylene glycol or a mixture thereof or an oily agent. As described herein, under normal storage and use conditions, such preparations may contain a preservative to prevent the growth of microorganisms.

[0099] The sterilized composition can be prepared by mixing the bioactive ingredient with any other optional ingredients (e.g., the aforementioned ingredients) in an appropriate amount of solvent and subjecting it to sterile filtration. The dispersion is usually prepared by dispersing various sterile bioactive ingredients in a sterile solvent containing a basic dispersion medium and other necessary raw materials (e.g., the aforementioned raw materials, etc.). In the case of a sterile powder for preparing a sterile injection solution, a method of pre-sterile filtering a solution containing the bioactive ingredient and the desired other raw materials and then vacuum drying or freeze-drying this solution to prepare a powder containing the bioactive ingredient and the desired other raw materials is preferred.

[0100] The oral composition may be in a liquid form such as, for example, a solution, syrup, suspension, etc., and may be provided as a pharmaceutical product to be reconstituted with water or other suitable solvents before use. Such liquid formulations may be prepared by conventional methods using pharmaceutically acceptable additives. Examples of such pharmaceutically acceptable additives include suspending agents (e.g., sorbitol syrup, cellulose derivatives or hardened edible fats); emulsifying agents (e.g., lecithin or gum arabic); non-aqueous solvents (e.g., almond oil, ester oil or fractionated vegetable oil); and preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate or sorbic acid). The compositions of the present disclosure may also be prepared in the form of tablets or capsules by conventional methods using pharmaceutically acceptable excipients. Examples of such pharmaceutically acceptable excipients include binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods known in the art.

[0101] The inhalation composition can be delivered in the form of an aerosol spray agent that is jetted from a pressurized pack or a nebulizer using an appropriate propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol agent, the dosage unit may be determined by providing a valve that delivers a fixed quantity. It may be formulated into capsules or cartridges (e.g., gelatin capsules or gelatin cartridges) for use in an inhaler or a nebulizer, and such capsules and cartridges contain a mixed powder composed of the bioactive ingredient described herein and an appropriate powder base such as lactose or starch.

[0102] Furthermore, examples of the compositions of the present disclosure include microchip devices (U.S. Patent No. 5,797,898), ophthalmic preparations (Bourlais et al., Prog Retin Eye Res, 17(1):33 - 58, 1998), transdermal matrices (U.S. Patent Nos. 5,770,219 and 5,783,208), and feedback - controlled delivery (U.S. Patent No. 5,697,899).

[0103] In addition, an auxiliary active ingredient can also be included in the composition of the present disclosure.

[0104] Generally, the composition of the present disclosure may contain at least 0.1% or more of the bioactive ingredient. Needless to say, the proportion of the bioactive ingredient can be varied in various ways. Briefly, it may be in the range of 1% or 2% to 70% or 80% or more, or in the range of 0.5 to 99% with respect to the total weight or total volume of the composition of the present disclosure. Naturally, the amount of the bioactive ingredient in each physiologically beneficial composition may be adjusted so that an appropriate dosage can be obtained according to the predetermined unit dosage of the said compound. Factors such as solubility, bioavailability, biological half - life, administration route, product shelf life, and other pharmacological considerations are examined by those skilled in the art responsible for the preparation of such pharmaceutical formulations, and thus, various compositions and dosages are considered desirable.

[0105] In certain embodiments, upon administration to humans, the compositions of the present disclosure are required to meet the criteria regarding sterility, pyrogenicity, general safety and purity in accordance with the requirements of the US Food and Drug Administration (FDA) or other competent regulatory authorities in other countries.

[0106] (iii) Cell lines containing artificial expression constructs The present disclosure includes cells comprising the artificial expression constructs described herein. Cells transformed with the artificial expression constructs can be used for various purposes such as neuroanatomical studies, evaluation of functional and / or non-functional proteins, and drug screening to evaluate the regulatory properties of enhancers.

[0107] A variety of host cell lines can be used, but in certain embodiments, the host cell is a mammalian cell.In certain embodiments, the artificial expression construct comprises an enhancer and / or vector sequence selected from eHGT_409h, eHGT_410h, eHGT_361h, eHGT_395m, eHGT_401h, eHGT_391h, eHGT_403h, 3xCore_eHGT_641m, 3xcore2_eHGT_400m, 3xcore2_eHGT_401h, 3xcore2_eHGT_409h, 3xcore2_eHGT 410h, eHGT_363h, eHGT_331h, eHGT_332h, eHGT_333h, eHGT_335h, eHGT_334h, eHGT_360h, eHGT_562h, eHGT_563h, eHGT_564h, eHGT_565h, eHGT_566m, eHGT_567m, eHGT_568m, eHGT_569m, eHGT_734m, eHGT_312h, eHGT_313h, eHGT_315h, eHGT_316h, eHGT_377m, eHGT_389m, eHGT_380h, eHGT_374h, 3xcore1_eHGT_387m, eHGT_385h, eHGT_386h, eHGT_378m, eHGT_642m, 3xcore2_eHGT_390h, 3xcore2_eHGT_380h, 3xcore2_eHGT_374m, 3xcore2_eHGT_375m, eHGT_358h, or eHGT_603m, and / or CN3062, CN3063, CN2979, CN2498, CN2677, CN2505, CN2499, CN4004, CN3575, CN4254, CN4255, CN4256, CN2806, CN2807, CN2808, CN2809, CN2812, CN2813, CN2678, CN2679, CN2680, CN2681, CN2833, CN2834, CN2835, CN2836, CN2627, CN2675, CN2344, CN3060, CN3061, CN3098, CN2493, CN3568, CN2495, CN2496, CN2827, CN2846, CN3572, CN4253, CN4257, CN2410, CN2345, CN2346, CN4268, CN4522, or CN4523, and the host cell line is a human cell, a primate cell or a mouse cell.Furthermore, as cell lines that can be used for gene transfer in the present disclosure, there are primary cell lines derived from living tissues such as the brains of rats and mice, and organotypic cell cultures such as brain slices derived from animals, such as neurosurgical tissues of rats, mice, non-human primates or humans.

[0108] In certain embodiments, non-neuronal cell lines such as mouse embryonic stem cells may be used. Cultured mouse embryonic stem cells can be transiently transfected with a plasmid construct to analyze the expression of the gene construct. Mouse embryonic stem cells are pluripotent undifferentiated cells. Mouse embryonic stem cells can be maintained in an undifferentiated state by leukemia inhibitory factor (LIF). By removing LIF, differentiation of mouse embryonic stem cells can be induced. Mouse embryonic stem cells form various types of differentiated cells by culture. Differentiation of mouse embryonic stem cells occurs by the expression of tissue-specific transcription factors, which enables the evaluation of the function of enhancer sequences (see, for example, Fiskerstrand et al., FEBS Lett 458: 171-174, 1999).

[0109] In certain embodiments, the term "neuronal cell" is used when describing any neuronal cell, something related to a neuronal cell, or something containing a neuronal cell. Neuronal cells are defined by the characteristic of having an axon and dendrites. The term "neuron-specific" refers to something that is found in neuronal cells or cells derived therefrom, but not or substantially not found in cells not derived from neuronal cells or non-neuronal cells (e.g., glial cells such as astrocytes and oligodendrocytes); or an activity that occurs in neuronal cells or cells derived therefrom, but does not or substantially does not occur in cells not derived from neuronal cells or non-neuronal cells (e.g., glial cells such as astrocytes and oligodendrocytes).

[0110] U.S. Patent Publication No. 2012 / 0329714 describes the use of prolactin to increase the number of neural stem cells, and U.S. Patent Publication No. 2012 / 0308530 describes a culture surface having an amino group that promotes neural differentiation into neurons, astrocytes, and oligodendrocytes. Thus, the fate of neural stem cells can be controlled by various extracellular factors. Commonly used extracellular factors include brain-derived neurotrophic factor (BDNF; Shetty and Turner, 1998, J. Neurobiol. 35:395-425); fibroblast growth factor (bFGF; U.S. Patent No. 5,766,948; FGF-1, FGF-2); neurotrophin 3 (nt-3) and neurotrophin 4 (nt-4) (Caldwell, et al., 2001, Nat. Biotechnol. 1;19:475-9); ciliary neurotrophic factor (CNTF); BMP-2 (U.S. Patent No. 5,948,428 and U.S. Patent No. 6,001,654); isobutyl-3-methylxanthine; leukemia inhibitory factor (LIF; U.S. Patent No. 6,103,530); somatostatin; amphiregulin; neurotrophin (e.g., cyclic adenosine monophosphate); epidermal growth factor (EGF); dexamethasone (glucocorticoid hormone); forskolin; ligand of GDNF family receptor; potassium; retinoic acid (U.S. Patent No. 6,395,546); tetanus toxin; and transforming growth factor α and TGF-β (U.S. Patent No. 5,851,832 and U.S. Patent No. 5,753,506).

[0111] In certain embodiments, a yeast one-hybrid system may be used to identify compounds that inhibit specific protein-DNA interactions, and examples of such compounds include transcription factors such as eHGT_409h, eHGT_410h, eHGT_361h, eHGT_395m, eHGT_401h, eHGT_391h, eHGT_403h, 3xCore_eHGT_641m, 3xcore2_eHGT_400m, 3xcore2_eHGT_401h, 3xcore2_eHGT_409h, 3xcore2_eHGT 410h, eHGT_363h, eHGT_331h, eHGT_332h, eHGT_333h, eHGT_335h, eHGT_334h, eHGT_360h, eHGT_562h, eHGT_563h, eHGT_564h, eHGT_565h, eHGT_566m, eHGT_567m, eHGT_568m, eHGT_569m, eHGT_734m, eHGT_312h, eHGT_313h, eHGT_315h, eHGT_316h, eHGT_377m, eHGT_389m, eHGT_380h, eHGT_374h, 3xcore1_eHGT_387m, eHGT_385h, eHGT_386h, eHGT_378m, eHGT_642m, 3xcore2_eHGT_390h, 3xcore2_eHGT_380h, 3xcore2_eHGT_374m, 3xcore2_eHGT_375m, eHGT_358h, or eHGT_603m.

[0112] Transgenic animals are described below. The cell line may be derived from such transgenic animals. For example, cell lines in which an artificial expression construct has been integrated into the genome can be obtained from primary tissue cultures derived from transgenic mice (for example, see MacKenzie & Quinn, Proc Natl Acad Sci USA 96: 15251-15255, 1999).

[0113] (iv) Transgenic animals Another aspect of the present disclosure includes transgenic animals whose genomes contain artificial expression constructs comprising eHGT_409h, eHGT_410h, eHGT_361h, eHGT_395m, eHGT_401h, eHGT_391h, eHGT_403h, 3xCore_eHGT_641m, 3xcore2_eHGT_400m, 3xcore2_eHGT_401h, 3xcore2_eHGT_409h, 3xcore2_eHGT 410h, eHGT_363h, eHGT_331h, eHGT_332h, eHGT_333h, eHGT_335h, eHGT_334h, eHGT_360h, eHGT_562h, eHGT_563h, eHGT_564h, eHGT_565h, eHGT_566m, eHGT_567m, eHGT_568m, eHGT_569m, eHGT_734m, eHGT_312h, eHGT_313h, eHGT_315h, eHGT_316h, eHGT_377m, eHGT_389m, eHGT_380h, eHGT_374h, 3xcore1_eHGT_387m, eHGT_385h, eHGT_386h, eHGT_378m, eHGT_642m, 3xcore2_eHGT_390h, 3xcore2_eHGT_380h, 3xcore2_eHGT_374m, 3xcore2_eHGT_375m, eHGT_358h, and / or eHGT_603m operably linked to a heterologous coding sequence. In certain embodiments, the genomes of the transgenic animals include CN3062, CN3063, CN2979, CN2498, CN2677, CN2505, CN2499, CN4004, CN3575, CN4254, CN4255, CN4256, CN2806, CN2807, CN2808, CN2809, CN2812, CN2813, CN2678, CN2679, CN2680, CN2681, CN2833, CN2834, CN2835, CN2836, CN2627, CN2675, CN2344, CN3060, CN3061, CN3098, CN2493, CN3568, CN2495, CN2496, CN2827, CN2846, CN3572, CN4253, CN4257, CN2410, CN2345, CN2346, CN4268, CN4522, and / or CN4523.In certain embodiments, when using a non-integrating vector, the transgenic animal contains one or more artificial expression constructs containing eHGT_409h, eHGT_410h, eHGT_361h, eHGT_395m, eHGT_401h, eHGT_391h, eHGT_403h, 3xCore_eHGT_641m, 3xcore2_eHGT_400m, 3xcore2_eHGT_401h, 3xcore2_eHGT_409h, 3xcore2_eHGT 410h, eHGT_363h, eHGT_331h, eHGT_332h, eHGT_333h, eHGT_335h, eHGT_334h, eHGT_360h, eHGT_562h, eHGT_563h, eHGT_564h, eHGT_565h, eHGT_566m, eHGT_567m, eHGT_568m, eHGT_569m, eHGT_734m, eHGT_312h, eHGT_313h, eHGT_315h, eHGT_316h, eHGT_377m, eHGT_389m, eHGT_380h, eHGT_374h, 3xcore1_eHGT_387m, eHGT_385h, eHGT_386h, eHGT_378m, eHGT_642m, 3xcore2_eHGT_390h, 3xcore2_eHGT_380h, 3xcore2_eHGT_374m, 3xcore2_eHGT_375m, eHGT_358h, and / or eHGT_603m, and / or CN3062, CN3063, CN2979, CN2498, CN2677, CN2505, CN2499, CN4004, CN3575, CN4254, CN4255, CN4256, CN2806, CN2807, CN2808, CN2809, CN2812, CN2813, CN2678, CN2679, CN2680, CN2681, CN2833, CN2834, CN2835, CN2836, CN2627, CN2675, CN2344, CN3060, CN3061, CN3098, CN2493, CN3568, CN2495, CN2496, CN2827, CN2846, CN3572, CN4253, CN4257, CN2410, CN2345, CN2346, CN4268, CN4522, and / or CN4523 in one or more cells.

[0114] A detailed description of the method for producing transgenic animals is described in U.S. Patent No. 4,736,866. The transgenic animal can be any non-human species, but is preferably a non-human primate (NHP), sheep, horse, cow, pig, goat, dog, cat, rabbit, chicken; or a rodent such as guinea pig, hamster, gerbil, rat, mouse, ferret, etc.

[0115] In certain embodiments, by producing a transgenic animal, an organism is obtained in which a recombinant construct has been introduced into the same genomic integration site in all cells. Thus, cell lines derived from such transgenic animals have a consistent characteristic in that they have a recombinant construct at the same genomic integration site in all cells, and thus all of these cells are subject to the same variegated position effect. In contrast, when genes are introduced into cell lines or primary cell cultures, heterogeneous expression of the construct is obtained. This method has the disadvantage that the expression of the introduced DNA is affected by the specific genetic background of the host animal.

[0116] As previously described in connection with cell lines, the artificial expression constructs of the present disclosure can be used for gene recombination of mouse embryonic stem cells using techniques known in the art. Typically, the artificial expression construct is introduced into cultured mouse embryonic stem cells. Next, the transformed ES cells are injected into a blastocyst derived from a host mother, and the host embryo is re-implanted into the host mother. By this operation, a chimeric mouse having a tissue composed of cells derived from both embryonic stem cells present in the cultured cell line and embryonic stem cells present in the host embryo is obtained. Usually, a mouse having a different coat color from the host mouse into which the transformed cells are injected into the embryo is selected to isolate the cultured ES cells used for gene transfer. Thus, the chimeric mouse has a mixed-color coat. If at least a part of the germ cell line tissue is derived from the gene-recombinant cells, then by mating the chimeric mouse with an appropriate strain, progeny having the transgene can be obtained.

[0117] In addition to the delivery methods described above, as another method of delivering an artificial expression construct to a target cell or target tissue or an organ of an animal, specifically, a cell, organ or tissue of a mammalian vertebrate, sonophoresis (e.g., ultrasound as described in U.S. Patent No. 5,656,016); intraosseous injection (U.S. Patent No. 5,779,708); microchip device (U.S. Patent No. 5,797,898); ophthalmic formulations (Bourlais et al., Prog Retin Eye Res, 17(1):33-58, 1998); transdermal matrix (U.S. Patent No. 5,770,219 and U.S. Patent No. 5,783,208); feedback controlled delivery (U.S. Patent No. 5,697,899), and other delivery methods available and / or other delivery methods otherwise described in the present disclosure are contemplated.

[0118] (v) Methods of use In certain embodiments, the composition comprising a bioactive ingredient described herein is administered to a subject to provide a physiological effect.

[0119] In certain embodiments, the present disclosure includes the use of the artificial expression constructs described herein for regulating the expression of a heterologous gene, a part or all of which is encoded downstream of an enhancer of a recombinant sequence. Accordingly, methods of using the artificial expression constructs of the present disclosure are provided in the investigation, study and future development of pharmaceuticals for the prevention, treatment or alleviation of the symptoms of a disease, dysfunction or disorder.

[0120] A method for inducing gene expression in a target type of cell by administering to an artificial expression construct, wherein the artificial expression construct is eHGT_409h, eHGT_410h, eHGT_361h, eHGT_395m, eHGT_401h, eHGT_391h, eHGT_403h, 3xCore_eHGT_641m, 3xcore2_eHGT_400m, 3xcore2_eHGT_401h, 3xcore2_eHGT_409h, 3xcore2_eHGT as described herein410h, eHGT_363h, eHGT_331h, eHGT_332h, eHGT_333h, eHGT_335h, eHGT_334h, eHGT_360h, eHGT_562h, eHGT_563h, eHGT_564h, eHGT_565h, eHGT_566m, eHGT_567m, eHGT_568m, eHGT_569m, eHGT_734m, eHGT_312h, eHGT_313h, eHGT_315h, eHGT_316h, eHGT_377m, eHGT_389m, eHGT_380h, eHGT_374h, 3xcore1_eHGT_387m, eHGT_385h, eHGT_386h, eHGT_378m, eHGT_642m, 3xcore2_eHGT_390h, 3xcore2_eHGT_380h, 3xcore2_eHGT_374m, 3xcore2_eHGT_375m, eHGT_358h, and / or eHGT_603m, and / or CN3062, CN3063, CN2979, CN2498, CN2677, CN2505, CN2499, CN4004, CN3575, CN4254, CN4255, CN4256, CN2806, CN2807, CN2808, CN2809, CN2812, CN2813, CN2678, CN2679, CN2680, CN2681, CN2833, CN2834, CN2835, CN2836, CN2627, CN2675, CN2344, CN3060, CN3061, CN3098, CN2493, CN3568, CN2495, CN2496, CN2827, CN2846, CN3572, CN4253, CN4257, CN2410, CN2345, CN2346, CN4268, CN4522, and / or CN4523. The method includes the step of using a method characterized by including the above. The subject may be an isolated cell, a cell network, a tissue section, an experimental animal, a veterinary animal or a human.

[0121] As is well known in the medical field, the dosage administered to a subject depends on various factors such as the size, surface area and age of the subject's body, the specific compound being administered, gender, administration period and route, general health status, and other drugs during combined administration. The dosage of the compounds of the present disclosure varies, but in certain embodiments, the dosage of the artificial expression construct of the present disclosure is 10 5 ~10 100 copies may be sufficient. In certain embodiments, for patients receiving intravenous administration, intracerebral administration, intraspinal administration, retrobulbar venous plexus administration or intrathecal administration, 10 6 ~10 22 copies of the artificial expression construct can be injected.

[0122] An "effective amount" is the amount of a composition necessary to cause a desired physiological change in a subject. An effective amount is often administered for research purposes. The effective amounts disclosed herein are amounts that can cause a statistically significant effect in animal models, human studies, in vivo assays or in vitro assays.

[0123] The dosage of the expression construct and the administration period of such a composition are determined by those skilled in the art who understand the advantages of the teachings of the present disclosure. However, it is contemplated that the administration of an effective amount of the composition of the present disclosure may be by a single administration, for example, by a single injection of a sufficient number of infectious particles to confer an effect on the subject. Alternatively, depending on the situation, it may be desirable to administer the artificial expression construct composition or other gene constructs multiple times or continuously over a relatively short or relatively long period, and the decision of whether to make such an administration may be determined by the person in charge of monitoring the administration of such a composition. For example, the number of infectious particles administered to a mammal is 10 7 per ml, 10 8 per ml, 10 9 per ml, 10 10 per ml, 10 11 per ml, 10 12 per ml, 10 13It may be a single administration or multiple divided administrations at a dose of / ml or more. In certain embodiments, in practice, it may be desirable to administer a combination of two or more expression constructs to obtain the desired effect.

[0124] In certain situations, the artificial expression construct may be delivered in the form of the compositions disclosed herein, appropriately formulated, using a pipette or by retro-orbital venous plexus injection, subcutaneous administration, intraocular administration, intravitreal administration, parenteral administration, subcutaneous administration, intravenous administration, intracerebral administration, intraventricular administration, intramuscular administration, intrathecal administration, intraspinal administration, intraperitoneal administration, oral administration, nasal inhalation, or direct administration or injection into one or more cells, tissues or organs. The administration methods may include those described in U.S. Patent No. 5,543,158; U.S. Patent No. 5,641,515 and U.S. Patent No. 5,399,363.

[0125] (vi) Kits and commercial packages Kits and commercial packages contain the artificial expression constructs described herein. This artificial expression construct can be isolated. In certain embodiments, the components of the expression product can be separated from each other. In certain embodiments, the expression product is found within a vector, within a viral vector, within a cell, within a tissue section or tissue sample, and / or within a transgenic animal. Such kits may further include one or more reagents, restriction enzymes, peptides, therapeutic agents, pharmaceutical compounds, or means for delivering the compositions of the present disclosure (e.g., syringes, injectors, etc.).

[0126] Embodiments of the kit or commercial package may further include instructions regarding the use of the components included in the kit or commercial package, for example, in basic research, electrophysiological research, neuroanatomical research, and / or research and / or treatment of disorders, diseases or conditions.

[0127] The following exemplary embodiments are described to illustrate specific embodiments of the present disclosure. Those skilled in the art who refer to the present disclosure will understand that various changes can be made to the specific embodiments disclosed herein, and that even if such changes are made, similar results or analogous results can be obtained without departing from the gist and scope of the present disclosure.

[0128] (vii) Exemplary embodiments 1. An artificial enhancer comprising a core region of the eHGT_374m enhancer, eHGT_375m enhancer, eHGT_380h enhancer, eHGT_387m enhancer, eHGT_390h enhancer, eHGT_400m enhancer, eHGT_401h enhancer, eHGT_409h enhancer, eHGT_410h enhancer or eHGT_641m enhancer. 2. The artificial enhancer according to embodiment 1, which is a human or mouse enhancer. 3. The artificial enhancer according to embodiment 1 or 2, wherein the core region of the enhancer comprises the sequence shown in SEQ ID NO: 8, SEQ ID NO: 159, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 39, SEQ ID NO: 47, SEQ ID NO: 51 or SEQ ID NO: 53, or a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 8, SEQ ID NO: 159, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 39, SEQ ID NO: 47, SEQ ID NO: 51 or SEQ ID NO: 53. 4. The artificial enhancer according to any one of embodiments 1 to 3, wherein the core region of the enhancer comprises 2 copies, 3 copies, 4 copies, 5 copies, 6 copies, 7 copies, 8 copies, 9 copies or 10 copies. 5. The artificial enhancer according to embodiment 4, comprising one copy, two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, nine copies or ten copies of the sequence shown in SEQ ID NO: 8, SEQ ID NO: 159, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 39, SEQ ID NO: 47, SEQ ID NO: 51 or SEQ ID NO: 53, or a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 8, SEQ ID NO: 159, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 39, SEQ ID NO: 47, SEQ ID NO: 51 or SEQ ID NO: 53. 6. The artificial enhancer according to embodiment 4 or 5, comprising one copy, two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, nine copies or ten copies of the sequence shown in SEQ ID NO: 8. 7. The artificial enhancer according to embodiment 4 or 5, comprising one copy, two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, nine copies or ten copies of the sequence shown in SEQ ID NO: 159. 8. The artificial enhancer according to embodiment 4 or 5, comprising one copy, two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, nine copies or ten copies of the sequence shown in SEQ ID NO: 10. 9. The artificial enhancer according to embodiment 4 or 5, comprising one copy, two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, nine copies or ten copies of the sequence shown in SEQ ID NO: 12. 10. The artificial enhancer according to embodiment 4 or 5, comprising one copy, two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, nine copies or ten copies of the sequence shown in SEQ ID NO: 14. 11. The artificial enhancer according to embodiment 4 or 5, comprising one copy, two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, nine copies or ten copies of the sequence shown in SEQ ID NO: 16. 12. An artificial enhancer according to embodiment 4 or 5, comprising 1 copy, 2 copies, 3 copies, 4 copies, 5 copies, 6 copies, 7 copies, 8 copies, 9 copies or 10 copies of the sequence shown in SEQ ID NO: 39. 13. An artificial enhancer according to embodiment 4 or 5, comprising 1 copy, 2 copies, 3 copies, 4 copies, 5 copies, 6 copies, 7 copies, 8 copies, 9 copies or 10 copies of the sequence shown in SEQ ID NO: 47. 14. An artificial enhancer according to embodiment 4 or 5, comprising 1 copy, 2 copies, 3 copies, 4 copies, 5 copies, 6 copies, 7 copies, 8 copies, 9 copies or 10 copies of the sequence shown in SEQ ID NO: 51. 15. An artificial enhancer according to embodiment 4 or 5, comprising 1 copy, 2 copies, 3 copies, 4 copies, 5 copies, 6 copies, 7 copies, 8 copies, 9 copies or 10 copies of the sequence shown in SEQ ID NO: 53. 16. An artificial enhancer according to embodiment 6, comprising 3 copies of the sequence shown in SEQ ID NO: 8. 17. An artificial enhancer according to embodiment 7, comprising 3 copies of the sequence shown in SEQ ID NO: 159. 18. An artificial enhancer according to embodiment 8, comprising 3 copies of the sequence shown in SEQ ID NO: 10. 19. An artificial enhancer according to embodiment 9, comprising 3 copies of the sequence shown in SEQ ID NO: 12. 20. An artificial enhancer according to embodiment 10, comprising 3 copies of the sequence shown in SEQ ID NO: 14. 21. An artificial enhancer according to embodiment 11, comprising 3 copies of the sequence shown in SEQ ID NO: 16. 22. An artificial enhancer according to embodiment 12, comprising 3 copies of the sequence shown in SEQ ID NO: 39. 23. An artificial enhancer according to embodiment 13, comprising 3 copies of the sequence shown in SEQ ID NO: 47. 24. An artificial enhancer according to embodiment 14, comprising 3 copies of the sequence shown in SEQ ID NO: 51. 25. An artificial enhancer according to embodiment 15, comprising 3 copies of the sequence shown in SEQ ID NO: 53. 26. The artificial enhancer according to embodiment 16, comprising the sequence shown in SEQ ID NO: 9, or a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 9. 27. The artificial enhancer according to embodiment 17, comprising the sequence shown in SEQ ID NO: 160, or a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 160. 28. The artificial enhancer according to embodiment 18, comprising the sequence shown in SEQ ID NO: 11, or a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 11. 29. The artificial enhancer according to embodiment 19, comprising the sequence shown in SEQ ID NO: 13, or a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 13. 30. The artificial enhancer according to embodiment 20, comprising the sequence shown in SEQ ID NO: 15, or a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 15. 31. The artificial enhancer according to embodiment 21, comprising the sequence shown in SEQ ID NO: 17, or a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 17. 32. The artificial enhancer according to embodiment 22, comprising the sequence shown in SEQ ID NO: 40, or a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 40. 33. The artificial enhancer according to embodiment 23, comprising the sequence shown in SEQ ID NO: 48, or a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 48. 34. The artificial enhancer according to embodiment 24, comprising the sequence shown in SEQ ID NO: 52, or a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 52. 35. The artificial enhancer according to embodiment 25, comprising the sequence shown in SEQ ID NO: 54, or a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 54. 36. An artificial expression construct, (i) enhancers selected from eHGT_409h, eHGT_410h, eHGT_361h, eHGT_395m, eHGT_401h, eHGT_391h, eHGT_403h, 3xCore_eHGT_641m, 3xcore2_eHGT_400m, 3xcore2_eHGT_401h, 3xcore2_eHGT_409h, 3xcore2_eHGT 410h, eHGT_363h, eHGT_331h, eHGT_332h, eHGT_333h, eHGT_335h, eHGT_334h, eHGT_360h, eHGT_562h, eHGT_563h, eHGT_564h, eHGT_565h, eHGT_566m, eHGT_567m, eHGT_568m, eHGT_569m, eHGT_734m, eHGT_312h, eHGT_313h, eHGT_315h, eHGT_316h, eHGT_377m, eHGT_389m, eHGT_380h, eHGT_374h, 3xcore1_eHGT_387m, eHGT_385h, eHGT_386h, eHGT_378m, eHGT_642m, 3xcore2_eHGT_390h, 3xcore2_eHGT_380h, 3xcore2_eHGT_374m, 3xcore2_eHGT_375m, eHGT_358h and eHGT_603m, (ii) a promoter, and (iii) a heterologous coding sequence An artificial expression construct comprising. 37. The artificial expression construct according to embodiment 36, wherein the heterologous coding sequence encodes an effector factor or an expressible factor. 38. The artificial expression construct according to embodiment 37, wherein the effector factor comprises a reporter protein or a functional molecule. 39. The artificial expression construct according to embodiment 38, wherein the reporter protein is a fluorescent protein. 40. The artificial expression construct according to embodiment 38, wherein the functional molecule is a functional ion transporter, a functional enzyme, a functional transcription factor, a functional receptor, a functional membrane protein, a functional cell transport protein, a functional signal transduction molecule, a functional neurotransmitter, a functional calcium reporter, a functional channelrhodopsin, a functional CRISPR / Cas molecule, a functional editase, a functional guide RNA molecule, a functional microRNA, a functional homologous recombination donor cassette, or a designer receptor exclusively activated by designer drugs (DREADD). 41. The artificial expression construct according to embodiment 37, wherein the factor capable of being expressed contains a non-functional molecule. 42. The artificial expression construct according to embodiment 41, wherein the non-functional molecule is a non-functional ion transporter, a non-functional enzyme, a non-functional transcription factor, a non-functional receptor, a non-functional membrane protein, a non-functional cell transport protein, a non-functional signal transduction molecule, a non-functional neurotransmitter, a non-functional calcium reporter, a non-functional channelrhodopsin, a non-functional CRISPR / Cas molecule, a non-functional editase, a non-functional guide RNA molecule, a non-functional microRNA, a non-functional homologous recombination donor cassette, or a non-functional designer receptor exclusively activated by designer drugs (DREADD). 43. The artificial expression construct according to any one of embodiments 36 to 42, accompanied by a capsid that passes through the blood-brain barrier. 44. The artificial expression construct according to embodiment 43, wherein the capsid is PHP.eB, AAV9, AAVrh.10, AAV-BR1, AAV-PHP.S, AAV-PHP.B, or AAV-PPS. 45. The artificial expression construct according to any one of embodiments 36 to 44, containing a skipping factor or encoding a skipping factor. 46. The artificial expression construct according to embodiment 45, wherein the skipping factor is a 2A peptide and / or an internal ribosome entry site (IRES). 47. The artificial expression construct according to embodiment 46, wherein the 2A peptide is T2A, P2A, E2A or F2A. 48. The artificial expression construct according to any one of embodiments 36 to 47, comprising a series of features selected from eHGT_409h, eHGT_410h, eHGT_361h, eHGT_395m, eHGT_401h, eHGT_391h, eHGT_403h, 3xCore_eHGT_641m, 3xcore2_eHGT_400m, 3xcore2_eHGT_401h, 3xcore2_eHGT_409h, 3xcore2_eHGT 410h, eHGT_363h, eHGT_331h, eHGT_332h, eHGT_333h, eHGT_335h, eHGT_334h, eHGT_360h, eHGT_562h, eHGT_563h, eHGT_564h, eHGT_565h, eHGT_566m, eHGT_567m, eHGT_568m, eHGT_569m, eHGT_734m, eHGT_312h, eHGT_313h, eHGT_315h, eHGT_316h, eHGT_377m, eHGT_389m, eHGT_380h, eHGT_374h, 3xcore1_eHGT_387m, eHGT_385h, eHGT_386h, eHGT_378m, eHGT_642m, 3xcore2_eHGT_390h, 3xcore2_eHGT_380h, 3xcore2_eHGT_374m, 3xcore2_eHGT_375m, eHGT_358h, eHGT_603m, AAV, scAAV, rAAV, pAAV, minBglobin, CMV, minCMV, minCMV*, minRho, minRho*, fluorescent proteins (e.g., EGFP, SYFP, GFP), hsA2, Cre, iCre, dgCre, FlpO, tTA2, SP10 (e.g., 3xSP10), tag cassette, 10aa, nuclear translocation protein, self-cleaving peptide, WPRE, WPRE3, hGHpA and / or BGHpA, or encoding a series of features selected from these. 49. eHGT_409h - minBglobin - [heterologous coding sequence] - [post-transcriptional regulatory factor]; eHGT_410h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_361h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_395m-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_401h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_391h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_403h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; 3xCore_eHGT_641m-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; 3xcore2_eHGT_400m-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; 3xcore2_eHGT_401h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; 3xcore2_eHGT_409h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; 3xcore2_eHGT_410h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_363h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_363h-minBglobin - [Heterologous coding sequence] - [Skipping factor] - [Tag cassette] - 10aa - [Nuclear translocation protein] - [Post-transcriptional regulator]; eHGT_331h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_332h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_333h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_335h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_334h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_360h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_562h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_563h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_564h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_565h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_566m-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_567m-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_568m-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_569m-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_734m-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_312h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_313h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_315h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_316h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_377m-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_389m-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_380h-minBglobin - [Heterologous coding sequence] - [Post-transcriptional regulator]; eHGT_374h-minBglobin-[Heterologous coding sequence]-[Post-transcriptional regulator]; 3xcore1_eHGT_387m-minBglobin-[Heterologous coding sequence]-[Post-transcriptional regulator]; eHGT_385h-minBglobin-[Heterologous coding sequence]-[Post-transcriptional regulator]; eHGT_386h-minBglobin-[Heterologous coding sequence]-[Post-transcriptional regulator]; eHGT_378m-minBglobin-[Heterologous coding sequence]-[Post-transcriptional regulator]; eHGT_642m-minBglobin-[Heterologous coding sequence]-[Post-transcriptional regulator]; 3xcore2_eHGT_390h-minBglobin-[Heterologous coding sequence]-[Post-transcriptional regulator]; 3xcore2_eHGT_380h-minBglobin-[Heterologous coding sequence]-[Post-transcriptional regulator]; 3xcore2_eHGT_374m-minBglobin-[Heterologous coding sequence]-[Post-transcriptional regulator]; 3xcore2_eHGT_375m-minBglobin-[Heterologous coding sequence]-[Post-transcriptional regulator]; eHGT_358h-minBglobin-[Heterologous coding sequence]-[Post-transcriptional regulator]; eHGT_358h-minBglobin-[Heterologous coding sequence]-[Skipping factor]-[Tag cassette]-10aa-[Nuclear translocation protein]-[Post-transcriptional regulator]; eHGT_603m-minBglobin-[Heterologous coding sequence]-[Post-transcriptional regulator]; eHGT_409h-[Minimal promoter]-[Heterologous coding sequence]-[Post-transcriptional regulator]; eHGT_410h-[Minimal promoter]-[Heterologous coding sequence]-[Post-transcriptional regulator]; eHGT_361h-[Minimal promoter]-[Heterologous coding sequence]-[Post-transcriptional regulator]; eHGT_395m-[Minimal promoter]-[Heterologous coding sequence]-[Post-transcriptional regulator]; eHGT_401h - [Minimal promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_391h - [Minimal promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_403h - [Minimal promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; 3xCore_eHGT_641m - [Minimal promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; 3xcore2_eHGT_400m - [Minimal promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; 3xcore2_eHGT_401h - [Minimal promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; 3xcore2_eHGT_409h - [Minimal promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; 3xcore2_eHGT_410h - [Minimal promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_363h - [Minimal promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_363h - [Minimal promoter] - [Heterologous coding sequence] - [Skipping factor] - [Tag cassette] - 10aa - [Nuclear translocation protein] - [Post - transcriptional regulator]; eHGT_331h - [Minimal promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_332h - [Minimal promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_333h - [Minimal promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_335h - [Minimal promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_334h - [Minimal promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_360h - [Minimal promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_562h - [Minimal promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_563h - [Minimum promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_564h - [Minimum promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_565h - [Minimum promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_566m - [Minimum promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_567m - [Minimum promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_568m - [Minimum promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_569m - [Minimum promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_734m - [Minimum promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_312h - [Minimum promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_313h - [Minimum promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_315h - [Minimum promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_316h - [Minimum promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_377m - [Minimum promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_389m - [Minimum promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_380h - [Minimum promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_374h - [Minimum promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; 3xcore1_eHGT_387m - [Minimum promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_385h - [Minimum promoter] - [Heterologous coding sequence] - [Post - transcriptional regulator]; eHGT_386h - [minimal promoter] - [heterologous coding sequence] - [post - transcriptional regulator]; eHGT_378m - [minimal promoter] - [heterologous coding sequence] - [post - transcriptional regulator]; eHGT_642m - [minimal promoter] - [heterologous coding sequence] - [post - transcriptional regulator]; 3xcore2_eHGT_390h - [minimal promoter] - [heterologous coding sequence] - [post - transcriptional regulator]; 3xcore2_eHGT_380h - [minimal promoter] - [heterologous coding sequence] - [post - transcriptional regulator]; 3xcore2_eHGT_374m - [minimal promoter] - [heterologous coding sequence] - [post - transcriptional regulator]; 3xcore2_eHGT_375m - [minimal promoter] - [heterologous coding sequence] - [post - transcriptional regulator]; eHGT_358h - [minimal promoter] - [heterologous coding sequence] - [post - transcriptional regulator]; eHGT_358h - [minimal promoter] - [heterologous coding sequence] - [skipping factor] - [tag cassette] - 10aa - [nuclear translocation protein] - [post - transcriptional regulator]; and eHGT_603m - [minimal promoter] - [heterologous coding sequence] - [post - transcriptional regulator] An artificial expression construct according to any one of Embodiments 36 to 48, comprising a series of features selected from, or encoding a series of features selected from, these. 50. A vector comprising the artificial expression construct according to any one of Embodiments 36 to 49. 51. The vector according to Embodiment 50, which is a viral vector. 52. The vector according to Embodiment 50 or 51, wherein the viral vector is a recombinant adeno - associated virus (AAV) vector. 53. An adeno-associated virus (AAV) vector comprising at least one heterologous coding sequence, wherein the heterologous coding sequence is under transcriptional control of an enhancer and a promoter selected from eHGT_409h, eHGT_410h, eHGT_361h, eHGT_395m, eHGT_401h, eHGT_391h, eHGT_403h, 3xCore_eHGT_641m, 3xcore2_eHGT_400m, 3xcore2_eHGT_401h, 3xcore2_eHGT_409h, 3xcore2_eHGT 410h, eHGT_363h, eHGT_331h, eHGT_332h, eHGT_333h, eHGT_335h, eHGT_334h, eHGT_360h, eHGT_562h, eHGT_563h, eHGT_564h, eHGT_565h, eHGT_566m, eHGT_567m, eHGT_568m, eHGT_569m, eHGT_734m, eHGT_312h, eHGT_313h, eHGT_315h, eHGT_316h, eHGT_377m, eHGT_389m, eHGT_380h, eHGT_374h, 3xcore1_eHGT_387m, eHGT_385h, eHGT_386h, eHGT_378m, eHGT_642m, 3xcore2_eHGT_390h, 3xcore2_eHGT_380h, 3xcore2_eHGT_374m, 3xcore2_eHGT_375m, eHGT_358h and eHGT_603m. 54. The AAV vector according to embodiment 53, wherein the heterologous coding sequence encodes an effector factor or an expressible factor. 55. The AAV vector according to embodiment 54, wherein the effector factor comprises a reporter protein or a functional molecule. 56. The AAV vector according to embodiment 55, wherein the reporter protein is a fluorescent protein. 57. The AAV vector according to embodiment 55, wherein the functional molecule is a functional ion transporter, a functional enzyme, a functional transcription factor, a functional receptor, a functional membrane protein, a functional cell transport protein, a functional signaling molecule, a functional neurotransmitter, a functional calcium reporter, a functional channelrhodopsin, a functional CRISPR / Cas molecule, a functional editase, a functional guide RNA molecule, a functional microRNA, a functional homologous recombination donor cassette, or a designer receptor exclusively activated by designer drugs (DREADD). 58. The AAV vector according to embodiment 54, wherein the expressible factor comprises a non-functional molecule. 59. The AAV vector according to embodiment 58, wherein the non-functional molecule is a non-functional ion transporter, a non-functional enzyme, a non-functional transcription factor, a non-functional receptor, a non-functional membrane protein, a non-functional cell transport protein, a non-functional signaling molecule, a non-functional neurotransmitter, a non-functional calcium reporter, a non-functional channelrhodopsin, a non-functional CRISPR / Cas molecule, a non-functional editase, a non-functional guide RNA molecule, a non-functional microRNA, a non-functional homologous recombination donor cassette, or a non-functional designer receptor exclusively activated by designer drugs (DREADD). 60. A transgenic cell comprising the artificial expression construct or vector according to any one of the preceding embodiments. 61. The transgenic cell according to embodiment 60, which is a non-neuronal cell. 62. The transgenic cell according to embodiment 60 or 61, which comprises glial cells. 63. The transgenic cell according to embodiment 62, wherein the glial cell is an oligodendrocyte, a microglia, or an astrocyte. 64. The transgenic cell according to embodiment 63, wherein the astrocyte is a layer 1 interlaminar astrocyte. 65. The transgenic cell according to embodiment 60 or 61, which is an endothelial cell. 66. The transgenic cell according to any one of embodiments 60 to 65, which is a mouse cell, a human cell, or a non-human primate cell. 67. A non-human transgenic animal comprising the artificial expression construct, vector, and / or transgenic cell according to any one of the preceding embodiments. 68. The non-human transgenic animal according to embodiment 67, which is a mouse or a non-human primate. 69. An administrable composition comprising the artificial expression construct, vector, and / or transgenic cell according to any one of the preceding embodiments. 70. A kit comprising the artificial expression construct, vector, transgenic cell, non-human transgenic animal, and / or administrable composition according to any one of the preceding embodiments. 71. A method for expressing a gene in a target population of non-neuronal cells, either in vivo or in vitro, the method comprising the step of expressing the gene in the target population of non-neuronal cells by providing the administrable composition according to embodiment 69 to a sample or subject comprising the target population of non-neuronal cells and other cells in a sufficient dose and for a sufficient period. 72. The method according to embodiment 71, wherein the gene encodes an effector factor or an expressible factor. 73. The method according to embodiment 72, wherein the effector factor comprises a reporter protein or a functional molecule. 74. The method according to embodiment 73, wherein the reporter protein is a fluorescent protein. 75. The method according to embodiment 73, wherein the functional molecule is a functional ion transporter, a functional enzyme, a functional transcription factor, a functional receptor, a functional membrane protein, a functional cell transport protein, a functional signaling molecule, a functional neurotransmitter, a functional calcium reporter, a functional channelrhodopsin, a functional CRISPR / Cas molecule, a functional editase, a functional guide RNA molecule, a functional microRNA, a functional homologous recombination donor cassette, or a functional designer receptor exclusively activated by designer drugs (DREADD). 76. The method according to embodiment 72, wherein the expressible factor comprises a non-functional molecule. 77. The method according to embodiment 76, wherein the non-functional molecule is a non-functional ion transporter, non-functional enzyme, non-functional transcription factor, non-functional receptor, non-functional membrane protein, non-functional cell transport protein, non-functional signaling molecule, non-functional neurotransmitter, non-functional calcium reporter, non-functional channelrhodopsin, non-functional CRISPR / Cas molecule, non-functional editase, non-functional guide RNA molecule, non-functional microRNA, non-functional homologous recombination donor cassette, or non-functional designer receptor exclusively activated by designer drugs (DREADD). 78. The method according to any one of embodiments 71-77, wherein the providing step comprises pipetting. 79. The method according to embodiment 78, wherein the pipetting is performed on a brain slice. 80. The method according to embodiment 79, wherein the brain slice comprises the target population of non-neuronal cells and neurons. 81. The method according to embodiment 79, wherein the brain slice comprises glial cells. 82. The method according to embodiment 81, wherein the glial cells comprise oligodendrocytes, microglia or astrocytes. 1. The method according to claim 82, wherein the astrocyte is a layer-intermediate astrocyte of layer 1. 83. The method according to embodiment 79, wherein the brain slice comprises endothelial cells. 84. The method according to any one of embodiments 79-83, wherein the brain slice is a brain slice of a mouse, human or non-human primate. 85. The method according to any one of embodiments 878-84, wherein the providing step comprises administration to a living subject. 86. The method according to embodiment 85, wherein the living subject is a human, non-human primate or mouse. 87. The method according to embodiment 85 or 86, wherein the administration to the living subject is performed by injection. 88. The method according to embodiment 87, wherein the injection is an intravenous injection, an intracerebral injection into brain tissue, an intracerebroventricular (ICV) injection, an intracisternal (ICM) injection, or an intrathecal injection. 89. An artificial expression construct, comprising a sequence shown in SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164 or SEQ ID NO: 165, or a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164 or SEQ ID NO: 165.

[0129] (viii) Conclusion The sequences disclosed in this specification and variants of the cited sequences are also included in the present application. Indicators for determining which amino acid residues can be substituted, inserted or deleted without losing biological activity are well-known computer programs in the art, such as DNASTAR TM software (Madison, Wisconsin, USA). The amino acid changes of the protein variants disclosed in this specification are preferably conservative amino acid changes, that is, substitutions between amino acids with similar charges or substitutions between uncharged amino acids are preferred. Conservative amino acid changes include substitutions by members of amino acid families with related side chains.

[0130] Suitable conservative substitutions of amino acids in peptides or proteins are known to those skilled in the art, and generally such conservative substitutions can be made without changing the biological activity of the ultimately obtained molecule. Those skilled in the art will generally be familiar with the fact that substituting one amino acid in an unimportant region of a polypeptide will not substantially change its biological activity (see, for example, Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Natural amino acids are generally classified into conservative substitution families, specifically: Group 1: alanine (Ala), glycine (Gly), serine (Ser) and threonine (Thr); Group 2: (acidic): aspartic acid (Asp) and glutamic acid (Glu); Group 3: (acidic; negatively charged residues with polarity and also classified as their amides): asparagine (Asn), glutamine (Gln), Asp and Glu; Group 4: Gln and Asn; Group 5: (basic; positively charged residues with polarity and also classified as such): arginine (Arg), lysine (Lys) and histidine (His); Group 6 (large aliphatic non-polar residues): isoleucine (Ile), leucine (Leu), methionine (Met), valine (Val) and cysteine (Cys); Group 7 (polar uncharged): tyrosine (Tyr), Gly, Asn, Gln, Cys, Ser and Thr; Group 8 (large aromatic residues): phenylalanine (Phe), tryptophan (Trp) and Tyr; Group 9 (non-polar): proline (Pro), Ala, Val, Leu, Ile, Phe, Met and Trp; Group 11 (aliphatic): Gly, Ala, Val, Leu and Ile; Group 10 (small aliphatic residues that are non-polar or slightly polar): Ala, Ser, Thr, Pro and Gly; and Group 12 (sulfur-containing residues): Met and Cys. Further information is described in Creighton (1984) Proteins, W.H. Freeman and Company.

[0131] When making such changes, the hydrophobicity index of amino acids may be taken into consideration. It is widely understood in the art that the hydrophobicity index of amino acids is important when conferring biological functions that interact with each other on a protein (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). The hydrophobicity indices of each amino acid are: Ile(+4.5); Val(+4.2); Leu(+3.8); Phe(+2.8); Cys(+2.5); Met(+1.9); Ala(+1.8); Gly(-0.4); Thr(-0.7); Ser(-0.8); Trp(-0.9); Tyr(-1.3); Pro(-1.6); His(-3.2); Glutamic acid(-3.5); Gln(-3.5); Aspartic acid(-3.5); Asn(-3.5); Lys(-3.9); and Arg(-4.5).

[0132] It is well known in the art that substituting a particular amino acid with another amino acid having a similar hydrophobicity index or degree of hydrophobicity results in a protein having similar biological activity, that is, a protein having biologically equivalent functionality. When making such changes, substitutions between amino acids with hydrophobicity indices within the range of ±2 are preferred, substitutions between amino acids with hydrophobicity indices within the range of ±1 are particularly preferred, and substitutions between amino acids with hydrophobicity indices within the range of ±0.5 are even more particularly preferred. Furthermore, it is also well known in the art that substitutions between similar amino acids can be effectively made based on hydrophilicity.

[0133] As detailed in U.S. Patent No. 4,554,101, a hydrophilicity value is assigned to each amino acid residue, and the hydrophilicity values of each amino acid residue are: Arg(+3.0); Lys(+3.0); aspartic acid(+3.0±1); glutamic acid(+3.0±1); Ser(+0.3); Asn(+0.2); Gln(+0.2); Gly(0); Thr(-0.4); Pro(-0.5±1); Ala(-0.5); His(-0.5); Cys(-1.0); Met(-1.3); Val(-1.5); Leu(-1.8); Ile(-1.8); Tyr(-2.3); Phe(-2.5); Trp(-3.4). Specific amino acids can be substituted with other amino acids having similar hydrophilicity values, and it is well known that even when such substitutions are made, biologically equivalent proteins can be obtained, particularly immunologically equivalent proteins. When making such changes, substitutions between amino acids with hydrophilicity values within the range of ±2 are preferred, substitutions between amino acids with hydrophilicity values within the range of ±1 are particularly preferred, and substitutions between amino acids with hydrophilicity values within the range of ±0.5 are even more particularly preferred.

[0134] As outlined above, amino acid substitutions may be made based on the relative similarity of the substituents of the amino acid side chains, such as their hydrophobicity, hydrophilicity, charge, size, etc.

[0135] As otherwise noted herein, variants of gene sequences include codon-optimized variants, sequence polymorphisms, splice variants, and / or mutations that do not have a statistically significant effect on the function of the encoded product.

[0136] Variants of the proteins, nucleic acids, and gene sequences disclosed herein include sequences having at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with the proteins, nucleic acids, or gene sequences disclosed herein.

[0137] "Sequence identity (%)" refers to the relationship of two or more sequences measured by comparing the sequences. In the art, "identity" also means the degree of relatedness between protein sequences, nucleic acid sequences, or gene sequences, measured by matching between protein sequence chains, nucleic acid sequence chains, or gene sequence chains. "Identity" (often also called "similarity") can be easily calculated by known methods, such as those described in Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). As a method for measuring identity, those designed to obtain the best match between the tested sequences are preferred. Methods for measuring identity and similarity are systematized in publicly available computer programs. The calculation of sequence alignment and identity may be performed using the Megalign program (DNASTAR, Madison, Wisconsin), which is included in the LASERGENE suite of bioinformatics calculation software.Multiple alignments of sequences can also be performed using the Clustal alignment method (Higgins and Sharp CABIOS, 5, 151-153 (1989), using default parameters (gap penalty = 10, gap length penalty = 10)). Related programs further include the GCG program suite (Wisconsin Package version 9.0, Genetics Computer Group (GCG), Madison, Wis.); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990)); DNASTAR (DNASTAR, Madison, Wis.); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y.). In the present disclosure, when sequence analysis software is used for analysis, the analysis results are interpreted based on the "default values" that are the standards of the program. As used herein, "default values" mean a series of numerical values or parameters that are pre-registered in the software at the time of software initialization.

[0138] Variants also include nucleic acid molecules that hybridize to the sequences disclosed herein under stringent hybridization conditions and have the same function as the reference sequence. Exemplary stringent hybridization conditions include incubation overnight at 42°C in a solution containing 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and fragmented and denatured salmon sperm DNA at 20 μg / ml, followed by washing the filter at 50°C with 0.1×SSC. Alterations in hybridization stringency and signal detection are mainly accomplished by adjusting the concentration of formamide (lowering the percentage of formamide results in lower stringency), salt conditions, or temperature. For example, moderately high stringent conditions include incubation overnight at 37°C in a solution containing 6×SSPE (20×SSPE = 3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, and 100 μg / ml salmon sperm DNA for blocking, followed by washing at 50°C with 1×SSPE and 0.1% SDS. Furthermore, even lower stringency is achieved by performing the wash after stringent hybridization at a high salt concentration (e.g., 5×SSC). The aforementioned conditions can be variously modified by adding and / or replacing with another blocking reagent used to reduce the background of the hybridization experiment. Common blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. When adding a specific blocking reagent, it may be necessary to partially modify the aforementioned hybridization conditions due to compatibility issues.

[0139] The term "concatemerize" is used in a broad sense and means to link in a chain or to link in a continuous sequence. This term is used when referring to linking multiple nucleotide sequences to obtain a single nucleotide sequence, or linking multiple amino acid sequences to obtain a single amino acid sequence. Also, "concatemerize" is to be interpreted as referring to "concatemerize".

[0140] As will be understood by those skilled in the art, each embodiment disclosed herein includes, consists essentially of, or consists of the specific components, steps, materials or ingredients described. Accordingly, the terms "comprising" or "including" should be interpreted to have the meaning of "comprising, consisting essentially of, or consisting of". The transitional phrase "comprising" means, but is not limited to, that even if the amount is large, components, steps, materials or ingredients not described are included. The transitional phrase "consisting of" excludes any components, steps, materials or ingredients not described. The transitional phrase "consisting essentially of" limits the scope of the embodiment to the described components, steps, materials or ingredients, and components, steps, materials or ingredients that do not materially affect the embodiment. Material effect means an effect in which the target expression measured by scRNA-Seq is statistically significantly reduced when the enhancer of the present disclosure is used in the target cell population, and as the combination of the target cell population and the enhancer, oligodendrocyte / eHGT_409h, eHGT_410h, eHGT_361h, eHGT_395m, eHGT_401h, eHGT_391h, eHGT_403h, 3xCore_eHGT_641m, 3xcore2_eHGT_400m, 3xcore2_eHGT_401h, 3xcore2_eHGT_409h, 3xcore2_eHGT 410h, and eHGT_363h; Microglia / eHGT_331h, eHGT_332h, eHGT_333h, eHGT_335h, eHGT_334h, eHGT_360h, eHGT_562h, eHGT_563h, eHGT_564h, eHGT_565h, eHGT_566m, eHGT_567m, eHGT_568m, eHGT_569m, and eHGT_734m; Astrocytes / eHGT_377m, eHGT_389m, eHGT_380h, eHGT_374h, 3xcore1_eHGT_387m, eHGT_385h, eHGT_386h, eHGT_378m, eHGT_642m, 3xcore2_eHGT_390h, 3xcore2_eHGT_380h, 3xcore2_eHGT_374m, 3xcore2_eHGT_375m, and eHGT_358h; Layer 1 Interlaminar Astrocytes / eHGT_312h, eHGT_313h, eHGT_315h, and eHGT_316h; and Endothelial Cells / eHGT_603m are included.

[0141] In certain embodiments, "artificial" means not a natural product.

[0142] Unless otherwise indicated, in this specification and the claims, all numerical values representing amounts or properties of materials, such as molecular weights and reaction conditions, are to be construed as being modified in all instances by the term "about". Accordingly, unless otherwise indicated, the numerical parameters set forth in this specification and the appended claims are approximations that vary depending on the desired properties sought to be obtained by the present invention. While not limiting the scope of the application of the principle of equivalents to the claims, each numerical parameter should be construed in light of the reported number of significant digits and by applying ordinary rounding procedures. More specifically, the term "about", when used in conjunction with a recited numerical value or range, has the meaning reasonably construed by one of ordinary skill in the art, i.e., within a range of ±20% of the recited numerical value; within a range of ±19% of the recited numerical value; within a range of ±18% of the recited numerical value; within a range of ±17% of the recited numerical value; within a range of ±16% of the recited numerical value; within a range of ±15% of the recited numerical value; within a range of ±14% of the recited numerical value; within a range of ±13% of the recited numerical value; within a range of ±12% of the recited numerical value; within a range of ±11% of the recited numerical value; within a range of ±10% of the recited numerical value; within a range of ±9% of the recited numerical value; within a range of ±8% of the recited numerical value; within a range of ±7% of the recited numerical value; within a range of ±6% of the recited numerical value; within a range of ±5% of the recited numerical value; within a range of ±4% of the recited numerical value; within a range of ±3% of the recited numerical value; within a range of ±2% of the recited numerical value; or within a range of ±1% of the recited numerical value, indicating that the recited numerical value or range is somewhat more or less than the recited value or range.

[0143] Although the numerical ranges and parameters setting forth the broad scope of the present invention are approximations and approximate ranges, the numerical values set forth in the specific examples are reported as accurately as possible. However, all numerical values inherently contain certain errors necessarily resulting from the standard deviation associated with each test measurement.

[0144] As used in the description of the present invention (especially in the description of the following claims), the articles "a", "an", "the" and similar designations shall be construed to include both singular and plural referents unless otherwise specified or the context clearly dictates otherwise. Numerical ranges set forth herein are intended to be presented in a shorthand manner that refers individually to each numerical value within the range. Unless otherwise specified, each numerical value is to be read as if it were individually recited herein. Unless otherwise specified or the context clearly dictates otherwise, any method described herein can be performed in any suitable order. Any use of examples provided herein, or language used to provide examples (such as "etc.") is for the sole purpose of better explaining the invention and is not intended to limit the scope of the invention as claimed. Terms described herein should not be construed as indicating any essential elements of the invention not recited in the claims.

[0145] Groupings of other components of the invention disclosed herein, or groupings of various embodiments of the invention, should not be construed as limiting the invention. Members of each group may be individually recited in this specification or in the claims, or may be recited in this specification or in the claims in combination with other members of the group described herein or with other components. For convenience and / or reasons of patentability, it is contemplated that one or more members of a group may be added to another group, or one or more members may be deleted from a group. When such additions or deletions are made, this specification includes groups that are constructed to satisfy the description of all Markush groups recited in the appended claims.

[0146] Certain embodiments of the present invention are described herein, including those that the inventors recognize as the best mode for carrying out the present invention. It will be readily understood by those skilled in the art that, upon perusal of the foregoing detailed description, various changes can be made to the embodiments described herein. The inventors anticipate that those skilled in the art can appropriately adopt such variations, and it is also intended that the present invention be implemented in modes other than those specifically described herein. Accordingly, the present invention includes, to the extent possible within the scope of the applicable law, any changes from the subject matter of the present invention recited in the appended claims and any equivalents of the subject matter of the present invention. Further, unless otherwise stated or the context clearly dictates a different meaning, any combination of the foregoing components in any variation is also included in the present invention.

[0147] Furthermore, throughout this specification, various patents, publications, journal articles, and other documents (references cited herein) are cited. Each document cited herein is hereby incorporated by reference in its entirety as if set forth herein in full, with the teachings thereof being individually incorporated herein by reference.

[0148] Finally, the embodiments of the present invention disclosed herein are to be construed as illustrative of the principles of the present invention. Other changes may be adopted within the scope of the present invention. Thus, by way of example, but not limitation, another configuration of the present invention may be used in accordance with the teachings herein. Accordingly, the present invention is not strictly limited to what is shown and described herein.

[0149] The details described in this specification are for example only and are intended only to illustrate the preferred embodiments of the present invention, to provide what is considered to be the most useful, and to make it possible to easily understand the principles and conceptual aspects of the various embodiments of the present invention. In this regard, the details of the structure of the present invention are not described in more detail than the information necessary for a basic understanding of the present invention, and those skilled in the art will be able to easily understand how to actually embody some forms of the present invention by carefully reading the description of the present invention with reference to the drawings and / or examples.

[0150] The definitions and explanations used in this disclosure are intended to control future interpretations, unless clear and explicit changes are made in the following examples, or unless the meaning of the terms becomes meaningless or substantially meaningless due to the meaning of the terms. If the definition of a term does not make sense or is substantially meaningless from the interpretation of the term, it is desired to cite the definition of the term from a dictionary known to those skilled in the art, such as Webster's Dictionary (3rd Edition) or Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

Claims

1. The sequence shown in SEQ ID NO: 47, SEQ ID NO: 8, SEQ ID NO: 159, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 39, SEQ ID NO: 51, or SEQ ID NO: 53 may contain 1 copy, 2 copies, 3 copies, 4 copies, 5 copies, 6 copies, 7 copies, 8 copies, 9 copies, or 10 copies, or An artificial enhancer containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of a sequence that has at least 90% sequence identity with the sequence shown in SEQ ID NO: 47, SEQ ID NO: 8, SEQ ID NO: 159, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 39, SEQ ID NO: 51, or SEQ ID NO: 53, and retains enhancer activity.

2. It is an artificial expression construct, (i) Enhancers selected from the sequences shown in SEQ ID NOs. 47, 48, 1, 2, 3, 4, 5, 6, 7, 9, 11, 13, 15, 17, 158, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 52, 54, 160, and 157; (ii) promoter; and (iii) Code array Artificial expression constructs, including those mentioned above.

3. (i) The coding sequence codes for a fluorescent protein, a functional molecule, or a non-functional molecule; (ii) accompanied by a capsid that crosses the blood-brain barrier; or (iii) containing or encoding a skipping factor The artificial expression construct according to claim 2.

4. (i) The functional molecule is a functional ion transporter, a functional enzyme, a functional transcription factor, a functional receptor, a functional membrane protein, a functional cell transport protein, a functional signaling molecule, a functional neurotransmitter, a functional calcium reporter, a functional channelrhodopsin, a functional CRISPR / Cas molecule, a functional editase, a functional guide RNA molecule, a functional microRNA, a functional homologous recombinant donor cassette, or a functional designer receptor (DREADD) that is activated solely by a designer drug; (ii) The non-functional molecule is a non-functional ion transporter, non-functional enzyme, non-functional transcription factor, non-functional receptor, non-functional membrane protein, non-functional cell transport protein, non-functional signaling molecule, non-functional neurotransmitter, non-functional calcium reporter, non-functional channelrhodopsin, non-functional CRISPR / Cas molecule, non-functional editase, non-functional guide RNA molecule, non-functional microRNA, non-functional homologous recombination donor cassette, or a non-functional designer receptor (DREADD) that is activated only by a designer drug; (iii) The capsid is PHP.eB, AAV9, AAVrh.10, AAV-BR1, AAV-PHP.S, AAV-PHP.B, or AAV-PPS; or (iv) The skipping factor is a T2A peptide, a P2A peptide, an E2A peptide, an F2A peptide, or an internal ribosome entry site (IRES), The artificial expression construct according to claim 3.

5. eHGT_409h(SEQ ID NO: 1)-minBglobin-SYFP2-WPRE3-BGHpA; eHGT_410h (SEQ ID NO: 2) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_361h (Sequence ID 3) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_395m(SEQ ID NO: 4)-minBglobin-SYFP2-WPRE3-BGHpA; eHGT_401h (Sequence ID 5) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_391h (SEQ ID NO: 6) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_403h (Sequence ID 7) - minBglobin-SYFP2-WPRE3-BGHpA; 3xCore_eHGT_641m (Sequence ID 9) -minBglobin-SYFP2-WPRE3-BGHpA; 3xcore2_eHGT_400m(SEQ ID NO: 11)-minBglobin-SYFP2-WPRE3-BGHpA; 3xcore2_eHGT_401h (SEQ ID NO: 13) -minBglobin-SYFP2-WPRE3-BGHpA; 3xcore2_eHGT_409h (SEQ ID NO: 15) -minBglobin-SYFP2-WPRE3-BGHpA; 3xcore2_eHGT_410h (SEQ ID NO: 17) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_331h (SEQ ID NO: 18) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_332h (SEQ ID NO: 19) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_333h (SEQ ID NO: 20) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_335h (SEQ ID NO: 21) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_334h (SEQ ID NO: 22) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_360h (SEQ ID NO: 23) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_562h (SEQ ID NO: 24) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_563h (SEQ ID NO: 25) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_564h (SEQ ID NO: 26) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_565h (Sequence ID 27) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_566m (SEQ ID NO: 28) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_567m (Sequence ID 29) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_568m (Sequence ID 30) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_569m (SEQ ID NO: 31) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_734m (Sequence ID 32) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_312h (Sequence ID 33) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_313h (SEQ ID NO: 34) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_377m (Sequence ID 35) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_389m (Sequence ID 36) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_380h (SEQ ID NO: 37) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_374h (SEQ ID NO: 38) -minBglobin-SYFP2-WPRE3-BGHpA; 3xcore1_eHGT_387m (SEQ ID NO: 40) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_385h (SEQ ID NO: 41) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_386h (SEQ ID NO: 42) - minBglobin-SYFP2-WPRE3-BGHpA; eHGT_378m (SEQ ID NO: 43) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_642m (Sequence ID 44) -minBglobin-SYFP2-WPRE3-BGHpA; 3xcore2_eHGT_390h (SEQ ID NO: 48) -minBglobin-SYFP2-WPRE3-BGHpA; 3xcore2_eHGT_380h (SEQ ID NO: 52) -minBglobin-SYFP2-WPRE3-BGHpA; 3xcore2_eHGT_374m (SEQ ID NO: 54) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_603m (SEQ ID NO: 55) - minBglobin-SYFP2-WPRE3-BGHpA; eHGT_315h (SEQ ID NO: 155) -minBglobin-SYFP2-WPRE3-BGHpA; eHGT_316h (SEQ ID NO: 156) -minBglobin-SYFP2-WPRE3-BGHpA; 3xcore2_eHGT_375m(SEQ ID NO: 160)-minBglobin-SYFP2-WPRE3-BGHpA; TeIN-LITR-eHGT_358h (SEQ ID NO: 157)-minBG-SYFP2-P2A-3XFLAG-10aa-H2B * -WPRE3-BGHpA-RITR-TeIN; and TeIN-LITR-eHGT_363h (Sequence ID 158)-minBG-SYFP2-P2A-3XFLAG-10aa-H2B * -WPRE3-BGHpA-RITR-TeIN The artificial expression construct according to claim 2, comprising or encoding a set of features selected from these.

6. A viral vector comprising the artificial expression construct described in claim 2.

7. A transgenic cell comprising the artificial expression construct described in claim 2.

8. (i) a non-neuronal cell, glial cell, oligodendrocyte, microglia, or interlaminar astrocyte of the first layer; or (ii) Mouse cells, human cells or non-human primate cells, The transgenic cell according to claim 7.

9. A non-human transgenic animal comprising the artificial expression construct according to claim 2, or the transgenic cells according to claim 7.

10. A non-human transgenic animal according to claim 9, which is a mouse or a non-human primate.

11. A method for expressing a coding sequence in a population of non-neuronal cells in vitro, comprising the step of providing a sample containing a population of non-neuronal cells with an artificial expression construct in a sufficient dose and for a sufficient period of time to express the coding sequence in the population of non-neuronal cells. The aforementioned artificial expression construct, (i) Enhancers selected from the sequences shown in SEQ ID NOs. 47, 48, 1, 2, 3, 4, 5, 6, 7, 9, 11, 13, 15, 17, 158, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 52, 54, 160, and 157; (ii) promoter; and (iii) Code array A method characterized by including

12. A method for expressing a coding sequence in vivo within a population of non-neuronal cells, comprising the step of providing a subject including a population of non-neuronal cells with an artificial expression construct in a sufficient dose and for a sufficient period of time, thereby expressing the coding sequence within the population of non-neuronal cells. The aforementioned artificial expression construct, (i) Enhancers selected from the sequences shown in SEQ ID NOs. 47, 48, 1, 2, 3, 4, 5, 6, 7, 9, 11, 13, 15, 17, 158, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 52, 54, 160, and 157; (ii) promoter; and (iii) Code array It is characterized by including, A method wherein the subject is a non-human subject.

13. The method according to claim 11 or 12, wherein the coding sequence codes for a fluorescent protein, a functional molecule, or a non-functional molecule.

14. (i) The functional molecule is a functional ion transporter, a functional enzyme, a functional transcription factor, a functional receptor, a functional membrane protein, a functional cell transport protein, a functional signaling molecule, a functional neurotransmitter, a functional calcium reporter, a functional channelrhodopsin, a functional CRISPR / Cas molecule, a functional editase, a functional guide RNA molecule, a functional microRNA, a functional homologous recombinant donor cassette, or a functional designer receptor (DREADD) that is activated only by a designer drug; or (ii) The non-functional molecule is a non-functional ion transporter, non-functional enzyme, non-functional transcription factor, non-functional receptor, non-functional membrane protein, non-functional cell transport protein, non-functional signaling molecule, non-functional neurotransmitter, non-functional calcium reporter, non-functional channelrhodopsin, non-functional CRISPR / Cas molecule, non-functional editase, non-functional guide RNA molecule, non-functional microRNA, non-functional homologous recombination donor cassette, or a non-functional designer receptor (DREADD) that is activated only by a designer drug. The method according to claim 13.

15. The method according to claim 11, wherein the provision includes pipetting performed on a brain section.

16. The method according to claim 12, wherein the provision includes administration to a living subject.

17. The method according to claim 16, wherein the living subject is a non-human primate or a mouse.

18. The administration to the aforementioned living subject is carried out by injection. The method according to claim 16, wherein the injection is an intravenous injection, an intraparenchymal injection into brain tissue, an intraventricular (ICV) injection, an intracisional (ICM) injection, or an intrathecal injection.