Artificial expression constructs that selectively regulate gene expression in non-neuronal brain cells

Abstract

To provide artificial expression constructs that selectively drive protein expression.SOLUTION: An artificial expression construct comprising a specific enhancer, a promoter, and a heterologous coding sequence, wherein in one aspect the heterologous coding sequence encodes an effector element or an expressible element, wherein the effector element comprises a reporter protein or a functional molecule, wherein the reporter protein comprises a fluorescent protein, An artificial expression construct is provided wherein the functional molecule comprises a functional ion transporter, an enzyme, a transcription factor, a receptor, a membrane protein, a cellular transport protein, a signaling molecule, a neurotransmitter, a calcium reporter, a channelrhodopsin, a CRISPR / CAS molecule, an editase, a guide RNA molecule, a microRNA, a homologous recombination donor cassette, or a designer receptor exclusively activated with a designer drug.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 001,159, filed March 27, 2020, the entire contents of which are incorporated herein by reference as if fully set forth herein.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with United States government support under grant numbers MH114126 and DA036909 from the National Institutes of Health. The United States government has certain rights in this invention.

[0003] Field of Disclosure The present disclosure provides artificial expression constructs that selectively regulate gene expression in select central nervous system cell types, which can be used to selectively express synthetic genes or modify gene expression in non-neuronal brain cells such as astrocytes, oligodendrocytes, microglia, pericytes, SMCs, and endothelial cells. [Background technology]

[0004] Background to the disclosure To fully understand brain biology, it is necessary to distinguish and define distinct cell types, and then to identify artificial expression constructs that can selectively label and perturb them for further study. In mice, recombinase driver strains have been used to great effect to label cell populations that share marker gene expression. However, the creation, maintenance, and use of such strains to label cell types with high specificity is expensive and often requires triple transgenic crosses, which results in low frequency of experimental animals. Furthermore, these tools require germline transgenic animals and are therefore not applicable to humans. Summary of the Invention

[0005] Disclosure Overview The present disclosure provides artificial expression constructs that selectively drive gene expression in target central nervous system cell populations, including non-neuronal brain cells such as astrocytes, oligodendrocytes, microglia, pericytes, SMCs, and endothelial cells.

[0006] Certain embodiments of the artificial expression construct utilize the following enhancers to selectively drive protein expression in targeted central nervous system cell populations as follows:

[0007] Astrocytes: eHGT_373m, 3xcore eHGT_373m, eHGT_375m, eHGT_379m, eHGT_372m, eHGT_384m, eHGT_386m, eHGT_390m, 3xcore eHGT_390m, eHGT_371m, eHGT_383m, eHGT_374m, eHGT_381m, eHGT_382m, eHGT_387m, eHGT_388m, eHGT_ 376m, eHGT_380m, eHGT_385m, eHGT_371h, eHGT_372h, eHGT_375h, eHGT_376h, eHGT_377h, eHGT_381h, e HGT_382h, eHGT_383h, eHGT_384h, eHGT_387h, eHGT_388h, eHGT_389h, eHGT_390h, eHGT_357h, eHGT_495m, eHGT_497m, mscRE1001, mscRE1002, mscRE1003, mscRE1004, mscRE1005, mscRE1006, and mscRE1007;

[0008] L1 interlaminar astrocytes: eHGT_267h, eHGT_268h, eHGT_269h, eHGT_270h, eHGT_271h, eHGT_272h, eHGT_273h, eHGT_274h, eHGT_275h, eHGT_276h, eHGT_315h, and eHGT_316h;

[0009] Oligodendrocytes: eHGT_391m, eHGT_398m, eHGT_402m, eHGT_409m, eHGT_396m, eHGT_393m, eHGT_399m, eHGT_400m, eHGT_405m, eHGT_406m, eHGT_410m, 3xcore eHGT_410m, eHGT_397m, eHGT_401m, eHGT_403m, eHGT_407m, eHGT_408m, eHGT_392h, eHGT_393h, eHGT_394h, eHGT_395h, eHGT_396h, eHGT_397h, eHGT_398h, eHGT_399h, eHGT_400h, eHGT_402h, eHGT_404h, eHGT_405h, eHGT_406h, eHGT_407h, eHGT_641m, and eHGT_408h;

[0010] Microglia: eHGT413m, eHGT_414m, eHGT_415m, eHGT_416m, eHGT_417m, eHGT_418m, eHGT_419m, eHGT_420m, eHGT_4 21m, eHGT_423m, eHGT_428m, eHGT_429m, eHGT_430m, eHGT_411m, eHGT_412m, eHGT_422m, eHGT_424m, eHGT_425 m, eHGT_426m, eHGT_427m, eHGT_411h, eHGT_412h, eHGT_413h, eHGT_414h, eHGT_417h, eHGT_418h, eHGT_419h, eHGT_420h, eHGT_423h, eHGT_424h, eHGT_425h, eHGT_426h, eHGT_427h, eHGT_428h, eHGT_429h, and eHGT_430h;

[0011] Pericytes: mscRE1023, mscRE1024, mscRE1025, mscRE1026, mscRE1027, mscRE1028, mscRE1029, mscRE1030, mscRE1031, mscRE1032, mscRE1033, mscRE1034, mscRE1035, mscRE1036, and mscRE1037;

[0012] SMC: mscRE1038, mscRE1039, mscRE1040, mscRE1041, mscRE1042, mscRE1043, mscRE1044, mscRE1045, mscRE1046, mscRE1047, mscRE1048, mscRE1049, mscRE1050, mscRE1051, and mscRE1052;

[0013] Endothelial cells: mscRE1008, mscRE1009, mscRE1010, mscRE1011, mscRE1012, mscRE1013, mscRE1014, mscRE1015, mscRE1016, mscRE1017, mscRE1018, mscRE1019, mscRE1020, mscRE1021, and mscRE1022.

[0014] Certain embodiments are directed to the vectors: CN1781, CN1782, CN1783, CN1784, CN1785, CN1786, CN1787, CN1788, CN1789, CN1790, CN2044, CN2082, CN2083, CN2084, CN2560, CN2085, CN2086, CN2087, CN2088, CN2089, CN2558, CN2090, CN2091, CN2092, CN2093, CN2094, CN2095, CN2096, CN2097, CN2098, CN2099, CN2100, CN2101, CN2102, CN2103, CN2104, CN2105, CN2106, CN2107, CN2108, CN2109, CN2110, CN2111, CN2112, CN2113, CN2114, CN2115, CN2116, CN2117, CN2118, CN2119, CN2120, CN2121, CN2122, CN2123, CN2124, CN2125, CN2126, CN2127, CN2128, CN2129, CN2130, CN2131, CN2132, CN2133, CN2134, CN2135, CN2136, CN2137, CN2138, CN2139, CN214 2, CN2103, CN2104, CN2105, CN2106, CN2107, CN2108, CN2109, CN2556, CN2110, CN2111, CN2112, CN2113, CN2114, CN2115, CN2116, CN2117, CN2118, CN21 19, CN2120, CN2121, CN2122, CN2123, CN2124, CN2125, CN2126, CN2127, CN2128, CN2129, CN2130, CN2131, CN2132, CN2133, CN2134, CN2141, CN2142, CN2 143, CN2144, CN2145, CN2146, CN2147, CN2148, CN2149, CN2150, CN2151, CN2152, CN2153, CN2154, CN2155, CN2156, CN2157, CN2158, CN2159, CN2160, CN 2161, CN2162, CN2163, CN2164, CN2165, CN2166, CN2167, CN2845, CN2168, CN2169, CN2170, CN2171, CN2172, CN2173, CN2174, CN2175, CN2176, CN2177, C N2178, CN2179, CN2180, CN2181, CN2182, CN2183, CN2184, CN2243, CN2268, CN2345, CN2346, 3001, 3002, 3003, 3004, 3005, 3006, 3007, 3008, 3009, 3010 , 3011, 3012, 3013, 3014, 3015, 3016, 3017, 3018, 3019, 3020, 3021, 3022, 30 23, 3024, 3025, 3026, 3027, 3028, 3029, 3030, 3031, 3032, 3033, 3034, 3035,Artificial expression constructs are provided that include features of the vectors described herein, such as 3036, 3037, 3038, 3039, 3040, 3041, 3042, 3043, 3044, 3045, 3046, 3047, 3048, 3049, 3050, 3051, and 3052.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS Many of the drawings submitted herein are best understood in color, and applicants consider the colored drawings to be a part of the application documents as originally filed and reserve the right to submit images of the colored drawings by following the procedure described below. [Brief explanation of the drawings]

[0016] [Figure 1] This is an outline of enhancer discovery for viral tools. To construct cell-type-specific labeling tools, we isolated cells from adult mouse cortex and performed single-cell assays for transposase-accessible chromatin using sequencing (scATAC-seq). Samples were clustered and compared with single-cell RNA sequencing (scRNA-seq) datasets to identify clusters. Single cells matching the same transcriptome type were then pooled, and the genomes were searched for type-specific putative enhancers. These regions were cloned upstream of a minimal promoter in the AAV genome backbone used to generate self-complementary adeno-associated viral vectors (scAAV) or recombinant adeno-associated viral vectors (rAAV). These viral tools were delivered to the retroorbital cavity to label specific cortical populations. In cells with matching cell types, enhancers recruit their cognate transcription factors to drive cell-type-specific expression. In other cells, the viral genome is present but no transcripts are expressed. However, not all enhancers behave as expected, so enhancer constructs need to be tested for specificity. [Figure 2-1] Brain regions and cell subclasses selectively labeled with different artificial expression constructs of the present disclosure. [Figure 2-2]Brain regions and cell subclasses selectively labeled with different artificial expression constructs of the present disclosure. [Figure 2-3] Brain regions and cell subclasses selectively labeled with different artificial expression constructs of the present disclosure. [Figure 2-4] Brain regions and cell subclasses selectively labeled with different artificial expression constructs of the present disclosure. [Figure 3A] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (3A) and visual cortex (3B) show selective expression of SYFP in cells with astrocytic morphology after retroorbital injection of CN2089 virus encapsidated with PHP.eB. The data demonstrate that the eHGT_390m enhancer selectively drives reporter expression in mouse astrocytes. [Figure 3B] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (3A) and visual cortex (3B) show selective expression of SYFP in cells with astrocytic morphology after retroorbital injection of CN2089 virus encapsidated with PHP.eB. The data demonstrate that the eHGT_390m enhancer selectively drives reporter expression in mouse astrocytes. [Figure 3C-1] Mapping of single-cell transcriptomic profiles of SYPF2+ cells sorted from the VISp region of mouse cortex after retroorbital injection of CN2089 virus encapsidated with PHP.eB. The number of cells mapped to the terminal lobe is shown in the bar graph below the dendrogram. Transcriptomic cell types are indicated at the bottom. The data demonstrate that eHGT_390m enhancer-driven reporter expression occurs selectively in mouse astrocytes (>95% of labeled cells) when VISp is assessed. [Figure 3C-2]Mapping of single-cell transcriptomic profiles of SYPF2+ cells sorted from the VISp region of mouse cortex after retroorbital injection of CN2089 virus encapsidated with PHP.eB. The number of cells mapped to the terminal lobe is shown in the bar graph below the dendrogram. Transcriptomic cell types are indicated at the bottom. The data demonstrate that eHGT_390m enhancer-driven reporter expression occurs selectively in mouse astrocytes (>95% of labeled cells) when VISp is assessed. [Figure 3D] This is a reversible plot of single-cell transcriptomic profiles of SYPF2+ cells sorted from the VISp region of mouse cortex after retroorbital injection of CN2089 virus encapsidated with PHP.eB. The data show class, subclass, and type mapping among cells. The data demonstrate that eHGT_390m enhancer-driven reporter expression occurs selectively in mouse astrocytes (>95% of labeled cells) when VISp is assessed. [Figure 3E] Native SYFP2 fluorescence after intraparenchymal injection of CN2089 encapsulated in PHP.eB into macaque non-human primate cortex is shown. Native SYFP2 alone (3E) and co-stained with DAPI and propidium iodide (3F) are shown. Note that most cells display prominent astrocytic morphology. This data demonstrates that enhancer eHGT_390m maintains astrocyte selectivity in the macaque neocortex. [Figure 3F] Native SYFP2 fluorescence after intraparenchymal injection of CN2089 encapsulated in PHP.eB into macaque non-human primate cortex is shown. Native SYFP2 alone (3E) and co-stained with DAPI and propidium iodide (3F) are shown. Note that most cells display prominent astrocytic morphology. This data demonstrates that enhancer eHGT_390m maintains astrocyte selectivity in the macaque neocortex. [Figure 3G]Native SYFP2 fluorescence after intraparenchymal injection of CN2089 encapsulated in PHP.eB into macaque non-human primate cortex is shown. Examples of native SYFP2 alone in an intact cortical column (3G) and labeled plasmalemmal (3H) and fibrous (3I) astrocytes are shown. The data clearly demonstrate that eHGT_390m enhancer-driven reporter expression in neocortical astrocytes displays diverse cellular morphologies. [Figure 3H] Native SYFP2 fluorescence after intraparenchymal injection of CN2089 encapsulated in PHP.eB into macaque non-human primate cortex is shown. Examples of native SYFP2 alone in an intact cortical column (3G) and labeled plasmalemmal (3H) and fibrous (3I) astrocytes are shown. The data clearly demonstrate that eHGT_390m enhancer-driven reporter expression in neocortical astrocytes displays diverse cellular morphologies. [Figure 3I] Native SYFP2 fluorescence after intraparenchymal injection of CN2089 encapsulated in PHP.eB into macaque non-human primate cortex is shown. Examples of native SYFP2 alone in an intact cortical column (3G) and labeled plasmalemmal (3H) and fibrous (3I) astrocytes are shown. The data clearly demonstrate that eHGT_390m enhancer-driven reporter expression in neocortical astrocytes displays diverse cellular morphologies. [Figure 3J]Molecular confirmation that SYFP2+ cells express astrocyte-selective but not neuron-selective genes. Native SYFP2 fluorescence is shown after intraparenchymal injection of CN2089 encapsulated in PHP.eB into macaque non-human primate cortex. Native SYFP2 (green) with mFISH labeling of glutamatergic (SLC17A7 - magenta), GABAergic (GAD1 - cyan), and astrocytic (FGFR3 - yellow) marker genes (3J), or with mFISH marker genes alone (3K). White arrows indicate SYFP2+ cells. Note that SYFP2+ cells almost always overlap with FGFR3 but not with GAD1 or SLC17A7. These data demonstrate that eHGT_390m enhancer-driven reporter expression occurs selectively in macaque astrocytes expressing the marker gene FGFR3. [Figure 3K] Molecular confirmation that SYFP2+ cells express astrocyte-selective but not neuron-selective genes. Native SYFP2 fluorescence is shown after intraparenchymal injection of CN2089 encapsulated in PHP.eB into the cortex of macaque non-human primates. Native SYFP2 (green) with mFISH labeling of glutamatergic (SLC17A7 - magenta), GABAergic (GAD1 - cyan), and astrocytic (FGFR3 - yellow) marker genes (3J), or with mFISH marker genes alone (3K). White arrows indicate SYFP2+ cells. Note that SYFP2+ cells almost always overlap with FGFR3 but not with GAD1 or SLC17A7. These data demonstrate that eHGT_390m enhancer-driven reporter expression occurs selectively in macaque astrocytes expressing the marker gene FGFR3. [Figure 3L] SYFP2 fluorescence in P21 mice after ICV injection of PHP.eB-encapsulated CN2089 into P2 mice is shown. Sagittal sections of the whole brain (L) and visual cortex (M) show strong expression in cells with astrocytic morphology within the brain, indicating that neonatal ICV injection of CN2089 results in particularly strong expression in forebrain structures. [Figure 3M]SYFP2 fluorescence in P21 mice after ICV injection of PHP.eB-encapsulated CN2089 into P2 mice is shown. Sagittal sections of the whole brain (L) and visual cortex (M) show strong expression in cells with astrocytic morphology within the brain, indicating that neonatal ICV injection of CN2089 results in particularly strong expression in forebrain structures. [Figure 4A] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (4A) and visual cortex (4B) showing selective expression of SYFP2 in cells with oligodendrocyte morphology after retroorbital injection of CN2109 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_410m enhancer selectively drives reporter expression in mouse oligodendrocytes. [Figure 4B] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (4A) and visual cortex (4B) showing selective expression of SYFP2 in cells with oligodendrocyte morphology after retroorbital injection of CN2109 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_410m enhancer selectively drives reporter expression in mouse oligodendrocytes. [Figure 4C] Native SYFP2 fluorescence in P21 mice after ICV injection of PHP.eB-encapsidated CN2109 virus into P2 mice is shown. Sagittal sections of the whole brain (4C) and cortex (4D) show strong expression in cells with oligodendrocyte morphology in the brain, with particularly prominent expression in the spinal cord. These data demonstrate that the eHGT_410m enhancer selectively drives reporter expression in mouse oligodendrocytes after neonatal delivery of CN2109 ICV. [Figure 4D]Native SYFP2 fluorescence in P21 mice after ICV injection of PHP.eB-encapsidated CN2109 virus into P2 mice is shown. Sagittal sections of the whole brain (4C) and cortex (4D) show strong expression in cells with oligodendrocyte morphology in the brain, with particularly prominent expression in the spinal cord. These data demonstrate that the eHGT_410m enhancer selectively drives reporter expression in mouse oligodendrocytes after neonatal delivery of CN2109 ICV. [Figure 4E] Native SYFP2 fluorescence is shown after intraparenchymal injection of CN2109 encapsulated in PHP.eB into macaque non-human primate cortex. Native SYFP2 alone (E) and co-staining with DAPI and propidium iodide (4F) are shown. A higher resolution image of (4E) (4G) shows SYFP2 and Nissl. Note that most cells exhibit prominent oligodendrocyte morphology. These data demonstrate that the eHGT_410m enhancer selectively drives reporter expression in macaque cells exhibiting oligodendrocyte morphology. [Figure 4F] Native SYFP2 fluorescence is shown after intraparenchymal injection of CN2109 encapsulated in PHP.eB into macaque non-human primate cortex. Native SYFP2 alone (E) and co-staining with DAPI and propidium iodide (4F) are shown. A higher resolution image of (4E) (4G) shows SYFP2 and Nissl. Note that most cells exhibit prominent oligodendrocyte morphology. These data demonstrate that the eHGT_410m enhancer selectively drives reporter expression in macaque cells exhibiting oligodendrocyte morphology. [Figure 4G]Native SYFP2 fluorescence is shown after intraparenchymal injection of CN2109 encapsulated in PHP.eB into macaque non-human primate cortex. Native SYFP2 alone (E) and co-staining with DAPI and propidium iodide (4F) are shown. A higher resolution image of (4E) (4G) shows SYFP2 and Nissl. Note that most cells exhibit prominent oligodendrocyte morphology. These data demonstrate that the eHGT_410m enhancer selectively drives reporter expression in macaque cells exhibiting oligodendrocyte morphology. [Figure 4H] Molecular confirmation that SYFP2+ cells express oligodendrocyte-selective genes, but not neuron-selective genes. Native SYFP2 fluorescence is shown after intraparenchymal injection of CN2109 encapsulated in PHP.eB into the cortex of macaque non-human primates. Native SYFP2 (green) with mFISH labeling of glutamatergic (SLC17A7 - magenta), GABAergic (GAD1 - cyan), and oligodendrocyte (SOX10 - yellow) marker genes (4H) or with mFISH marker genes alone (4K). White arrows indicate SYFP2+ cells. Note that SYFP2+ cells almost always overlap with SOX10 but not with GAD1 or SLC17A7. This indicates that the eHGT_410m enhancer selectively drives reporter expression in macaque neocortical cells, representing the oligodendrocyte-selective marker gene SOX10. [Figure 4I]Molecular confirmation that SYFP2+ cells express oligodendrocyte-selective genes, but not neuron-selective genes. Native SYFP2 fluorescence is shown after intraparenchymal injection of CN2109 encapsulated in PHP.eB into the cortex of macaque non-human primates. Native SYFP2 (green) with mFISH labeling of glutamatergic (SLC17A7 - magenta), GABAergic (GAD1 - cyan), and oligodendrocyte (SOX10 - yellow) marker genes (4H) or with mFISH marker genes alone (4K). White arrows indicate SYFP2+ cells. Note that SYFP2+ cells almost always overlap with SOX10 but not with GAD1 or SLC17A7. This indicates that the eHGT_410m enhancer selectively drives reporter expression in macaque neocortical cells, representing the oligodendrocyte-selective marker gene SOX10. [Figure 4J] The oligodendrocyte-targeted enhancer AAV vector CN2109 (enhancer: eHGT_410m) drives SYFP2 reporter expression in white matter-enriched human neocortical oligodendrocytes. (4J) Fluorescent reporter expression in human rapid postmortem neocortical slice cultures at day 5 in vitro and 5 days after infection with AAV vector CN2109 serotype PHPeB. The postmortem interval between tissue culture and postmortem was 10 hours. Scale bar: 1 mm. (4K) Higher magnification of the boxed area (4J) shows native SYFP2 signal in cells with oligodendrocyte morphology in the white matter (below the dotted line in (4J)). Scale bar: 200 microns. Demonstration of oligodendrocyte labeling in human postmortem slice cultures is strong evidence for targeting non-neuronal cell types in the human / primate brain. [Figure 4K]The oligodendrocyte-targeted enhancer AAV vector CN2109 (enhancer: eHGT_410m) drives SYFP2 reporter expression in white matter-enriched human neocortical oligodendrocytes. (4J) Fluorescent reporter expression in human rapid postmortem neocortical slice cultures at day 5 in vitro and 5 days after infection with AAV vector CN2109 serotype PHPeB. The postmortem interval between tissue culture and postmortem was 10 hours. Scale bar: 1 mm. (4K) Higher magnification of the boxed area (4J) shows native SYFP2 signal in cells with oligodendrocyte morphology in the white matter (below the dotted line in (4J)). Scale bar: 200 microns. Demonstration of oligodendrocyte labeling in human postmortem slice cultures is strong evidence for targeting non-neuronal cell types in the human / primate brain. [Figure 5A] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (5A) and visual cortex (5B) showing selective expression of SYFP2 in cells with astrocytic morphology after retroorbital injection of CN2153 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_390h enhancer selectively drives reporter expression in mouse astrocytes. [Figure 5B] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (5A) and visual cortex (5B) showing selective expression of SYFP2 in cells with astrocytic morphology after retroorbital injection of CN2153 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_390h enhancer selectively drives reporter expression in mouse astrocytes. [Figure 6A] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (A) and visual cortex (B) showing dim but selective expression of SYFP2 in cells with astrocytic morphology after retroorbital injection of CN2145 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_377h enhancer selectively drives reporter expression in mouse cells with astrocytic morphology. [Figure 6B] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (A) and visual cortex (B) showing dim but selective expression of SYFP2 in cells with astrocytic morphology after retroorbital injection of CN2145 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_377h enhancer selectively drives reporter expression in mouse cells with astrocytic morphology. [Figure 7A] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (7A) and visual cortex (7B) showing selective expression of SYFP2 in cells with astrocytic morphology after retroorbital injection of CN2144 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_376h enhancer selectively drives reporter expression in mouse cells with astrocytic morphology. [Figure 7B] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (7A) and visual cortex (7B) showing selective expression of SYFP2 in cells with astrocytic morphology after retroorbital injection of CN2144 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_376h enhancer selectively drives reporter expression in mouse cells with astrocytic morphology. [Figure 8A] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (8A) and visual cortex (8B) showing dim but selective expression of SYFP2 in cells with astrocytic morphology after retroorbital injection of CN2147 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_382h enhancer selectively drives reporter expression in mouse cells with astrocytic morphology. [Figure 8B]Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (8A) and visual cortex (8B) showing dim but selective expression of SYFP2 in cells with astrocytic morphology after retroorbital injection of CN2147 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_382h enhancer selectively drives reporter expression in mouse cells with astrocytic morphology. [Figure 9A] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (9A) and visual cortex (9B) showing selective expression of SYFP2 in cells with astrocytic morphology after retroorbital injection of CN2084 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_373m enhancer selectively drives reporter expression in mouse cells with astrocytic morphology. [Figure 9B] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (9A) and visual cortex (9B) showing selective expression of SYFP2 in cells with astrocytic morphology after retroorbital injection of CN2084 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_373m enhancer selectively drives reporter expression in mouse cells with astrocytic morphology. [Figure 10A] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (10A) and visual cortex (10B) showing selective expression of SYFP2 in cells with astrocytic morphology after retroorbital injection of CN2088 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_386m enhancer selectively drives reporter expression in mouse cells with astrocytic morphology. [Figure 10B]Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (10A) and visual cortex (10B) showing selective expression of SYFP2 in cells with astrocytic morphology after retroorbital injection of CN2088 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_386m enhancer selectively drives reporter expression in mouse cells with astrocytic morphology. [Figure 11A] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (11A) and visual cortex (11B) showing dim but selective expression of SYFP2 in cells with astrocytic morphology after retroorbital injection of CN2097 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_383m enhancer selectively drives reporter expression in mouse cells with astrocytic morphology. [Figure 11B] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (11A) and visual cortex (11B) showing dim but selective expression of SYFP2 in cells with astrocytic morphology after retroorbital injection of CN2097 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_383m enhancer selectively drives reporter expression in mouse cells with astrocytic morphology. [Figure 12A] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (12A), visual cortex (12B), and cerebellum (12C) show selective expression of SYFP2 in cells with astrocytic and Bergmann glial morphologies in the cerebellum after retroorbital injection of CN2102 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_387m enhancer selectively drives reporter expression in mouse cells with astrocytic and Bergmann glial morphologies in the cerebellum. [Figure 12B]Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (12A), visual cortex (12B), and cerebellum (12C) show selective expression of SYFP2 in cells with astrocytic and Bergmann glial morphologies in the cerebellum after retroorbital injection of CN2102 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_387m enhancer selectively drives reporter expression in mouse cells with astrocytic and Bergmann glial morphologies in the cerebellum. [Figure 12C] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (12A), visual cortex (12B), and cerebellum (12C) show selective expression of SYFP2 in cells with astrocytic and Bergmann glial morphologies in the cerebellum after retroorbital injection of CN2102 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_387m enhancer selectively drives reporter expression in mouse cells with astrocytic and Bergmann glial morphologies in the cerebellum. [Figure 13A] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (13A) and visual cortex (13B) showing dim but selective expression of SYFP2 in cells with astrocytic morphology after retroorbital injection of CN2103 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_388m enhancer selectively drives reporter expression in mouse cells with astrocytic morphology. [Figure 13B] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (13A) and visual cortex (13B) showing dim but selective expression of SYFP2 in cells with astrocytic morphology after retroorbital injection of CN2103 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_388m enhancer selectively drives reporter expression in mouse cells with astrocytic morphology. [Figure 14A]Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (14A) and visual cortex (14B) showing dim but selective expression of SYFP2 in cells with oligodendrocyte and OPC morphology after retroorbital injection of CN2099 virus encapsidated with PHP.eB. This data indicates that the eHGT_396m enhancer selectively drives reporter expression in mouse cells with astrocytic morphology. [Figure 14B] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (14A) and visual cortex (14B) showing dim but selective expression of SYFP2 in cells with oligodendrocyte and OPC morphology after retroorbital injection of CN2099 virus encapsidated with PHP.eB. This data indicates that the eHGT_396m enhancer selectively drives reporter expression in mouse cells with astrocytic morphology. [Figure 15A] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (15A) and visual cortex (15B) showing dim but selective expression of SYFP2 in cells with oligodendrocyte and OPC morphology after retroorbital injection of CN2107 virus encapsidated with PHP.eB. This data indicates that the eHGT_405m enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology. [Figure 15B] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (15A) and visual cortex (15B) showing dim but selective expression of SYFP2 in cells with oligodendrocyte and OPC morphology after retroorbital injection of CN2107 virus encapsidated with PHP.eB. This data indicates that the eHGT_405m enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology. [Figure 16A]Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (16A) and visual cortex (16B) showing selective expression of SYFP2 in cells with oligodendrocyte morphology after retroorbital injection of CN2093 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_409m enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology. [Figure 16B] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (16A) and visual cortex (16B) showing selective expression of SYFP2 in cells with oligodendrocyte morphology after retroorbital injection of CN2093 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_409m enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology. [Figure 17A] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (17A) and visual cortex (17B) showing dim but selective expression of SYFP2 in cells with oligodendrocyte and OPC morphology after retroorbital injection of CN2091 virus encapsidated with PHP.eB. This data indicates that the eHGT_398m enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology. [Figure 17B] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (17A) and visual cortex (17B) showing dim but selective expression of SYFP2 in cells with oligodendrocyte and OPC morphology after retroorbital injection of CN2091 virus encapsidated with PHP.eB. This data indicates that the eHGT_398m enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology. [Figure 18A]Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (18A) and visual cortex (18B) showing selective expression of SYFP2 in cells with oligodendrocyte morphology after retroorbital injection of CN2106 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_400m enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology. [Figure 18B] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (18A) and visual cortex (18B) showing selective expression of SYFP2 in cells with oligodendrocyte morphology after retroorbital injection of CN2106 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_400m enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology. [Figure 19A] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (19A) and visual cortex (19B) showing dim but selective expression of SYFP2 in cells with oligodendrocyte and OPC morphology after retroorbital injection of CN2092 virus encapsidated with PHP.eB. This data indicates that the eHGT_402m enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology. [Figure 19B] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (19A) and visual cortex (19B) showing dim but selective expression of SYFP2 in cells with oligodendrocyte and OPC morphology after retroorbital injection of CN2092 virus encapsidated with PHP.eB. This data indicates that the eHGT_402m enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology. [Figure 20A]Native SYFP2 fluorescence montages of sagittal sections of the mouse whole brain (20A), visual cortex (20B), and cerebellum and brainstem (20C) show selective expression of SYFP2 in cells with oligodendrocyte morphology after retroorbital injection of CN2157 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_395h enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology in multiple brain regions, including midbrain and hindbrain structures. [Figure 20B] Native SYFP2 fluorescence montages of sagittal sections of the mouse whole brain (20A), visual cortex (20B), and cerebellum and brainstem (20C) show selective expression of SYFP2 in cells with oligodendrocyte morphology after retroorbital injection of CN2157 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_395h enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology in multiple brain regions, including midbrain and hindbrain structures. [Figure 20C] Native SYFP2 fluorescence montages of sagittal sections of the mouse whole brain (20A), visual cortex (20B), and cerebellum and brainstem (20C) show selective expression of SYFP2 in cells with oligodendrocyte morphology after retroorbital injection of CN2157 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_395h enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology in multiple brain regions, including midbrain and hindbrain structures. [Figure 21A] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (21A) and visual cortex (21B) showing dim but selective expression of SYFP2 in cells with oligodendrocyte and OPC morphology after retroorbital injection of CN2167 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_407h enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology. [Figure 21B]Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (21A) and visual cortex (21B) showing dim but selective expression of SYFP2 in cells with oligodendrocyte and OPC morphology after retroorbital injection of CN2167 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_407h enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology. [Figure 22A] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (22A) and visual cortex (22B) showing dim but selective expression of SYFP2 in cells with oligodendrocyte and OPC morphology after retroorbital injection of CN2159 virus encapsidated with PHP.eB. This data indicates that the eHGT_397h enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology. [Figure 22B] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (22A) and visual cortex (22B) showing dim but selective expression of SYFP2 in cells with oligodendrocyte and OPC morphology after retroorbital injection of CN2159 virus encapsidated with PHP.eB. This data indicates that the eHGT_397h enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology. [Figure 23A] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (23A), visual cortex (23B), hippocampus (23C), striatum (23D), and cerebellum (23E) show selective expression of SYFP2 in cells with oligodendrocyte morphology after retroorbital injection of CN2845 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_641m enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology in the cortex, hippocampus, striatum, and brainstem. [Figure 23B]Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (23A), visual cortex (23B), hippocampus (23C), striatum (23D), and cerebellum (23E) show selective expression of SYFP2 in cells with oligodendrocyte morphology after retroorbital injection of CN2845 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_641m enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology in the cortex, hippocampus, striatum, and brainstem. [Figure 23C] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (23A), visual cortex (23B), hippocampus (23C), striatum (23D), and cerebellum (23E) show selective expression of SYFP2 in cells with oligodendrocyte morphology after retroorbital injection of CN2845 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_641m enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology in the cortex, hippocampus, striatum, and brainstem. [Figure 23D] Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (23A), visual cortex (23B), hippocampus (23C), striatum (23D), and cerebellum (23E) show selective expression of SYFP2 in cells with oligodendrocyte morphology after retroorbital injection of CN2845 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_641m enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology in the cortex, hippocampus, striatum, and brainstem. [Figure 23E]Native SYFP2 fluorescence montages of sagittal sections of mouse whole brain (23A), visual cortex (23B), hippocampus (23C), striatum (23D), and cerebellum (23E) show selective expression of SYFP2 in cells with oligodendrocyte morphology after retroorbital injection of CN2845 virus encapsidated with PHP.eB. This data demonstrates that the eHGT_641m enhancer selectively drives reporter expression in mouse cells with oligodendrocyte morphology in the cortex, hippocampus, striatum, and brainstem. [Figure 24A] Validation of the optimized oligodendrocyte enhancer AAV vector CN2556 in adult macaque neocortical slice cultures. (24A, 24B) SYFP2 reporter expression was enhanced in white matter oligodendrocytes under matched conditions with the full-length enhancer eHGT_410m (24A-CN2109 serotype PHP.eB) and the 3xCore-eHGT_410m enhancer (24B-CN2556 serotype PHP.eB). Scale bar: 1 mm. (24C, 24D) Higher magnification images show native SYFP2 signal in cells with oligodendrocyte morphology in white matter regions under matched conditions with the full-length enhancer eHGT_410m (24C-CN2109 serotype PHP.eB) and the 3xCore-eHGT_410m enhancer (24D-CN2556 serotype PHP.eB). Scale bar: 100 microns. [Figure 24B]Validation of the optimized oligodendrocyte enhancer AAV vector CN2556 in adult macaque neocortical slice cultures. (24A, 24B) SYFP2 reporter expression was enhanced in white matter oligodendrocytes under matched conditions with the full-length enhancer eHGT_410m (24A-CN2109 serotype PHP.eB) and the 3xCore-eHGT_410m enhancer (24B-CN2556 serotype PHP.eB). Scale bar: 1 mm. (24C, 24D) Higher magnification images show native SYFP2 signal in cells with oligodendrocyte morphology in white matter regions under matched conditions with the full-length enhancer eHGT_410m (24C-CN2109 serotype PHP.eB) and the 3xCore-eHGT_410m enhancer (24D-CN2556 serotype PHP.eB). Scale bar: 100 microns. [Figure 24C] Validation of the optimized oligodendrocyte enhancer AAV vector CN2556 in adult macaque neocortical slice cultures. (24A, 24B) SYFP2 reporter expression was enhanced in white matter oligodendrocytes under matched conditions with the full-length enhancer eHGT_410m (24A-CN2109 serotype PHP.eB) and the 3xCore-eHGT_410m enhancer (24B-CN2556 serotype PHP.eB). Scale bar: 1 mm. (24C, 24D) Higher magnification images show native SYFP2 signal in cells with oligodendrocyte morphology in white matter regions under matched conditions with the full-length enhancer eHGT_410m (24C-CN2109 serotype PHP.eB) and the 3xCore-eHGT_410m enhancer (24D-CN2556 serotype PHP.eB). Scale bar: 100 microns. [Figure 24D]Validation of the optimized oligodendrocyte enhancer AAV vector CN2556 in adult macaque neocortical slice cultures. (24A, 24B) SYFP2 reporter expression was enhanced in white matter oligodendrocytes under matched conditions with the full-length enhancer eHGT_410m (24A-CN2109 serotype PHP.eB) and the 3xCore-eHGT_410m enhancer (24B-CN2556 serotype PHP.eB). Scale bar: 1 mm. (24C, 24D) Higher magnification images show native SYFP2 signal in cells with oligodendrocyte morphology in white matter regions under matched conditions with the full-length enhancer eHGT_410m (24C-CN2109 serotype PHP.eB) and the 3xCore-eHGT_410m enhancer (24D-CN2556 serotype PHP.eB). Scale bar: 100 microns. [Figure 25-1] Enhancer name, length, and sequence. [Figure 25-2] Enhancer name, length, and sequence. [Figure 25-3] Enhancer name, length, and sequence. [Figure 25-4] Enhancer name, length, and sequence. [Figure 25-5] Enhancer name, length, and sequence. [Figure 25-6] Enhancer name, length, and sequence. [Figure 25-7] Enhancer name, length, and sequence. [Figure 25-8] Enhancer name, length, and sequence. [Figure 25-9] Enhancer name, length, and sequence. [Figure 25-10] Enhancer name, length, and sequence. [Figure 25-11] Enhancer name, length, and sequence. [Figure 25-12] Enhancer name, length, and sequence. [Figure 25-13] Enhancer name, length, and sequence. [Figure 25-14] Enhancer name, length, and sequence. [Figure 25-15] Enhancer name, length, and sequence. [Figure 25-16] Enhancer name, length, and sequence. [Figure 25-17] Enhancer name, length, and sequence. [Figure 25-18] Enhancer name, length, and sequence. [Figure 25-19] Enhancer name, length, and sequence. [Figure 25-20] Enhancer name, length, and sequence. [Figure 25-21] Enhancer name, length, and sequence. [Figure 25-22] Enhancer name, length, and sequence. [Figure 25-23] Enhancer name, length, and sequence. [Figure 25-24] Enhancer name, length, and sequence. [Figure 25-25] Enhancer name, length, and sequence. [Figure 25-26] Enhancer name, length, and sequence. [Figure 25-27] Enhancer name, length, and sequence. [Figure 25-28] Enhancer name, length, and sequence. [Figure 25-29] Enhancer name, length, and sequence. [Figure 25-30] Enhancer name, length, and sequence. [Figure 25-31] Enhancer name, length, and sequence. [Figure 25-32] Enhancer name, length, and sequence. [Figure 25-33] Enhancer name, length, and sequence. [Figure 25-34] Enhancer name, length, and sequence. [Figure 25-35] Enhancer name, length, and sequence. [Figure 25-36] Enhancer name, length, and sequence. [Figure 25-37]Enhancer name, length, and sequence. [Figure 25-38] Enhancer name, length, and sequence. [Figure 25-39] Enhancer name, length, and sequence. [Figure 25-40] Enhancer name, length, and sequence. [Figure 25-41] Enhancer name, length, and sequence. [Figure 25-42] Enhancer name, length, and sequence. [Figure 26-1] Vector name, length (between ITRs), and sequence. [Figure 26-2] Vector name, length (between ITRs), and sequence. [Figure 26-3] Vector name, length (between ITRs), and sequence. [Figure 26-4] Vector name, length (between ITRs), and sequence. [Figure 26-5] Vector name, length (between ITRs), and sequence. [Figure 26-6] Vector name, length (between ITRs), and sequence. [Figure 26-7] Vector name, length (between ITRs), and sequence. [Figure 26-8] Vector name, length (between ITRs), and sequence. [Figure 26-9] Vector name, length (between ITRs), and sequence. [Figure 26-10] Vector name, length (between ITRs), and sequence. [Figure 26-11] Vector name, length (between ITRs), and sequence. [Figure 26-12] Vector name, length (between ITRs), and sequence. [Figure 26-13] Vector name, length (between ITRs), and sequence. [Figure 26-14] Vector name, length (between ITRs), and sequence. [Figure 26-15] Vector name, length (between ITRs), and sequence. [Figure 26-16] Vector name, length (between ITRs), and sequence. [Figure 26-17] Vector name, length (between ITRs), and sequence. [Figure 26-18] Vector name, length (between ITRs), and sequence. [Figure 26-19] Vector name, length (between ITRs), and sequence. [Figure 26-20] Vector name, length (between ITRs), and sequence. [Figure 26-21] Vector name, length (between ITRs), and sequence. [Figure 26-22] Vector name, length (between ITRs), and sequence. [Figure 26-23] Vector name, length (between ITRs), and sequence. [Figure 26-24] Vector name, length (between ITRs), and sequence. [Figure 26-25] Vector name, length (between ITRs), and sequence. [Figure 26-26] Vector name, length (between ITRs), and sequence. [Figure 26-27] Vector name, length (between ITRs), and sequence. [Figure 26-28] Vector name, length (between ITRs), and sequence. [Figure 26-29] Vector name, length (between ITRs), and sequence. [Figure 26-30] Vector name, length (between ITRs), and sequence. [Figure 26-31] Vector name, length (between ITRs), and sequence. [Figure 26-32] Vector name, length (between ITRs), and sequence. [Figure 26-33] Vector name, length (between ITRs), and sequence. [Figure 26-34] Vector name, length (between ITRs), and sequence. [Figure 26-35] Vector name, length (between ITRs), and sequence. [Figure 26-36] Vector name, length (between ITRs), and sequence. [Figure 26-37] Vector name, length (between ITRs), and sequence. [Figure 26-38] Vector name, length (between ITRs), and sequence. [Figure 26-39] Vector name, length (between ITRs), and sequence. [Figure 26-40] Vector name, length (between ITRs), and sequence. [Figure 26-41] Vector name, length (between ITRs), and sequence. [Figure 26-42] Vector name, length (between ITRs), and sequence. [Figure 26-43] Vector name, length (between ITRs), and sequence. [Figure 26-44] Vector name, length (between ITRs), and sequence. [Figure 26-45] Vector name, length (between ITRs), and sequence. [Figure 26-46] Vector name, length (between ITRs), and sequence. [Figure 26-47] Vector name, length (between ITRs), and sequence. [Figure 26-48] Vector name, length (between ITRs), and sequence. [Figure 26-49] Vector name, length (between ITRs), and sequence. [Figure 26-50] Vector name, length (between ITRs), and sequence. [Figure 26-51] Vector name, length (between ITRs), and sequence. [Figure 26-52] Vector name, length (between ITRs), and sequence. [Figure 26-53] Vector name, length (between ITRs), and sequence. [Figure 26-54] Vector name, length (between ITRs), and sequence. [Figure 26-55] Vector name, length (between ITRs), and sequence. [Figure 26-56] Vector name, length (between ITRs), and sequence. [Figure 26-57] Vector name, length (between ITRs), and sequence. [Figure 26-58] Vector name, length (between ITRs), and sequence. [Figure 26-59] Vector name, length (between ITRs), and sequence. [Figure 26-60] Vector name, length (between ITRs), and sequence. [Figure 26-61] Vector name, length (between ITRs), and sequence. [Figure 26-62] Vector name, length (between ITRs), and sequence. [Figure 26-63] Vector name, length (between ITRs), and sequence. [Figure 26-64] Vector name, length (between ITRs), and sequence. [Figure 26-65] Vector name, length (between ITRs), and sequence. [Figure 26-66] Vector name, length (between ITRs), and sequence. [Figure 26-67] Vector name, length (between ITRs), and sequence. [Figure 26-68] Vector name, length (between ITRs), and sequence. [Figure 26-69] Vector name, length (between ITRs), and sequence. [Figure 26-70] Vector name, length (between ITRs), and sequence. [Figure 26-71] Vector name, length (between ITRs), and sequence. [Figure 26-72] Vector name, length (between ITRs), and sequence. [Figure 26-73] Vector name, length (between ITRs), and sequence. [Figure 26-74] Vector name, length (between ITRs), and sequence. [Figure 26-75] Vector name, length (between ITRs), and sequence. [Figure 26-76] Vector name, length (between ITRs), and sequence. [Figure 26-77] Vector name, length (between ITRs), and sequence. [Figure 26-78] Vector name, length (between ITRs), and sequence. [Figure 26-79] Vector name, length (between ITRs), and sequence. [Figure 26-80] Vector name, length (between ITRs), and sequence. [Figure 26-81] Vector name, length (between ITRs), and sequence. [Figure 26-82] Vector name, length (between ITRs), and sequence. [Figure 26-83] Vector name, length (between ITRs), and sequence. [Figure 26-84] Vector name, length (between ITRs), and sequence. [Figure 26-85] Vector name, length (between ITRs), and sequence. [Figure 26-86] Vector name, length (between ITRs), and sequence. [Figure 26-87] Vector name, length (between ITRs), and sequence. [Figure 26-88] Vector name, length (between ITRs), and sequence. [Figure 26-89] Vector name, length (between ITRs), and sequence. [Figure 26-90] Vector name, length (between ITRs), and sequence. [Figure 26-91] Vector name, length (between ITRs), and sequence. [Figure 26-92] Vector name, length (between ITRs), and sequence. [Figure 26-93] Vector name, length (between ITRs), and sequence. [Figure 26-94] Vector name, length (between ITRs), and sequence. [Figure 26-95] Vector name, length (between ITRs), and sequence. [Figure 26-96] Vector name, length (between ITRs), and sequence. [Figure 26-97] Vector name, length (between ITRs), and sequence. [Figure 26-98] Vector name, length (between ITRs), and sequence. [Figure 26-99] Vector name, length (between ITRs), and sequence. [Figure 26-100] Vector name, length (between ITRs), and sequence. [Figure 26-101] Vector name, length (between ITRs), and sequence. [Figure 26-102] Vector name, length (between ITRs), and sequence. [Figure 26-103] Vector name, length (between ITRs), and sequence. [Figure 26-104] Vector name, length (between ITRs), and sequence. [Figure 26-105] Vector name, length (between ITRs), and sequence. [Figure 26-106] Vector name, length (between ITRs), and sequence. [Figure 26-107] Vector name, length (between ITRs), and sequence. [Figure 26-108] Vector name, length (between ITRs), and sequence. [Figure 26-109] Vector name, length (between ITRs), and sequence. [Figure 26-110] Vector name, length (between ITRs), and sequence. [Figure 26-111] Vector name, length (between ITRs), and sequence. [Figure 26-112] Vector name, length (between ITRs), and sequence. [Figure 26-113] Vector name, length (between ITRs), and sequence. [Figure 26-114] Vector name, length (between ITRs), and sequence. [Figure 26-115] Vector name, length (between ITRs), and sequence. [Figure 26-116] Vector name, length (between ITRs), and sequence. [Figure 26-117] Vector name, length (between ITRs), and sequence. [Figure 26-118] Vector name, length (between ITRs), and sequence. [Figure 26-119] Vector name, length (between ITRs), and sequence. [Figure 26-120] Vector name, length (between ITRs), and sequence. [Figure 26-121] Vector name, length (between ITRs), and sequence. [Figure 26-122] Vector name, length (between ITRs), and sequence. [Figure 26-123] Vector name, length (between ITRs), and sequence. [Figure 26-124] Vector name, length (between ITRs), and sequence. [Figure 26-125] Vector name, length (between ITRs), and sequence. [Figure 26-126] Vector name, length (between ITRs), and sequence. [Figure 26-127] Vector name, length (between ITRs), and sequence. [Figure 26-128] Vector name, length (between ITRs), and sequence. [Figure 26-129] Vector name, length (between ITRs), and sequence. [Figure 26-130] Vector name, length (between ITRs), and sequence. [Figure 26-131] Vector name, length (between ITRs), and sequence. [Figure 26-132] Vector name, length (between ITRs), and sequence. [Figure 26-133] Vector name, length (between ITRs), and sequence. [Figure 27-1] 1. Exemplary sequences of subcomponents used in the artificial expression constructs disclosed herein. [Figure 27-2]1. Exemplary sequences of subcomponents used in the artificial expression constructs disclosed herein. [Figure 27-3] 1. Exemplary sequences of subcomponents used in the artificial expression constructs disclosed herein. [Figure 27-4] 1. Exemplary sequences of subcomponents used in the artificial expression constructs disclosed herein. [Figure 27-5] 1. Exemplary sequences of subcomponents used in the artificial expression constructs disclosed herein. [Figure 27-6] 1. Exemplary sequences of subcomponents used in the artificial expression constructs disclosed herein. [Figure 27-7] 1. Exemplary sequences of subcomponents used in the artificial expression constructs disclosed herein. DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description To fully understand brain biology, we need to distinguish and define distinct cell types, and then identify artificial expression constructs that can selectively label and perturb them for further study. (Tasic, Curr. Opin. Neurobiol. 50, 242-249 (2018); Zeng & Sanes, Nat. Rev. Neurosci. 18, 530-546 (2017)) In mice, recombinase driver lines have been used to great effect to label 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 lines to label cell types with high specificity is expensive and often requires triple transgenic breeding, which results in low frequency of experimental animals. Furthermore, these tools require germline transgenic animals and are therefore not applicable to humans.

[0018] The present disclosure provides artificial expression constructs that selectively drive gene expression in target central nervous system cell populations, including non-neuronal brain cells such as astrocytes, oligodendrocytes, microglia, pericytes, SMCs, and endothelial cells.

[0019] Certain embodiments of the artificial expression construct utilize the following enhancers to selectively drive protein expression in targeted central nervous system cell populations as follows:

[0020] Astrocytes: eHGT_373m, 3xcore eHGT_373m, eHGT_375m, eHGT_379m, eHGT_372m, eHGT_384m, eHGT_386m, eHGT_390m, 3xcore eHGT_390m, eHGT_371m, eHGT_383m, eHGT_374m, eHGT_381m, eHGT_382m, eHGT_387m, eHGT_388m, eHGT_ 376m, eHGT_380m, eHGT_385m, eHGT_371h, eHGT_372h, eHGT_375h, eHGT_376h, eHGT_377h, eHGT_381h, e HGT_382h, eHGT_383h, eHGT_384h, eHGT_387h, eHGT_388h, eHGT_389h, eHGT_390h, eHGT_357h, eHGT_495m, eHGT_497m, mscRE1001, mscRE1002, mscRE1003, mscRE1004, mscRE1005, mscRE1006, and mscRE1007;

[0021] L1 interlaminar astrocytes: eHGT_267h, eHGT_268h, eHGT_269h, eHGT_270h, eHGT_271h, eHGT_272h, eHGT_273h, eHGT_274h, eHGT_275h, eHGT_276h, eHGT_315h, and eHGT_316h;

[0022] Oligodendrocytes: eHGT_391m, eHGT_398m, eHGT_402m, eHGT_409m, eHGT_396m, eHGT_393m, eHGT_399m, eHGT_400m, eHGT_405m, eHGT_406m, eHGT_410m, 3xcore eHGT_410m, eHGT_397m, eHGT_401m, eHGT_403m, eHGT_407m, eHGT_408m, eHGT_392h, eHGT_393h, eHGT_394h, eHGT_395h, eHGT_396h, eHGT_397h, eHGT_398h, eHGT_399h, eHGT_400h, eHGT_402h, eHGT_404h, eHGT_405h, eHGT_406h, eHGT_407h, eHGT_641m, and eHGT_408h;

[0023] Microglia: eHGT413m, eHGT_414m, eHGT_415m, eHGT_416m, eHGT_417m, eHGT_418m, eHGT_419m, eHGT_420m, eHGT_4 21m, eHGT_423m, eHGT_428m, eHGT_429m, eHGT_430m, eHGT_411m, eHGT_412m, eHGT_422m, eHGT_424m, eHGT_425 m, eHGT_426m, eHGT_427m, eHGT_411h, eHGT_412h, eHGT_413h, eHGT_414h, eHGT_417h, eHGT_418h, eHGT_419h, eHGT_420h, eHGT_423h, eHGT_424h, eHGT_425h, eHGT_426h, eHGT_427h, eHGT_428h, eHGT_429h, and eHGT_430h;

[0024] Pericytes: mscRE1023, mscRE1024, mscRE1025, mscRE1026, mscRE1027, mscRE1028, mscRE1029, mscRE1030, mscRE1031, mscRE1032, mscRE1033, mscRE1034, mscRE1035, mscRE1036, and mscRE1037;

[0025] SMC: mscRE1038, mscRE1039, mscRE1040, mscRE1041, mscRE1042, mscRE1043, mscRE1044, mscRE1045, mscRE1046, mscRE1047, mscRE1048, mscRE1049, mscRE1050, mscRE1051, and mscRE1052;

[0026] Endothelial cells: mscRE1008, mscRE1009, mscRE1010, mscRE1011, mscRE1012, mscRE1013, mscRE1014, mscRE1015, mscRE1016, mscRE1017, mscRE1018, mscRE1019, mscRE1020, mscRE1021, and mscRE1022.

[0027] Certain embodiments are directed to the vectors: CN1781, CN1782, CN1783, CN1784, CN1785, CN1786, CN1787, CN1788, CN1789, CN1790, CN2044, CN2082, CN2083, CN2084, CN2560, CN2085, CN2086, CN2087, CN2088, CN2089, CN2558, CN2090, CN2091, CN2092, CN2093, CN2094, CN2095, CN2096, CN2097, CN2098, CN2099, CN2100, CN2101, CN2102, CN2103, CN2104, CN2105, CN2106, CN2107, CN2108, CN2109, CN2110, CN2111, CN2112, CN2113, CN2114, CN2115, CN2116, CN2117, CN2118, CN2119, CN2120, CN2121, CN2122, CN2123, CN2124, CN2125, CN2126, CN2127, CN2128, CN2129, CN2130, CN2131, CN2132, CN2133, CN2134, CN2135, CN2136, CN2137, CN2138, CN2139, CN214 2, CN2103, CN2104, CN2105, CN2106, CN2107, CN2108, CN2109, CN2556, CN2110, CN2111, CN2112, CN2113, CN2114, CN2115, CN2116, CN2117, CN2118, CN21 19, CN2120, CN2121, CN2122, CN2123, CN2124, CN2125, CN2126, CN2127, CN2128, CN2129, CN2130, CN2131, CN2132, CN2133, CN2134, CN2141, CN2142, CN2 143, CN2144, CN2145, CN2146, CN2147, CN2148, CN2149, CN2150, CN2151, CN2152, CN2153, CN2154, CN2155, CN2156, CN2157, CN2158, CN2159, CN2160, CN 2161, CN2162, CN2163, CN2164, CN2165, CN2166, CN2167, CN2845, CN2168, CN2169, CN2170, CN2171, CN2172, CN2173, CN2174, CN2175, CN2176, CN2177, C N2178, CN2179, CN2180, CN2181, CN2182, CN2183, CN2184, CN2243, CN2268, CN2345, CN2346, 3001, 3002, 3003, 3004, 3005, 3006, 3007, 3008, 3009, 3010 , 3011, 3012, 3013, 3014, 3015, 3016, 3017, 3018, 3019, 3020, 3021, 3022, 30 23, 3024, 3025, 3026, 3027, 3028, 3029, 3030, 3031, 3032, 3033, 3034, 3035,Artificial expression constructs are provided that include features of the vectors described herein, such as 3036, 3037, 3038, 3039, 3040, 3041, 3042, 3043, 3044, 3045, 3046, 3047, 3048, 3049, 3050, 3051, and 3052.

[0028] Aspects of the present disclosure are described in the following further options and details: (i) Artificial Expression Constructs and Vectors for Selective Expression of Genes in Selected Cell Types, (ii) Compositions for Administration, (iii) Cell Lines Comprising Artificial Expression Constructs, (iv) Transgenic Animals, (v) Methods of Use, (vi) Kits and Commercial Products, (vii) Exemplary Embodiments, (viii) Experimental Examples, and (ix) Closing Paragraphs. These headings are provided for organizational purposes only and are not intended to limit the scope or interpretation of the disclosure.

[0029] (i) Artificial expression constructs and vectors for selective expression of genes in selected cell types. The artificial expression constructs disclosed herein include (i) an enhancer sequence that confers selective expression of a coding sequence in a target central nervous system cell type, (ii) the coding sequence to be expressed, and (iii) a promoter. The expression constructs can also include other regulatory elements, if necessary or beneficial.

[0030] In certain embodiments, an "enhancer" or "enhancer element" is a cis-acting sequence that increases the level of transcription associated with a promoter, can function in either orientation relative to the promoter and coding sequence to transcribe, and can be located upstream or downstream relative to the promoter or coding sequence to transcribe. Methods and techniques for measuring the function(s) of an enhancer element sequence exist and are recognized in the art. Specific examples of enhancer sequences for use in the artificial expression constructs disclosed herein include: eHGT_267h, eHGT_268h, eHGT_269h, eHGT_270h, eHGT_271h, eHGT_272h, eHGT_273h, eHGT_274h, eHGT_275h, eHGT_276h, eHGT_315h, eHGT_316h, eHGT_357h, eHGT_371h, eHGT_371m, eHGT_372h, eHGT_372m, eHGT_373m, 3xcore eHGT_373m, eHGT_374m, eHGT_375h, eHGT_375m, eHGT_376h, eHGT_376m, eHGT_3 77h, eHGT_379m, eHGT_380m, eHGT_381h, eHGT_381m, eHGT_382h, eHGT_382m, eHG T_383h, eHGT_383m, eHGT_384h, eHGT_384m, eHGT_385m, eHGT_386m, eHGT_387h , eHGT_387m, eHGT_388h, eHGT_388m, eHGT_389h, eHGT_390h, eHGT_390m, 3xcoreeHGT_390m、eHGT_391m、eHGT_392h、eHGT_393h、eHGT_393m、eHGT_394h、eHGT_395h、eHGT_396h、eHGT_396m、eHGT_397h、eHGT_397m、eHGT_398h、eHGT_398m、eHGT_399h、eHGT_399m、eHGT_400h、eHGT_400m、eHGT_401m、eHGT_402h、eHGT_402m、eHGT_403m、eHGT_404h、eHGT_405h、eHGT_405m、eHGT_406h、eHGT_406m、eHGT_407h、eHGT_641m、eHGT_407m、eHGT_408h、eHGT_408m、eHGT_409m、eHGT_410m、3xcoreeHGT_410m, eHGT_411h, eHGT_411m, eHGT_412h, eHGT_412m, eHGT_413h, eHGT_414h, eHGT_414m, eHGT_415m, eHGT_416m, eHGT_417h, eHGT _417m, eHGT_418h, eHGT_418m, eHGT_419h, eHGT_419m, eHGT_420h, eHGT_420m, eHGT_421m, eHGT_422m, eHGT_423h, eHGT_423m, eHGT_424 h, eHGT_424m, eHGT_425h, eHGT_425m, eHGT_426h, eHGT_426m, eHGT_427h, eHGT_427m, eHGT_428h, eHGT_428m, eHGT_429h, eHGT_429m, eH GT_430h, eHGT_430m, eHGT_495m, eHGT_497m, eHGT413m, mscRE1001, mscRE1002, mscRE1003, mscRE1004, mscRE1005, mscRE1006, mscRE100 7, mscRE1008, mscRE1009, mscRE1010, mscRE1011, mscRE1012, mscRE1013, mscRE1014, mscRE1015, mscRE1016, mscRE1017, mscRE1018, ms cRE1019, mscRE1020, mscRE1021, mscRE1022, mscRE1023, mscRE1024, mscRE1025, mscRE1026, mscRE1027, mscRE1028, mscRE1029, mscRE1 030, mscRE1031, mscRE1032, mscRE1033, mscRE1034, mscRE1035, mscRE1036, mscRE1037, mscRE1038, mscRE1039, mscRE1040, mscRE1041, mscRE1042, mscRE1043, mscRE1044, mscRE1045, mscRE1046, mscRE1047, mscRE1048, mscRE1049, mscRE1050, mscRE1051, and mscRE1052.

[0031] In certain embodiments, a target central nervous system cell type enhancer is an enhancer that is uniquely or primarily utilized by the target central nervous system cell type. The target central nervous system cell type enhancer enhances expression of a gene in the target central nervous system cell type but does not substantially induce expression of the gene in other, non-target cell types, and thus has cell-type-specific transcriptional activity.

[0032] A product of a coding sequence is preferentially expressed in a selected cell type if the coding sequence is selectively expressed in the selected cell and substantially not expressed in other cell types. In certain embodiments, preferential expression is greater than 50% compared to a reference cell type; greater than 60% compared to a reference cell type; greater than 70% compared to a reference cell type; greater than 80% compared to a reference cell type; or greater than 90% compared to a reference cell type. In certain embodiments, the reference cell type refers to non-targeted cells. Non-targeted cells are located within the same anatomical structure and / or occur in a common anatomical region as the targeted cells. In certain embodiments, the reference cell type is located in an anatomical structure adjacent to the anatomical structure containing the targeted cell type. In certain embodiments, the reference cell type is a non-targeted cell that has a gene expression profile different from that of the targeted cells.

[0033] In certain embodiments, the product of the coding sequence may be expressed in a non-selected cell type at a low level, for example, less than 1%, or 1%, 2%, 3%, 5%, 10%, 15%, or 20% of the level at which the product is expressed in a selected cell. In certain embodiments, the target central nervous system cell type is the only cell type that expresses the appropriate combination of transcription factors that bind to the enhancers disclosed herein to drive gene expression. Thus, in certain embodiments, expression occurs only within the target cell type.

[0034] In certain embodiments, target cell types (e.g., non-neuronal cell types described herein) can be identified based on transcriptional profiles, such as those described in Tasic et al., 2018 and Hodge et al., Nature, 573, 61-68 (2019). For reference, a description of cell types and identifying characteristics is provided as follows:

[0035] Neocortical GABAergic subclasses: ●All: express the GABA synthesis genes Gad1 / GAD1 and Gad2 / GAD2. • Lamp5, Sncg, Serpinf1, and Vip GABAergic neurons: developmentally derived from neuronal precursors from the caudal ganglion luminal (CGE) or preoptic area (POA). • Sst and Pvalb GABAergic neurons: are developmentally derived from neuronal precursors in the medial ganglionic emittance (MGE). ●Lamp5 GABAergic neurons: Found in many cortical layers, especially the upper layers (L1-L2 / 3), and mainly have glial and single plexus morphologies. ●Sncg GABAergic neurons: Found in many cortical layers and have molecular overlap with Lamp5 and Vip cells, but they express Lamp5 or Vip inconsistently and Sncg more consistently. Serpinf1 GABAergic neurons: found in many cortical layers and have molecular overlap with Sncg and Vip cells, but expression of Sncg or Vip is inconsistent and expression of Serpinf1 is more consistent. ●Vip GABAergic neurons: Found in many cortical layers, but particularly abundant in the upper layers (L1-L4), they highly express the neurotransmitter vasoactive intestinal peptide (Vip). Sst GABAergic neurons are found in many cortical layers, but are particularly frequent in the lower layers (L5-L6). They highly express the neurotransmitter somatostatin (Sst) and frequently block dendritic input to postsynaptic neurons. This subclass includes sleep-active Sst Chodl neurons (which also express Nos1 and Tacr1), which are very distinct from other Sst neurons but express several shared marker genes, including Sst. In humans, Sst gene expression is often detected in layer 1 LAMP5+ cells. Pvalb GABAergic neurons are found in many cortical layers, but are particularly frequent in the lower layers (L5-L6). They highly express the calcium-binding protein parvalbumin (Pvalb), express the neuropeptide Tac1, and often attenuate the output of postsynaptic neurons. Most fast-spiking inhibitory cells strongly express Pvalb. This subclass includes chandelier cells, which have a distinct chandelier-like morphology and express the markers Cpne5 and Vipr2 in mice and NOG and UNC5B in humans. Meis2: A distinct subclass defined as only the neocortical GABAergic type expressing the Meis2 gene, but not expressing some other genes expressed by all other neocortical GABAergic types (e.g., Thy1 and Scn2b). This type is found in L6b and subcortical white matter.

[0036] Neocortical glutamatergic subclasses: All glutamatergic neurons express the glutamate transmitters Slc17a6 and / or Slc17a7. They all express Snap25 and lack Gad1 / Gad2 expression. L2 / 3 IT glutamatergic neurons: Predominantly present in layer 2 / 3 and have primarily intratelencephalic (cortico-cortical) projections. • L4 IT glutamatergic neurons: Predominantly located in layer 4 and have either primarily local or intratelencephalic (cortico-cortical) projections. L5 IT glutamatergic neurons: Predominantly located in layer 5 and with predominantly intratelencephalic (cortico-cortical) projections. Also called L5a. L5 PT glutamatergic neurons: Predominantly located in layer 5 and primarily possess cortico-subcortical (pyramidal or corticosubduction) projections. Also referred to as L5b or L5 CF (corticofugal), or L5 ET (extratelencephalic). This subclass includes cells that are corticospinal projection neurons located in the primary motor cortex and adjacent areas and associated with motor neuron / movement disorders such as ALS. This subclass includes thick-tufted pyramidal neurons, including distinctive subtypes found only in specialized regions, such as Betz cells, Meynert cells, and von Economo cells. ●L5 NP glutamatergic neurons: reside mainly in layer 5 and have projections mainly in the vicinity. • L6 CT glutamatergic neurons: Predominantly located in layer 6 and have predominantly corticothalamic projections. • L6 IT glutamatergic neurons: Predominantly present in layer 6 and have predominantly intracranial (cortico-cortical) projections. This subclass includes L6 IT Car3 cells, which are very similar to the intracranial cortical projection cells. • L6b glutamatergic neurons: Predominantly located in the neocortical subplate (L6b), with local (near the cell body) projections and some cortico-cortical projections from the VISp to the anterior cisternae and cortico-subcortical projections to the thalamus. ●CR glutamatergic neurons: Cajal-Retzius cells, a distinct subclass defined as a single type in L1, express distinct molecular markers Lhx5 and Trp73.

[0037] Cerebellar Purkinje cells: large GABAergic neurons, the only projection neurons and sole product of the cerebellum. Their cell bodies form a single layer called the "Purkinje cell layer" and express parvalbumin.

[0038] Deep cerebellar nucleus neurons: Neurons located in the deep cerebellar nucleus structures. These include glutamatergic and GABAergic cells that express the Pvalb gene.

[0039] Non-neuronal subclasses: Astrocytes: Neuroectodermal-derived glial cells that express the marker Aqp4 and often GFAP, but not the neuronal marker SNAP25. They can have a distinct star-shaped morphology and are involved in the metabolic support of other cells in the brain. Multiple astrocyte morphologies are found in mice and humans. Oligodendrocytes: Glial cells derived from the neuroectoderm that express the marker Sox10. This category includes oligodendrocyte precursor cells (OPCs). Oligodendrocytes are a subclass of cells primarily responsible for myelination of neurons. • VLMC: Vascular leptomeningeal cells (VLMC) are part of the meninges that surround the outer layer of the cortex and express the marker genes Lum and Col1a1. Pericytes: Blood vessel-associated cells that express the marker genes Kcnj8 and Abcc9. Pericytes surround endothelial cells and are important in regulating capillary blood flow and are involved in blood-brain barrier permeability. SMCs: Specialized smooth muscle cells that are associated with blood vessels and express the marker gene Acta2. SMCs line the arteries of the brain and are involved in blood-brain barrier permeability. Endothelial cells: Cells that line the blood vessels in the brain. Endothelial cells express the markers Tek and PDGF-B. Microglia: Brain-resident macrophages and perivascular macrophages (PVMs) are hematopoietic-derived immune cells that may be transiently associated with brain tissue or included as a by-product of brain dissection. Microglia are known to express Cx3cr1, Tmem119, and PTPRC (CD45).

[0040] In certain embodiments, the coding sequence is a heterologous coding sequence that encodes an effector element. An effector element is a sequence that is expressed to achieve, and actually achieve, a desired effect. Examples of effector elements include reporter genes / proteins and functional genes / proteins.

[0041] Exemplary reporter genes / proteins include those expressed by Addgene ID numbers 83894 (pAAV-hDlx-Flex-DTomato-Fishell_7), 83895 (pAAV-hDlx-Flex-GFP-Fishell_6), 83896 (pAAV-hDlx-GiDREADD-dTomato-Fishell-5), 83898 (pAAV-mDlx-ChR2-mCherry-Fishell-3), 83899 (pAAV-mDlx-GCaMP6f-Fishell-2), 83900 (pAAV-mDlx-GFP-Fishell-1), and 89897 (pcDNA3-FLAG-mTET2(N500)).Exemplary reporter genes include those encoding expressible fluorescent proteins or expressible biotin, among others: 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, ZsGreenl, Oregon Green™ (Thermo Fisher Scientific)); luciferase; orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato, dTomato); red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRuby, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred, Texas Red™ (Thermo Fisher Scientific)); far-red fluorescent proteins (e.g., mPlum and mNeptune); yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, SYFP2, Venus, YPet, PhiYFP, ZsYellowl); and tandem conjugates.

[0042] GFP, composed of 238 amino acids (26.9 kDa), was originally isolated from the jellyfish Aequorea victoria / Aequorea aequorea / Aequorea forskalea, which fluoresces green when exposed to blue light. GFP from A. victoria has a major excitation peak at 395 nm and a minor excitation peak at 475 nm. Its emission peak is at 509 nm, in the lower green portion of the visible spectrum. GFP from the sea pansy (Renilla reniformis) has a single major excitation peak at 498 nm. Due to its widespread potential and evolving research needs, many different variants of GFP have been genetically engineered. 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, resulting in increased fluorescence, photostability, and a shift in the main excitation peak to 488 nm, with peak emission maintained at 509 nm. Adding the 37°C folding efficiency (F64L) point mutation to this backbone resulted in enhanced GFP (EGFP). EGFP has a 9.13 × 10-21 m, also referred to as 55,000 L / (mol cm). 2 The extinction coefficient (ε), also known as the optical cross section of the molecule, is ε. In 2006, we reported superfolder GFP, a series of mutations that allowed GFP to rapidly fold and mature even when fused to poorly folding peptides.

[0043] "Yellow fluorescent protein" (YFP) is a genetic variant of the green fluorescent protein derived from the jellyfish Aequorea victoria. Its excitation peak is 514 nm and its emission peak is 527 nm.

[0044] Exemplary functional molecules include functional ion transporters, cellular transport proteins, enzymes, transcription factors, neurotransmitters, calcium reporters, channelrhodopsins, guide RNAs, nucleases, microRNAs, or designer receptors activated exclusively by designer drugs (DREADDs).

[0045] Ion transporters are transmembrane proteins that mediate the transport of ions across cell membranes. These ion transporters are widespread throughout most cell types and are important for regulating cellular excitability and homeostasis. Ion transporters are involved in numerous cellular processes, such as action potentials, synaptic transmission, hormone secretion, and muscle contraction. Many important biological processes in living cells involve the movement of cations, such as calcium (Ca), potassium (K), and sodium (Na), ions, through such ion channels. In certain embodiments, ion transporters include voltage-gated sodium channels (e.g., SCN1A), potassium channels (e.g., KCNQ2), and calcium channels (e.g., CACNA1C).

[0046] Exemplary enzymes, transcription factors, receptors, membrane proteins, cellular transport proteins, signaling molecules, and neurotransmitters include enzymes such as lactase, lipase, helicase, alpha-glucosidase, amylase; transcription factors such as SP1, AP-1, heat shock factor protein 1, C / EBP (CCAA-T / enhancer binding protein), and Oct-1; receptors such as transforming growth factor receptor beta 1, platelet-derived growth factor receptor, epidermal growth factor receptor, vascular endothelial growth factor receptor, and interleukin-8 receptor alpha; membrane proteins, cellular transport proteins such as clathrin, dynamin, caveolin, Rab-4A, and Rab-11A; signaling molecules such as nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor beta (TGFβ), epidermal growth factor (EGF), GTPases, and HRas; and neurotransmitters such as cocaine- and amphetamine-regulated transcription factors, substance P, oxytocin, and somatostatin.

[0047] In certain embodiments, functional molecules include reporters of cell function and status, such as calcium reporters. Intracellular calcium concentration is an important predictor of numerous cellular activities, including neuronal activation, muscle cell contraction, and second messenger signaling. A sensitive and convenient technique for monitoring intracellular calcium levels is via genetically encoded calcium indicators (GECIs). Among GECIs, a green fluorescent protein (GFP)-based calcium sensor, designated GCaMP, is an efficient and widely used tool. GCaMP is formed by fusing M13 and calmodulin proteins to the N- and C-termini of a circular mutant GFP. Some GCaMPs exhibit distinct fluorescence emission spectra (Zhao et al., Science, 2011, 333(6051):1888-1891). Exemplary GECIs with green fluorescence include GCaMP3, GCaMP5G, GCaMP6s, GCaMP6m, GCaMP6f, jGCaMP7s, jGCaMP7c, jGCaMP7b, and jGCaMP7f. Additionally, GECIs with red fluorescence include jRGECO1a and jRGECO1b. AAV products containing GECIs are commercially available.For example, Vigene Biosciences offers AAV8-CAG-GCaMP3 (catalog number: BS4-CX3AAV8), AAV8-Syn-FLEX-GCaMP6s-WPRE (catalog number: BS1-NXSAAV8), AAV8-Syn-FLEX-GCaMP6s-WPRE (catalog number: BS1-NXSAAV8), AAV9-CAG-FLEX-GCaMP6m-WPRE (catalog number: BS2-CXMAAV9), AAV9-Syn-FLEX-jGCaMP7s-WPRE (catalog number: BS12-NXSAAV9), and AAV9-CAG-FLEX We provide AAV products including AAV9-Syn-FLEX-jGCaMP7f-WPRE (Cat. No.: BS12-CXFAAV9), AAV9-Syn-FLEX-jGCaMP7b-WPRE (Cat. No.: BS12-NXBAAV9), AAV9-Syn-FLEX-jGCaMP7c-WPRE (Cat. No.: BS12-NXCAAV9), AAV9-Syn-FLEX-NES-jRGECO1a-WPRE (Cat. No.: BS8-NXAAAV9), and AAV8-Syn-FLEX-NES-jRCaMP1b-WPRE (Cat. No.: BS7-NXBAAV8).

[0048] In certain embodiments, the calcium reporter includes a genetically encoded calcium indicator GECI, NTnC; a myosin light chain kinase, GFP, calmodulin chimera; the calcium indicator TN-XXL; a BRET-type autoluminescent calcium indicator; and / or the calcium indicator protein OeNL(Ca2+)-18u).

[0049] In certain embodiments, functional molecules include modulators of neuronal activity, such as channelrhodopsins (e.g., channelrhodopsin-1, channelrhodopsin-2, and their variants). Channelrhodopsins are a subfamily of retinylidene ion-gated proteins (rhodopsins) 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) described the creation of chimeras of the transmembrane domains of ChR1 and ChR2 combined with site-directed mutagenesis. Zhang et al. (Nat Neurosci, 2008, 11(6):631-3) described a red-shifted channelrhodopsin variant, VChR1. VChR1 has low light sensitivity and poor membrane trafficking and expression. Other known channelrhodopsin mutants include the ChR2 mutants 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, JY et al., Biophys J, 2009, 96(5):1803-14), which are activated by blue light (470 nm) but are insensitive to orange / red light. Further mutants are described in Lin, Experimental Physiology, 2010, 96.1:19-25, and Knopfel et al., The Journal of Neuroscience, 2010, 30(45):14998-15004.

[0050] In certain embodiments, functional molecules include DNA and RNA editing tools such as CRISPR / CAS (e.g., guide RNA and a nuclease such as Cas, Cas9, or cpfl). Functional molecules can also include engineered Cpfl, such as those described in US2018 / 0030425, US2016 / 0208243, WO / 2017 / 184768, and Zetsche et al. (2015) Cell 163:759-771; a single gRNA (see, e.g., Jinek et al. (2012) Science 337:816-821; Jinek et al. (2013) eLife 2:e00471; Segal (2013) eLife 2:e00563), or an editase, guide RNA molecule, microRNA, or homologous recombination donor cassette.

[0051] Sequences 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), alpha-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 proteins. 1 (e.g., UniProtKB / Swiss-Prot: Q00613.1), CCAAT / enhancer-binding protein (C / EBP) beta isoform A (e.g., NP_005185.2), Oct-1 (e.g., UniProtKB / Swiss-Prot: P14859.2), TGFβ (e.g., GenBank: CAF02096.2), platelet-derived growth factor receptor (e.g., GenBank: AAA60049.1), epidermal growth factor receptor (e.g., GenBank: CAA25240.1), vascular endothelial growth factor receptor (VEGF) (e.g., GenBank: AAC16449.2), interleukin-8 receptor alpha (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 Ras-related protein Rab-4A isoform 1 (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-beta 3 (e.g., GenBank: AAA61161.1), nerve growth factor (e.g., GenBank: CAA37703.1), and EGF (e.g., GenBank: CAA34902).2), cocaine- and amphetamine-regulated transcript (chain A) (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 (chain A) [synthetic construct] (e.g., PDB:5MWC_A), calcium indicator TN-XXL [synthetic construct] (e.g., GenBank:ACF93133.1), BRET-based self-luminescent calcium indicator [synthetic construct] (e.g., GenBank ADF42668.1), calcium indicator protein OeNL(Ca2+)-18u [synthetic construct] (e.g., GenBank BBB18812.1), myosin light chain kinase, green fluorescent protein, calmodulin chimera (chain A) [synthetic construct] (e.g., PDB: 3EKJ_A), channellopsin 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 endonucleases (ECs) Examples include nuclease Cpf1 (e.g., UniProtKB / Swiss-Prot: U2UMQ6.1), ribonuclease 4 or ribonuclease L (e.g., UniProtKB / Swiss-Prot: Q05823.2), deoxyribonuclease II beta (e.g., GenBank: AAF76893.1), sodium channel protein type 1 subunit alpha (e.g., UniProtKB-P35498), potassium voltage-gated channel subfamily KQT member 2 (e.g., UniProtKB-O43526), ​​and voltage-gated L-type calcium channel subunit alpha-1C (e.g., UniProtKB-Q13936).

[0052] Additional effector elements include Cre, iCre, dgCre, FlpO, and tTA2. iCre refers to codon-improved Cre. dgCre refers to an enhanced GFP / Cre recombinase fusion gene with an N-terminal fusion of the first 159 amino acids of the Escherichia coli K-12 chromosomal dihydrofolate reductase gene (DHFR or folA) containing a G67S mutation, modified to also contain an R12Y / Y100I instability domain mutation. FlpO refers to a codon-optimized form of FLPe, which significantly increases protein expression and FRT recombination efficiency in mouse cells. Similar to the Cre / LoxP system, the FLP / FRT system is widely used for gene expression (and for generating conditional knockout mice mediated by the FLP / FRT system). tTA2 refers to the tetracycline transactivator.

[0053] Exemplary expressible elements are expression products that do not contain effector elements, such as, for example, non-functional or defective proteins. In certain embodiments, expressible elements can provide a method for studying the effects of their functional counterparts. In certain embodiments, expressible elements are non-functional or defective due to engineered mutations that render them non-functional. In these aspects, non-expressible elements are as similar in structure as possible to their functional counterparts.

[0054] An exemplary self-cleaving peptide is the 2A peptide, which leads to the production of two proteins from a single mRNA. Because 2A sequences are short (e.g., 20 amino acids), they allow for more use in size-constrained constructs. Specific examples include P2A, T2A, E2A, and F2A. In certain embodiments, the artificial expression construct comprises an internal ribosome entry site (IRES) sequence. The IRES allows ribosomes to initiate translation at a second internal site on the mRNA molecule, leading to the production of two proteins from a single mRNA.

[0055] Coding sequences encoding the molecules (e.g., RNA, proteins) described herein can be obtained from publicly available databases and publications. Coding sequences can further contain various sequence polymorphisms, mutations, and / or sequence variants; such modifications do not affect the function of the encoded molecule. The terms "encode" or "encoding" refer to the property of a nucleic acid sequence, such as a vector, plasmid, gene, cDNA, mRNA, etc., that serves as a template for the synthesis of another molecule, such as a protein.

[0056] The term "gene" can include not only coding sequences but also regulatory regions such as promoters, enhancers, insulators, and / or termination regions. The term can further include all introns and other DNA sequences spliced ​​from the mRNA transcript, as well as variants resulting from alternative splice sites. A sequence can also include degenerate codons of a reference sequence, or sequences that may be introduced to provide codon preference in a particular organism or cell type.

[0057] Promoters can include general promoters, tissue-specific promoters, cell-specific promoters, and / or cytoplasm-specific promoters. Promoters can be strong promoters, weak promoters, constitutive promoters, and / or inducible promoters. Inducible promoters induce expression in response to specific conditions, signals, or cellular events. For example, a promoter can be an inducible promoter that requires a specific ligand, small molecule, transcription factor, or hormone protein to effect transcription from the promoter. Specific examples of promoters include minBglobin, CMV, minCMV, and minCMV. * (minCMV * is minCMV with the SacI restriction site removed), minRho, minRho * (minRho *These include the SV40 immediate-early promoter, the Hsp68 minimal promoter (proHSP68), and the Rous sarcoma virus (RSV) long repeat (LTR) promoter. Minimal promoters do not have the activity to drive gene expression by themselves, but can be activated to drive gene expression when linked to proximal enhancer elements.

[0058] In certain embodiments, the expression construct is provided within a vector. The term vector refers to a nucleic acid molecule that can transfer or transport another nucleic acid molecule, such as an artificial expression construct. The transferred nucleic acid is generally linked, e.g., inserted, into the vector nucleic acid molecule. The vector may contain a sequence that directs autonomous replication within the cell, or may contain a sequence that allows integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.

[0059] The term "viral vector" is broadly used to refer to nucleic acid molecules containing virally derived nucleic acid elements that facilitate the transfer and expression of non-naturally occurring nucleic acid molecules into cells. The term adeno-associated viral vector primarily refers to viral vectors or plasmids containing structural and functional genetic elements, or portions thereof, derived from AAV. The term "retroviral vector" primarily refers to viral vectors or plasmids containing structural and functional genetic elements, or portions thereof, derived from retroviruses, etc. The term "lentiviral vector" primarily refers to viral vectors or plasmids containing structural and functional genetic elements, or portions thereof, derived from lentiviruses, etc. The term "hybrid vector" refers to vectors containing structural and / or functional genetic elements from two or more viral types.

[0060] An adenoviral vector is a construct containing sufficient adenoviral sequences to (a) support the packaging of an artificial expression construct and (b) express a coding sequence cloned therein in either the sense or antisense orientation. Recombinant adenoviral vectors contain genetically engineered forms of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kb linear, double-stranded DNA virus, allows for the replacement of large elements of adenoviral DNA with up to 7 kb of foreign sequence. In contrast to retroviruses, adenoviral DNA can replicate episomalally without potential genotoxicity, so adenoviral infection of host cells does not result in chromosomal integration. Adenoviruses are also structurally stable, and no genome rearrangements have been detected after extensive amplification.

[0061] Adenoviruses are particularly suitable for use as gene transfer vectors due to their medium-sized genome, ease of manipulation, high titer, wide target cell range, and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis-receptors necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain distinct transcription units that are separated by the initiation of viral DNA replication. The E1 region (E1A and E1B) encodes proteins involved in regulating the transcription of the viral genome and several cellular genes. Expression of the E2 region (E2A and E2B) results in the synthesis of proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shutoff. Late gene products, including the majority of viral capsid proteins, are expressed only after significant processing of a single primary transcript derived from the major late promoter (MLP). MLP is particularly efficient during the late stages of infection, and all mRNAs derived from this promoter contain a 5'-triple leader (TPL) sequence, making them preferred mRNAs for translation.

[0062] Other than the requirement that the adenoviral vector be replication-defective, or at least conditionally defective, the nature of the adenoviral vector is not believed to be critical to the successful practice of certain embodiments disclosed herein. The adenovirus can be any of the 42 different known serotypes or subgroups A-F. In certain embodiments, adenovirus type 5 of subgroup C is the preferred starting material for obtaining the conditionally replication-defective adenoviral vectors used in certain embodiments, since adenovirus type 5 is a human adenovirus for which a great deal of biochemical and genetic information is known and has historically been used for most constructs using adenoviruses as vectors.

[0063] As indicated, typical vectors are replication-defective and lack the adenovirus E1 region. Therefore, it will be most convenient to introduce a polynucleotide encoding a gene of interest into the location where the E1 coding sequence has been removed. However, the insertion location of the construct within the adenovirus sequence is not critical. A polynucleotide encoding a gene of interest can also be inserted into an E3 replacement vector or helper cell line, or into the E4 region where the helper virus complements the E4 deficiency, replacing the deleted E3 region.

[0064] Adeno-associated virus (AAV) is a parvovirus discovered as a contaminant in adenovirus strains. It is a ubiquitous virus not associated with any disease (antibodies are present in 85% of the US human population). It is classified as a helper virus because its replication depends on the presence of a helper virus, such as adenovirus. Various serotypes have been isolated, of which AAV-2 is the most well-characterized. AAV contains single-stranded linear DNA packaged within capsid proteins VP1, VP2, and VP3 to form icosahedral virions with a diameter of 20–24 nm.

[0065] AAV DNA is 4.7 kilobases long. It contains two open reading frames flanked by two ITRs. The AAV genome contains two major genes, rep and cap. The rep gene encodes proteins involved in viral replication, while cap encodes the capsid proteins VP1-VP3. Each ITR forms a T-shaped hairpin structure. These terminal repeats are the only essential cis-terminal components of AAV for chromosomal integration. Therefore, AAV can be used as a vector for delivery by removing all viral coding sequences and replacing them with gene cassettes. Three AAV viral promoters have been identified and named p5, p19, and p40 according to their map locations. Transcription from p5 and p19 leads to the production of rep proteins, while transcription from p40 produces capsid proteins.

[0066] AAVs are prominent for use within the present disclosure due to their excellent safety profile and the fact that their capsids and genomes can be tailored to allow expression in selected cell populations. scAAV refers to self-complementary AAV. pAAV refers to plasmid adeno-associated virus. rAAV refers to recombinant adeno-associated virus.

[0067] Other viral vectors may be employed, such as those derived from viruses such as vaccinia virus, poliovirus, and herpesvirus, which offer several attractive features for a variety of mammalian cells.

[0068] Retroviruses are common tools for gene delivery. The term "retrovirus" refers to an RNA virus that reverse-transcribes its genomic RNA into a linear, double-stranded DNA copy and then covalently integrates the genomic DNA into the host genome. Once the virus is integrated into the host genome, it is called a "provirus." The provirus serves as a template for RNA polymerase II, which induces the expression of RNA molecules encoding the structural proteins and enzymes required to produce new viral particles.

[0069] Exemplary retroviruses suitable for use in certain embodiments include: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentiviruses.

[0070] "Lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include: HIV (including human immunodeficiency virus, HIV types 1 and 2); visnamesi virus (VMV); capric 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., HIV cis-acting sequence elements) can be used.

[0071] Improved safety for the use of some vectors can be achieved by replacing the U3 region of the 5'LTR with a heterologous promoter to drive transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used for this purpose include the simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate-early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters. Typical promoters are capable of driving high levels of transcription in a Tat-independent manner. This substitution reduces the possibility of recombination to generate replication-competent virus due to the absence of the complete U3 sequence in the viral production system. In certain embodiments, heterologous promoters have the added advantage of controlling how the viral genome is transcribed. For example, the heterologous promoter can be inducible, so that transcription of all or part of the viral genome occurs only in the presence of an inducer. Inducers can include one or more chemical compounds or physiological conditions under which the host cells are cultured, such as temperature or pH.

[0072] In certain embodiments, the viral vector comprises a TAR element. The term "TAR" refers to the "transactivation response" gene element located in the R region of the lentiviral LTR. This element interacts with the lentiviral transactivator (tat) gene element to enhance viral replication. However, this element is not required in embodiments where the U3 region of the 5'LTR is replaced with a heterologous promoter.

[0073] The "R region" refers to the region within a retroviral LTR that begins at the beginning of the capping sequence (i.e., at the beginning of transcription) and ends just before the beginning of the poly(A) tract. The R region is also defined as being adjacent to the U3 and U5 regions. The R region serves to allow the transfer of nascent DNA from one end of the genome to the other during reverse transcription.

[0074] In certain embodiments, expression of heterologous sequences in viral vectors is enhanced by incorporating posttranscriptional regulatory elements, efficient polyadenylation sites, and, optionally, transcription termination signals into the vector. Various posttranscriptional regulatory elements can enhance expression of heterologous nucleic acids. Examples include the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE, Zufferey et al., 1999, J. Virol., 73:2886); the posttranscriptional regulatory element present in hepatitis B virus (HPRE) (Smith et al., Nucleic Acids Res. 26(21):4818-4827, 1998); and the like (Liu et al., 1995, Genes Dev., 9:1766). In certain embodiments, vectors contain posttranscriptional regulatory elements such as WPRE or HPRE. In certain embodiments, vectors lack or do not contain posttranscriptional regulatory elements such as WPRE or HPRE.

[0075] Elements that direct efficient termination and polyadenylation of heterologous nucleic acid transcripts can enhance heterologous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. In certain embodiments, vectors contain a polyadenylation sequence 3' of a polynucleotide encoding a molecule (e.g., a protein) to be expressed. The term "poly(A) site" or "poly(A) sequence" refers to a DNA sequence that directs both the termination and polyadenylation of a nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by adding a poly(A) tail to the 3' end of the coding sequence, thus contributing to improved translation efficiency. Certain embodiments may utilize BGHpA or SV40pA. In certain embodiments, preferred artificial expression constructs contain terminator elements. These elements can help increase transcription levels and minimize readthrough from the construct to other plasmid sequences.

[0076] In certain embodiments, the viral vector further comprises one or more insulator elements. Insulator elements can help protect viral vector expression sequences, such as effector elements or expressible elements, from integration site effects (i.e., position effects; see, e.g., Burgess-Beusse et al., PNAS., USA, 99:16433, 2002, and Zhan et al., Hum. Genet., 109:471, 2001), which can result in deregulated expression of the imported sequence, mediated by cis-acting elements present in genomic DNA. In certain embodiments, the viral import vector comprises one or more insulator elements in the 3' LTR, and upon integration of the provirus into the host genome, the provirus replicates the 3' LTR to include one or more insulators in both the 5' LTR and the 3' LTR. Insulators suitable for use in certain embodiments include the chicken β-globin insulator (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).

[0077] Beyond the foregoing description, a wide range of suitable expression vector types is known to those skilled in the art. These can include commercially available expression vectors designed for common recombinant procedures, such as plasmids containing one or more reporter genes and regulatory elements necessary for reporter gene expression in cells. Numerous vectors are commercially available, for example, from Invitrogen, Stratagene, Clontech, etc., and are described in many relevant guides. In certain embodiments, suitable expression vectors include any plasmid, cosmid, or phage construct capable of supporting the expression of a coding gene in mammalian cells, such as the pUC or Bluescript plasmid series.

[0078] Particular embodiments of the vectors disclosed herein include: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0079] Those skilled in the art can readily identify the sequences of subcomponents within a larger vector sequence, and can readily identify them based on the teachings of this disclosure (see Figures 2C-2E). Nucleotides between the identifiable and listed subcomponents reflect restriction enzyme recognition sites used in construct assembly (cloning), and in some cases, additional nucleotides convey no discernible function. These segments of the complete vector sequence can be adjusted based on different cloning strategies and / or vector uses. Generally, short six-nucleotide palindromic sequences reflect vector construction artifacts that are not critical to vector function.

[0080] In certain embodiments, a vector (e.g., AAV) is selected that has a capsid that crosses the blood-brain barrier (BBB). In certain embodiments, the vector is modified to include a capsid that crosses the BBB. Examples of AAVs with 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), and 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 PHP.eB capsid is distinct from and references AAV9, with the amino acids beginning at residue 586: S-AQ-A (SEQ ID NO:1) changed to S-DGTLAVPFK-A (SEQ ID NO:2). In certain embodiments, PHP.eb refers to a sequence such as that specified in Figure 27.

[0081] AAV9 is a naturally occurring AAV serotype that, unlike many other naturally occurring serotypes, can cross the BBB after intravenous injection. It transforms large portions of the central nervous system (CNS), thus enabling minimally invasive treatments, as described, for example, in connection with clinical trials for the treatment of spinal muscular atrophy (SMA) syndrome with AveXis (AVXS-101, NCT03505099) and CLN3 gene-associated neuronal ceroid-lipofusion syndrome (NCT03770572) (Naso et al., BioDrugs. 2017;31(4):317).

[0082] AAVrh.10 was originally isolated from rhesus macaques and has shown low seropositivity in humans compared to other common serotypes used in gene delivery applications (see Selot et al., Front Pharmacol. 2017;8:441), and is being evaluated in the LYS-SAF302, LYSOGENE, and NCT03612869 clinical trials.

[0083] Two variants isolated from a library of chimeric AAV vectors, AAV1R6 and AAV1R7 (in which the AAV1 capsid domain was swapped with AAVrh.10), retain the ability to cross the BBB and transduce the CNS while showing significantly reduced liver and vascular endothelial transduction.

[0084] Similarly, rAAVrh.8 isolated from rhesus macaques demonstrates global transduction of glial and neuronal cell types in clinically important regions after peripheral administration and also exhibits reduced peripheral tissue tropism compared to other vectors.

[0085] AAV-BR1 is an AAV2 variant displaying the NRGTEWD ​​(SEQ ID NO: 3) epitope isolated during in vivo screening of a random AAV-display peptide library. It exhibits high specificity with high transgene expression in the brain and minimal off-target affinity (including the liver) (Korbelin et al., EMBO Mol Med. 2016;8(6):609).

[0086] AAV-PHP.S (Addgene, Watertown, MA) is a CREATE-generated variant of AAV9 that encodes the 7-mer sequence QAVRTSL (SEQ ID NO: 4) and potently transduces neurons within the enteric nervous system and peripheral sensory afferents entering the spinal cord and brainstem.

[0087] AAV-PHP.B (Addgene, Watertown, MA) is a CREATE-generated variant of AAV9 that encodes the 7-mer sequence TLAVPFK (SEQ ID NO: 5). It transfers genes throughout the CNS more efficiently than AAV9, transducing a large proportion of astrocytes and neurons across multiple CNS regions.

[0088] AAV-PPS was created by inserting the DSPAHPS (SEQ ID NO: 6) epitope into the capsid of AAV2, and exhibits dramatically improved brain tropism compared to AAV2.

[0089] For more information regarding capsids crossing the blood-brain barrier, see Chan et al., Nat. Neurosci, 2017 Aug:20(8):1172-1179.

[0090] (ii) Compositions for Administration. The artificial expression constructs and vectors (herein referred to as physiologically active components) of the present disclosure can be formulated with a carrier suitable for administration to cells, tissue slices, animals (e.g., mice, non-human primates), or humans. The physiologically active components in the compositions described herein can be prepared in neutral form, as a free base, or as a pharmacologically acceptable salt.

[0091] Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like, and salts formed with the free carboxyl groups are derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like.

[0092] Carriers for physiologically active ingredients can include solvents, dispersion media, vehicles, coatings, diluents, isotonic and absorption delaying agents, buffers, solutions, suspensions, colloids, etc. The use of such carriers for physiologically active ingredients is well known in the art. Except insofar as any conventional media or agent is incompatible with the physiologically active ingredient, it can be used with the compositions described herein.

[0093] The phrase "pharmaceutically acceptable carrier" refers to a carrier that does not produce an allergic or similar untoward reaction when administered to a human, and in certain embodiments, when administered intravenously (e.g., into the retroorbital plexus).

[0094] In certain embodiments, the compositions can be formulated for intravenous, intraparenchymal, intraocular, intravitreal, parenteral, subcutaneous, intracerebroventricular, intramuscular, intrathecal, intraspinal, intraperitoneal, oral or intranasal inhalation, or for direct injection or application to one or more cells, tissues, or organs.

[0095] The composition may comprise liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, and / or nanoparticles.

[0096] The formation and use of liposomes is generally known to those skilled in the art. Liposomes have been developed with improved serum stability and circulatory half-lives (see, e.g., U.S. Pat. No. 5,741,516). Furthermore, various methods for preparing liposomes and liposome-like preparations as potential drug carriers have been described (see, e.g., U.S. Pat. Nos. 5,567,434; 5,552,157; ​​5,565,213; 5,738,868; and 5,795,587).

[0097] The present disclosure also provides pharmaceutically acceptable nanocapsule formulations of physiologically active ingredients. Nanocapsules generally 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 intracellular polymer overload, such ultrafine particles can be designed using polymers that can be degraded in vivo. Biodegradable polyalkylcyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present disclosure. Such particles can be readily produced as described in Couvreuret al., J Pharm Sci 69(2):199-202, 1980; Couvreuret et al., Crit Rev Ther Drug Carrier Syst. 5(1)1-20, 1988; zur Muhlenet 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. Pat. No. 5,145,684.

[0098] Injectable compositions may include sterile aqueous solutions, or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468). For injectable delivery, the form is sterile and fluid to the extent that it can be delivered by syringe. In certain embodiments, the composition is stable under the conditions of manufacture and storage and, optionally, contains one or more preservatives to protect against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and / or by the use of surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and / or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In various embodiments, the preparation includes isotonic agent(s), such as sugar(s) or sodium chloride. Prolonged absorption of the injectable composition can be achieved by including an agent that delays absorption into the composition, for example, aluminum monostearate and gelatin. The injectable composition can be suitably buffered, if necessary, and the liquid diluent can be first rendered isotonic with sufficient saline or glucose, for example.

[0099] Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. As indicated, under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0100] Sterile compositions can be prepared by incorporating the physiologically active ingredient in an appropriate amount of a solvent containing other optional ingredients (e.g., as described above), followed by filtration sterilization. Generally, dispersions are prepared by incorporating various sterilized physiologically active ingredients into a sterile vehicle containing a basic dispersion medium and other required ingredients (e.g., as described above). In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method can be vacuum drying and freeze-drying technology, which yields a powder of the physiologically active ingredient and any additional desired ingredients from the solution previously sterile-filtered.

[0101] Oral compositions can be in liquid form, for example, as solution, syrup or suspension, or can be presented as a pharmaceutical product to be reconstituted with water or other suitable vehicle before use.Such liquid preparations can be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (for example, lecithin or acacia);non-aqueous vehicles (for example, almond oil, oily esters or fractionated vegetable oils);preservatives (for example, methyl or propyl-p-hydroxybenzoate or sorbic acid). The compositions may take the form of tablets or capsules prepared with pharmaceutically acceptable excipients such as 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); or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods well known in the art.

[0102] Inhalable compositions can be delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve that delivers a metered amount. Capsules and cartridges, for example, of gelatin, for use in an inhaler or filler can be formulated to contain a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0103] Compositions also include microchip devices (U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al., Prog Retin Eye Res, 17(1):33-58, 1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208), and feedback-controlled delivery (U.S. Pat. No. 5,697,899), and any other delivery methods available and / or described elsewhere in this disclosure.

[0104] Supplementary active ingredients can also be incorporated into the compositions.

[0105] Typically, the composition will contain at least 0.1% of the physiologically active ingredient, although the percentage of the physiologically active ingredient may, of course, vary, and may conveniently be 1 or 2% to 70% or 80% or more, or 0.5 to 99% by weight or volume of the total composition. Naturally, the amount of physiologically active ingredient in each physiologically useful composition can be prepared in such a way that a suitable dosage is obtained in any given unit dose of compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be considered by those skilled in the art of preparing such pharmaceutical formulations, and accordingly, various compositions and dosages may be desirable.

[0106] In certain embodiments, for human administration, compositions should meet sterility, pyrogenicity, and general safety and purity standards as required by the U.S. Food and Drug Administration (FDA) or other applicable regulatory agencies in other countries.

[0107] (iii) Cell lines containing the artificial expression constructs. The present disclosure includes cells containing the artificial expression constructs described herein. Cells transformed with artificial expression constructs can be used for many purposes, including neuroanatomical studies, evaluation of functional and / or non-functional proteins, and drug screens to evaluate the regulatory properties of enhancers.

[0108] While a variety of host cell lines can be used, in certain embodiments, the cells are mammalian non-neuronal cells. In certain embodiments, the artificial expression constructs are selected from the group consisting of eHGT_267h, eHGT_268h, eHGT_269h, eHGT_270h, eHGT_271h, eHGT_272h, eHGT_273h, eHGT_274h, eHGT_275h, eHGT_276h, eHGT_315h, eHGT_316h, eHGT_357h, eHGT_371h, eHGT_371m, eHGT_372h, eHGT_372m, eHGT_373m, 3xcore eHGT_373m, eHGT_374m, eHGT_375h, eHGT_375m, eHGT_376h, eHGT_376m, eHGT_3 77h, eHGT_379m, eHGT_380m, eHGT_381h, eHGT_381m, eHGT_382h, eHGT_382m, eHG T_383h, eHGT_383m, eHGT_384h, eHGT_384m, eHGT_385m, eHGT_386m, eHGT_387h , eHGT_387m, eHGT_388h, eHGT_388m, eHGT_389h, eHGT_390h, eHGT_390m, 3xcore eHGT_390m, eHGT_391m, eHGT_392h, eHGT_393h, eHGT_393m, eHGT_394h, eHGT_395h, eHGT_396h, eHGT _396m, eHGT_397h, eHGT_397m, eHGT_398h, eHGT_398m, eHGT_399h, eHGT_399m, eHGT_400h, eHGT_400 m, eHGT_401m, eHGT_402h, eHGT_402m, eHGT_403m, eHGT_404h, eHGT_405h, eHGT_405m, eHGT_406h, eH GT_406m, eHGT_407h, eHGT_641m, eHGT_407m, eHGT_408h, eHGT_408m, eHGT_409m, eHGT_410m, 3xcore eHGT_410m, eHGT_411h, eHGT_411m, eHGT_412h, eHGT_412m, eHGT_413h, eHGT_414h, eHGT_414m, eHGT_415m, eHGT_416m, eHGT_417h, eHGT_417m, eHGT_418h, eHGT_418m,eHGT_419h、eHGT_419m、eHGT_420h、eHGT_420m、eHGT_421m、eHGT_422m、eHGT_423h、eHGT_423m、eHGT_424h、eHGT_424m、eHGT_425h、eHGT_425m、eHGT_426h、eHGT_426m、eHGT_427h、eHGT_427m、eHGT_428h、eHGT_428m、eHGT_429h、eHGT_429m、eHGT_430h、eHGT_430m、eHGT_495m、eHGT_497m、eHGT413m、mscRE1001、mscRE1002、mscRE1003、mscRE1004、mscRE1005、mscRE1006、mscRE1007、mscRE1008、mscRE1009、mscRE1010、mscRE1011、mscRE1012、mscRE1013、mscRE1014、mscRE1015、mscRE1016、mscRE1017、mscRE1018、mscRE1019、mscRE1020、mscRE1021、mscRE1022、mscRE1023、mscRE1024、mscRE1025、mscRE1026、mscRE1027、mscRE1028、mscRE1029、mscRE1030、mscRE1031、mscRE1032、mscRE1033、mscRE1034、mscRE1035、mscRE1036、mscRE1037、mscRE1038、mscRE1039、mscRE1040、mscRE1041、mscRE1042、mscRE1043、mscRE1044、mscRE1045、mscRE1046、mscRE1047、mscRE1048、mscRE1049、mscRE1050、mscRE1051、or mscRE1052 and / or CN1781、CN1782、CN1783、CN1784、CN1785、CN1786、CN1787、CN1788、CN1789、CN1790、CN2044、CN2082、CN2083、CN2084、CN2560、CN2085、CN2086、CN2087、CN2088、CN2089、CN2558、CN2090、CN2091、CN2092、CN2093、CN2094、CN2095、CN2096、CN2097、CN2098、CN2099、CN2100, CN2101, CN2102, CN2103, CN2104, CN2105, CN2106, CN2107, CN2108, CN2109, CN2556, CN2110, CN2111, CN2112, CN2113, CN2114, C N2115, CN2116, CN2117, CN2118, CN2119, CN2120, CN2121, CN2122, CN2123, CN2124, CN2125, CN2126, CN2127, CN2128, CN2129, CN2130, CN2 131, CN2132, CN2133, CN2134, CN2141, CN2142, CN2143, CN2144, CN2145, CN2146, CN2147, CN2148, CN2149, CN2150, CN2151, CN2152, CN21 53, CN2154, CN2155, CN2156, CN2157, CN2158, CN2159, CN2160, CN2161, CN2162, CN2163, CN2164, CN2165, CN2166, CN2167, CN2845, CN2168 , CN2169, CN2170, CN2171, CN2172, CN2173, CN2174, CN2175, CN2176, CN2177, CN2178, CN2179, CN2180, CN2181, CN2182, CN2183, CN2184, CN2243, CN2268, CN2345, CN2346, 3001, 3002, 3003, 3004, 3005, 3006, 3007, 3008, 3009, 3010, 3011, 3012, 3013, 3014, 3015, 3016, 3017, 3 3040, 3041, 3042, 3043, 3044, 3045, 3046, 3047, 3048, 3049, 3050, 3051, or 3052, and the cell line is a human, primate, or murine non-neuronal cell line. Cell lines that can be used for transfection in the present disclosure include primary cell lines derived from biological tissues such as rat or mouse brain,and organic cell cultures, including brain slices from animals such as rats or mice.

[0109] In certain embodiments, non-neuronal cell lines, including mouse embryonic stem cells, can be used. Cultured mouse embryonic stem cells can be used to analyze the expression of gene constructs using transient transfection with a plasmid construct. Mouse embryonic stem cells are pluripotent and undifferentiated. These cells can be maintained in this undifferentiated state by leukemia inhibitory factor (LIF). Withdrawal of LIF induces differentiation of embryonic stem cells. In culture, stem cells form various differentiated cell types. Differentiation is triggered by the expression of tissue-specific transcription factors, allowing the function of enhancer sequences to be evaluated. (See, for example, Fiskerstrand et al., FEBS Lett 458:171-174, 1999.)

[0110] In certain embodiments, "neuronal" describes something that is, relates to, or includes a nerve cell. A nerve cell is defined by the presence of an axon and a dendrite. The term "neuronal-specific" refers to activity that is found or occurs in nerve cells or cells derived from nerve cells, but that is not found, occurs, or substantially not found, or substantially occurs, in non-neuronal cells or cells not derived from nerve cells, e.g., glial cells such as astrocytes or oligodendrocyte cells.

[0111] A method for differentiating stem cells into different cell types involves replacing the stem cell culture medium with a medium containing basic fibroblast growth factor (bFGF), heparin, N2 supplements (e.g., transferrin, insulin, progesterone, putrescine, and selenate), laminin, and polyornithine. A process for producing myelinating oligodendrocyte cells from stem cells is described in Hu, et al., 2009, Nat. Protoc. 4:1614-22. U.S. Publication No. 2012 / 0308530 describes a culture surface with amino groups that promotes differentiation into neurons, astrocytes, and oligodendrocytes. Thus, the fate of stem cells can be controlled by various extracellular factors. Commonly used factors include brain-derived growth 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 Nos. 5,948,428 and 6,001,654); isobutyl methylcellulose (BMP-2); 3-methylxanthine; leukemia inhibitory growth factor (LIF; U.S. Patent No. 6,103,530); somatostatin; amphiregulin; neurotrophins (e.g., cyclic adenosine monophosphate; epidermal growth factor (EGF); dexamethasone (a glucocorticoid hormone); forskolin; GDNF family receptor ligands; potassium; retinoic acid (U.S. Patent No. 6,395,546); tetanus toxin; and transforming growth factor-α and TGF-β (U.S. Patent Nos. 5,851,832 and 5,753,506).

[0112] In certain embodiments, the yeast one-hybrid system is used to identify eHGT_267h, eHGT_268h, eHGT_269h, eHGT_270h, eHGT_271h, eHGT_272h, eHGT_273h, eHGT_274h, eHGT_275h, eHGT_276h, eHGT_315h, eHGT_316h, eHGT_357h, eHGT_371h, eHGT_371m, eHGT_372h, eHGT_372m, eHGT_373m, 3xcore eHGT_373m, eHGT_374m, eHGT_375h, eHGT_375m, eHGT_376h, eHGT_376m, eHGT_3 77h, eHGT_379m, eHGT_380m, eHGT_381h, eHGT_381m, eHGT_382h, eHGT_382m, eHG T_383h, eHGT_383m, eHGT_384h, eHGT_384m, eHGT_385m, eHGT_386m, eHGT_387h , eHGT_387m, eHGT_388h, eHGT_388m, eHGT_389h, eHGT_390h, eHGT_390m, 3xcore eHGT_390m, eHGT_391m, eHGT_392h, eHGT_393h, eHGT_393m, eHGT_394h, eHGT_395h, eHGT_396h, eHGT _396m, eHGT_397h, eHGT_397m, eHGT_398h, eHGT_398m, eHGT_399h, eHGT_399m, eHGT_400h, eHGT_400 m, eHGT_401m, eHGT_402h, eHGT_402m, eHGT_403m, eHGT_404h, eHGT_405h, eHGT_405m, eHGT_406h, eH GT_406m, eHGT_407h, eHGT_641m, eHGT_407m, eHGT_408h, eHGT_408m, eHGT_409m, eHGT_410m, 3xcoreeHGT_410m, eHGT_411h, eHGT_411m, eHGT_412h, eHGT_412m, eHGT_413h, eHGT_414h, eHGT_414m, eHGT_415m, eHGT_416m, eHGT_417h, eHGT_417 m, eHGT_418h, eHGT_418m, eHGT_419h, eHGT_419m, eHGT_420h, eHGT_420m, eHGT_421m, eHGT_422m, eHGT_423h, eHGT_423m, eHGT_424h, eHGT_42 4m, eHGT_425h, eHGT_425m, eHGT_426h, eHGT_426m, eHGT_427h, eHGT_427m, eHGT_428h, eHGT_428m, eHGT_429h, eHGT_429m, eHGT_430h, eHGT_4 30m, eHGT_495m, eHGT_497m, eHGT413m, mscRE1001, mscRE1002, mscRE1003, mscRE1004, mscRE1005, mscRE1006, mscRE1007, mscRE1008, mscRE1 009, mscRE1010, mscRE1011, mscRE1012, mscRE1013, mscRE1014, mscRE1015, mscRE1016, mscRE1017, mscRE1018, mscRE1019, mscRE1020, mscR E1021, mscRE1022, mscRE1023, mscRE1024, mscRE1025, mscRE1026, mscRE1027, mscRE1028, mscRE1029, mscRE1030, mscRE1031, mscRE1032, msc Compounds that inhibit specific protein / DNA interactions, such as transcription factors RE1033, mscRE1034, mscRE1035, mscRE1036, mscRE1037, mscRE1038, mscRE1039, mscRE1040, mscRE1041, mscRE1042, mscRE1043, mscRE1044, mscRE1045, mscRE1046, mscRE1047, mscRE1048, mscRE1049, mscRE1050, mscRE1051, or mscRE1052, may also be identified.

[0113] Transgenic animals are described below. Cell lines can also be derived from such transgenic animals. For example, primary tissue cultures from transgenic mice (e.g., as also described below) can provide cell lines that have artificial expression constructs already integrated into their genomes (see, e.g., MacKenzie and Quinn, Proc Natl Acad Sci USA 96:15251-15255, 1999).

[0114] (iv) Transgenic Animals. Another aspect of the present disclosure includes transgenic animals, the genome of which comprises an artificial expression construct comprising one of: eHGT_267h, eHGT_268h, eHGT_269h, eHGT_270h, eHGT_271h, eHGT_272h, eHGT_273h, eHGT_274h, eHGT_275h, eHGT_276h, eHGT_315h, eHGT_316h, eHGT_357h, eHGT_371h, eHGT_371m, eHGT_372h, eHGT_372m, eHGT_373m, 3xcore eHGT_373m, eHGT_374m, eHGT_375h, eHGT_375m, eHGT_376h, eHGT_376m, eHGT_3 77h, eHGT_379m, eHGT_380m, eHGT_381h, eHGT_381m, eHGT_382h, eHGT_382m, eHG T_383h, eHGT_383m, eHGT_384h, eHGT_384m, eHGT_385m, eHGT_386m, eHGT_387h , eHGT_387m, eHGT_388h, eHGT_388m, eHGT_389h, eHGT_390h, eHGT_390m, 3xcore eHGT_390m, eHGT_391m, eHGT_392h, eHGT_393h, eHGT_393m, eHGT_394h, eHGT_395h, eHGT_396h, eHGT _396m, eHGT_397h, eHGT_397m, eHGT_398h, eHGT_398m, eHGT_399h, eHGT_399m, eHGT_400h, eHGT_400 m, eHGT_401m, eHGT_402h, eHGT_402m, eHGT_403m, eHGT_404h, eHGT_405h, eHGT_405m, eHGT_406h, eH GT_406m, eHGT_407h, eHGT_641m, eHGT_407m, eHGT_408h, eHGT_408m, eHGT_409m, eHGT_410m, 3xcore eHGT_410m, eHGT_411h, eHGT_411m, eHGT_412h, eHGT_412m, eHGT_413h, eHGT_414h, eHGT_414m, eHGT_415m, eHGT_416m, eHGT_417h, eHGT_417m, eHGT_418h,eHGT_418m, eHGT_419h, eHGT_419m, eHGT_420h, eHGT_420m, eHGT_421m, eHGT_422m, eHGT_423h, eHGT_423m, eHGT_424 h, eHGT_424m, eHGT_425h, eHGT_425m, eHGT_426h, eHGT_426m, eHGT_427h, eHGT_427m, eHGT_428h, eHGT_428m, eHGT_4 29h, eHGT_429m, eHGT_430h, eHGT_430m, eHGT_495m, eHGT_497m, eHGT413m, mscRE1001, mscRE1002, mscRE1003, mscRE 1004, mscRE1005, mscRE1006, mscRE1007, mscRE1008, mscRE1009, mscRE1010, mscRE1011, mscRE1012, mscRE1013, msc RE1014, mscRE1015, mscRE1016, mscRE1017, mscRE1018, mscRE1019, mscRE1020, mscRE1021, mscRE1022, mscRE1023, m scRE1024, mscRE1025, mscRE1026, mscRE1027, mscRE1028, mscRE1029, mscRE1030, mscRE1031, mscRE1032, mscRE1033 , mscRE1034, mscRE1035, mscRE1036, mscRE1037, mscRE1038, mscRE1039, mscRE1040, mscRE1041, mscRE1042, mscRE1043, mscRE1044, mscRE1045, mscRE1046, mscRE1047, mscRE1048, mscRE1049, mscRE1050, mscRE1051, or mscRE1052. In certain embodiments, the genome of the transgenic animal is selected from the group consisting of CN1781, CN1782, CN1783, CN1784, CN1785, CN1786, CN1787, CN1788, CN1789, CN1790, CN2044, CN2082, CN2083, CN2084, CN2560, CN2085, CN2086, CN2087, CN2088, CN2089, CN2558, CN2090, CN2091, CN2092, CN2093, CN2094, CN2095,CN2096, CN2097, CN2098, CN2099, CN2100, CN2101, CN2102, CN2103, CN2104, CN2105, CN2106, CN2107, CN2108, CN2109, CN2556, CN2110 , CN2111, CN2112, CN2113, CN2114, CN2115, CN2116, CN2117, CN2118, CN2119, CN2120, CN2121, CN2122, CN2123, CN2124, CN2125, CN212 6, CN2127, CN2128, CN2129, CN2130, CN2131, CN2132, CN2133, CN2134, CN2141, CN2142, CN2143, CN2144, CN2145, CN2146, CN2147, CN21 48, CN2149, CN2150, CN2151, CN2152, CN2153, CN2154, CN2155, CN2156, CN2157, CN2158, CN2159, CN2160, CN2161, CN2162, CN2163, CN2 164, CN2165, CN2166, CN2167, CN2845, CN2168, CN2169, CN2170, CN2171, CN2172, CN2173, CN2174, CN2175, CN2176, CN2177, CN2178, CN 2179, CN2180, CN2181, CN2182, CN2183, CN2184, CN2243, CN2268, CN2345, CN2346, 3001, 3002, 3003, 3004, 3005, 3006, 3007, 3008, 300 9, 3010, 3011, 3012, 3013, 3014, 3015, 3016, 3017, 3018, 3019, 3020, 3021, 3022, 3023, 3024, 3025, 3026, 3027, 3028, 3029, 3030, 3031, 3032, 3033, 3034, 3035, 3036, 3037, 3038, 3039, 3040, 3041, 3042, 3043, 3044, 3045, 3046, 3047, 3048, 3049, 3050, 3051, or 3052. In certain embodiments, when non-integrating vectors are used, the transgenic animal contains within one or more of its cells eHGT_267h, eHGT_268h, eHGT_269h, eHGT_270h, eHGT_271h,eHGT_272h、eHGT_273h、eHGT_274h、eHGT_275h、eHGT_276h、eHGT_315h、eHGT_316h、eHGT_357h、eHGT_371h、eHGT_371m、eHGT_372h、eHGT_372m、eHGT_373m、3xcore eHGT_373m、eHGT_374m、eHGT_375h、eHGT_375m、eHGT_376h、eHGT_376m、eHGT_377h、eHGT_379m、eHGT_380m、eHGT_381h、eHGT_381m、eHGT_382h、eHGT_382m、eHGT_383h、eHGT_383m、eHGT_384h、eHGT_384m、eHGT_385m、eHGT_386m、eHGT_387h、eHGT_387m、eHGT_388h、eHGT_388m、eHGT_389h、eHGT_390h、eHGT_390m、3xcore eHGT_390m、eHGT_391m、eHGT_392h、eHGT_393h、eHGT_393m、eHGT_394h、eHGT_395h、eHGT_396h、eHGT_396m、eHGT_397h、eHGT_397m、eHGT_398h、eHGT_398m、eHGT_399h、eHGT_399m、eHGT_400h、eHGT_400m、eHGT_401m、eHGT_402h、eHGT_402m、eHGT_403m、eHGT_404h、eHGT_405h、eHGT_405m、eHGT_406h、eHGT_406m、eHGT_407h、eHGT_641m、eHGT_407m、eHGT_408h、eHGT_408m、eHGT_409m、eHGT_410m、3xcore eHGT_410m、eHGT_411h、eHGT_411m、eHGT_412h、eHGT_412m、eHGT_413h、eHGT_414h、eHGT_414m、eHGT_415m、eHGT_416m、eHGT_417h、eHGT_417m、eHGT_418h、eHGT_418m、eHGT_419h、eHGT_419m、eHGT_420h、eHGT_420m、eHGT_421m、eHGT_422m、eHGT_423h、eHGT_423m、eHGT_424h、eHGT_424m、eHGT_425h、eHGT_425m、eHGT_426h, eHGT_426m, eHGT_427h, eHGT_427m, eHGT_428h, eHGT_428m, eHGT_429h, eHGT_429m, eHGT_430h, eHGT_430m, eHGT_495m, eHGT_497m, eHGT4 13m, mscRE1001, mscRE1002, mscRE1003, mscRE1004, mscRE1005, mscRE1006, mscRE1007, mscRE1008, mscRE1009, mscRE1010, mscRE1011, mscRE1012, m scRE1013, mscRE1014, mscRE1015, mscRE1016, mscRE1017, mscRE1018, mscRE1019, mscRE1020, mscRE1021, mscRE1022, mscRE1023, mscRE1024, mscRE1 025, mscRE1026, mscRE1027, mscRE1028, mscRE1029, mscRE1030, mscRE1031, mscRE1032, mscRE1033, mscRE1034, mscRE1035, mscRE1036, mscRE1037, m scRE1038, mscRE1039, mscRE1040, mscRE1041, mscRE1042, mscRE1043, mscRE1044, mscRE1045, mscRE1046, mscRE1047, mscRE1048, mscRE1049, mscRE1050, mscRE1051, or mscRE1052 and / or CN1781, CN1782, CN1783, CN1784, CN1785, CN1786, CN1787, CN1788, CN1789, CN1790, CN2044, CN2082, CN2083, CN 2084, CN2560, CN2085, CN2086, CN2087, CN2088, CN2089, CN2558, CN2090, CN2091, CN2092, CN2093, CN2094, CN2095, CN2096, CN2097, CN2098, CN2099, C N2100, CN2101, CN2102, CN2103, CN2104, CN2105, CN2106, CN2107, CN2108, CN2109, CN2556, CN2110, CN2111, CN2112, CN2113, CN2114, CN2115, CN2116,CN2117, CN2118, CN2119, CN2120, CN2121, CN2122, CN2123, CN2124, CN2125, CN2126, CN2127, CN2128, CN2129, CN2130, CN2131, CN2132, CN2133, CN2134, CN2141, , CN2142, CN2143, CN2144, CN2145, CN2146, CN2147, CN2148, CN2149, CN2150, CN2151, CN215 2, CN2153, CN2154, CN2155, CN2156, CN2157, CN2158, CN2159, CN2160, CN2161, CN2162, CN216 3, CN2164, CN2165, CN2166, CN2167, CN2845, CN2168, CN2169, CN2170, CN2171, CN2172, CN21 73, CN2174, CN2175, CN2176, CN2177, CN2178, CN2179, CN2180, CN2181, CN2182, CN2183, CN21 84, CN2243, CN2268, CN2345, CN2346, 3001, 3002, 3003, 3004, 3005, 3006, 3007, 3008, 3009, 3010, 3011, 3012, 3013, 3014, 3015, 3016, 3017, 3018, 3019, 3020, 3021, 3022, 3023, 3024, 30 25, 3026, 3027, 3028, 3029, 3030, 3031, 3032, 3033, 3034, 3035, 3036, 3037, 3038, 3039, 3040, 3041, 3042, 3043, 3044, 3045, 3046, 3047, 3048, 3049, 3050, 3051, or 3052.

[0115] Detailed methods for producing transgenic animals are described in U.S. Patent No. 4,736, 866. The transgenic animals can be of any non-human species, but preferably include non-human primates (NHPs), sheep, horses, cows, pigs, goats, dogs, cats, rabbits, chickens, and rodents, such as guinea pigs, hamsters, gerbils, rats, mice, and ferrets.

[0116] In certain embodiments, the construction of transgenic animals results in organisms with genetically engineered constructs present in all cells within the same genomic integration site.Therefore, cell lines derived from such transgenic animals will be as consistent as if the genetically engineered constructs were present in all cells at the same genomic integration site, and therefore will undergo the same position effect mutations.In contrast, introducing genes into cell lines or primary cell cultures can result in heterologous expression of constructs.The disadvantage of this approach is that the expression of introduced DNA can be affected by the specific genetic background of the host animal.

[0117] As noted above in connection with cell lines, the artificial expression constructs of the present disclosure can be used to genetically modify mouse embryonic stem cells using techniques known in the art. Typically, the artificial expression construct is introduced into cultured mouse embryonic stem cells. Transformed ES cells are then injected into a blastocyst from a host mother, and the host embryo is reimplanted into the mother. This results in chimeric mice whose tissues are composed of cells derived from both the embryonic stem cells present in the cultured cell line and the embryonic stem cells present in the host embryo. Typically, the mice from which the cultured ES cells used for transfer are derived are selected to have a coat color different from that of the host mouse whose embryo is injected with the transformed cells. The chimeric mice then have a variety of coat colors. The chimeric mice are bred with an appropriate strain to produce offspring carrying the transgene, as long as at least some of their germline tissue is derived from the genetically modified cells.

[0118] In addition to the delivery methods described above, the following techniques are also contemplated as alternative methods for delivering artificial expression constructs to target cells or selected tissues and organs of animals, particularly cells, organs, or tissues of vertebrates: ultrasound (e.g., ultrasound as described in U.S. Pat. No. 5,656,016); intraosseous injection (U.S. Pat. No. 5,779,708); microchip devices (U.S. Pat. No. 5,797,898); ophthalmic formulations (Bourlais et al., Prog Retin Eye Res, 17(1):33-58, 1998); transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208); feedback-controlled delivery (U.S. Pat. No. 5,697,899), and other delivery methods available and / or described elsewhere in this disclosure.

[0119] (v) Methods of Use. In certain embodiments, compositions comprising physiologically active ingredients described herein are administered to a subject to produce a physiological effect.

[0120] In certain embodiments, the present disclosure includes the use of the artificial expression constructs described herein to regulate the expression of a heterologous gene partially or completely encoded at a position downstream of an enhancer in an engineered sequence. Accordingly, provided herein are methods of using the disclosed artificial expression constructs in the research, clinical trials, and potential development of pharmaceuticals to prevent, treat, or alleviate symptoms of a disease, dysfunction, or disorder.

[0121] Specific embodiments include eHGT_373m, 3xcore eHGT_373m, eHGT_375m, eHGT_379m, eHGT_372m, eHGT_384m, eHGT_386m, eHGT_390m, 3xcore eHGT_390m, eHGT_371m, eHGT_383m, eHGT_374m, eHGT_381m, eHGT_382m, eHGT_387m, eHGT_388m, eHGT_376m, eHGT_380m, eHGT_385m, eHGT_371h, eHGT_372h, eHGT_375h, eHGT _376h, eHGT_377h, eHGT_381h, eHGT_382h, eHGT_383h, eHGT_384h, eHGT_387h, eHGT_3 88h, eHGT_389h, eHGT_390h, eHGT_357h, eHGT_495m, eHGT_497m, mscRE1001, mscRE100 2, mscRE1003, mscRE1004, mscRE1005, mscRE1006, mscRE1007, eHGT_267h, eHGT_268h, eHGT_269h, eHGT_270h, eHGT_271h, eHGT_272h, eHGT_273h, eHGT_274h, eHGT_275h, eH GT_276h, eHGT_315h, eHGT_316h, eHGT_391m, eHGT_398m, eHGT_402m, eHGT_409m, eHGT _396m, eHGT_393m, eHGT_399m, eHGT_400m, eHGT_405m, eHGT_406m, eHGT_410m, 3xcore eHGT_410m, eHGT_397m, eHGT_401m, eHGT_403m, eHGT_407m, eHGT_408m, eHGT_392h, eHGT_393h , eHGT_394h, eHGT_395h, eHGT_396h, eHGT_397h, eHGT_398h, eHGT_399h, eHGT_400h, eHGT_402h , eHGT_404h, eHGT_405h, eHGT_406h, eHGT_407h, eHGT_641m, eHGT_408h, eHGT413m, eHGT_414m, eHGT_415m, eHGT_416m, eHGT_417m, eHGT_418m, eHGT_419m, eHGT_420m, eHGT_421m, eHGT_423m,eHGT_428m, eHGT_429m, eHGT_430m, eHGT_411m, eHGT_412m, eHGT_422m, eHGT_424m, eHGT_425m, eHGT_426m, eHGT_427m, eHGT_411h, eHGT_412h, eHGT _413h, eHGT_414h, eHGT_417h, eHGT_418h, eHGT_419h, eHGT_420h, eHGT_423h, eHGT_424h, eHGT_425h, eHGT_426h, eHGT_427h, eHGT_428h, eHGT_429h , eHGT_430h, mscRE1023, mscRE1024, mscRE1025, mscRE1026, mscRE1027, mscRE1028, mscRE1029, mscRE1030, mscRE1031, mscRE1032, mscRE1033, mscR E1034, mscRE1035, mscRE1036, mscRE1037, mscRE1038, mscRE1039, mscRE1040, mscRE1041, mscRE1042, mscRE1043, mscRE1044, mscRE1045, mscRE1046 , mscRE1047, mscRE1048, mscRE1049, mscRE1050, mscRE1051, mscRE1052, mscRE1008, mscRE1009, mscRE1010, mscRE1011, mscRE1012, mscRE1013, mscRE1014, mscRE1015, mscRE1016, mscRE1017, mscRE1018, mscRE1019, mscRE1020, mscRE1021, or mscRE1022, and / or CN1781, CN1782, CN1783, CN1784, CN 1785, CN1786, CN1787, CN1788, CN1789, CN1790, CN2044, CN2082, CN2083, CN2084, CN2560, CN2085, CN2086, CN2087, CN2088, CN2089, CN2558, CN2090, C N2091, CN2092, CN2093, CN2094, CN2095, CN2096, CN2097, CN2098, CN2099, CN2100, CN2101, CN2102, CN2103, CN2104, CN2105, CN2106, CN2107, CN2108,CN2109, CN2556, CN2110, CN2111, CN2112, CN2113, CN2114, CN2115, CN2116, CN2117, CN2118, CN2119, CN2120, CN2121, CN2122, CN2123, CN2124, CN2125, CN2126, CN2127, CN2128, CN2129, CN2130, CN2131, CN2132, CN2133, CN2134, CN2141, CN2142, CN2143, CN2144, CN2145, CN2146, CN2147, CN2148, CN2149, CN2150, CN2151, CN2152, CN2153, CN2154, CN2155, CN2156, CN2157, CN2158, CN2159, CN2160, CN2161, CN2162, CN2163, CN2164, CN2165, CN2166, CN2167, CN2845, CN2168, CN2169, CN2170, CN2171, CN2172, C N2173, CN2174, CN2175, CN2176, CN2177, CN2178, CN2179, CN2180, CN2181, CN2182, CN2183, CN2184, CN2243, CN2268, CN2345, C N2346, 3001, 3002, 3003, 3004, 3005, 3006, 3007, 3008, 3009, 3010, 3011, 3012, 3013, 3014, 3015, 3016, 3017, 3018, 3019, 3020 , 3021, 3022, 3023, 3024, 3025, 3026, 3027, 3028, 3029, 3030, 3031, 3032, 3033, 3034, 3035, 3036, 3037, 3038, 3039, 3040, 3041, 3042, 3043, 3044, 3045, 3046, 3047, 3048, 3049, 3050, 3051, or 3052 to a subject to drive selective expression of a gene in a selected cell type. The subject can be an isolated cell, a cell network, a tissue slice, a laboratory animal, a veterinary animal, or a human.

[0122] As is well known in the medical arts, the dosage for any one subject will depend on many factors, including the subject's size, surface area, age, the particular compound being administered, sex, time and route of administration, general health, and other drugs being administered concomitantly. Doses of compounds of the present disclosure will vary, but in certain embodiments, the dosage will be approximately 10 times the dosage of an artificial expression construct of the present disclosure. 5 ~10 100 In certain embodiments, patients receiving intravenous, intraparenchymal, intraspinal, retro-orbital, or intrathecal administration may receive 10 6 ~10 22 A copy of the artificial expression construct can be injected.

[0123] An "effective amount" is the amount of a composition required to produce a desired physiological change in a subject. Effective amounts are often administered for research purposes. The effective amounts disclosed herein may produce statistically significant effects in animal models or in vitro assays.

[0124] The amount of expression construct and the time of administration of such compositions will be within the purview of one of ordinary skill in the art having the benefit of the present teachings. However, administration of an effective amount of the disclosed compositions can likely be achieved by a single administration, such as, for example, a single injection of a sufficient number of infectious particles to produce an effect in a subject. Alternatively, in some situations, it may be desirable to provide multiple or sequential administrations of an artificial expression construct composition or other genetic construct over either a relatively short or relatively long period of time, as may be determined by the individual overseeing the administration of such compositions. For example, the number of infectious particles administered to a mammal may be as little as 10 or as many as 10, given as a single dose or divided into two or more administrations, as may be required to achieve the intended effect. 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13, or even higher infectious particles / ml. Indeed, in certain embodiments, it may be desirable to administer two or more different expression constructs in combination to achieve a desired effect.

[0125] In certain circumstances, it may be desirable to deliver the artificial expression construct in an appropriately formulated composition as disclosed herein either by pipette, retro-orbital injection, subcutaneous, intraocular, intravitreal, parenteral, subcutaneous, intravenous, intraparenchymal, intracerebroventricular, intramuscular, intrathecal, intraspinal, intraperitoneal, oral or nasal inhalation, or by direct application or injection into one or more cells, tissues, or organs. Methods of administration may also include those forms described in U.S. Patent Nos. 5,543,158, 5,641,515, and 5,399,363.

[0126] (vi) Kits and Commercial Packages. Kits and commercial packages contain the artificial expression constructs described herein. The artificial expression constructs can be sequestered. In certain embodiments, components of the expression product can be sequestered from one another. In certain embodiments, the expression product can be in a vector, a viral vector, a cell, a tissue slice or sample, and / or a transgenic animal. Such kits can further include one or more reagents, restriction enzymes, peptides, therapeutic agents, pharmaceutical compounds, or a means of delivery of the composition, such as a syringe, injection, etc.

[0127] Kit or commercial package embodiments also include instructions for use of the included components, for example, in basic research, electrophysiological studies, neuroanatomical studies, and / or in the study and / or treatment of disorders, diseases, or conditions.

[0128] The following illustrative embodiments and experimental examples are included to demonstrate specific embodiments of the present disclosure. Those of skill in the art will, in light of the present disclosure, appreciate that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0129] (vii) Exemplary embodiments. 1. Artificial expression constructs comprising: (i) 3xcore eHGT_390m, eHGT_390m, eHGT_373m, 3xcore eHGT_373m, eHGT_375m, eHGT_379m, eHGT_372m, eHGT_384m, eHGT_386m, eHGT_371m, eHGT_383m, eHGT_374m, eHGT_381m, eHGT_382m, eHGT_387m, eHGT_388m, eHGT_376m, eHGT_380m, eHGT_3 85m, eHGT_371h, eHGT_372h, eHGT_375h, eHGT_376h, eHGT_377h, eHGT_381h, eHGT_382h, eHGT _383h, eHGT_384h, eHGT_387h, eHGT_388h, eHGT_389h, eHGT_390h, eHGT_357h, eHGT_495m, eHG T_497m, mscRE1001, mscRE1002, mscRE1003, mscRE1004, mscRE1005, mscRE1006, mscRE1007, e HGT_267h, eHGT_268h, eHGT_269h, eHGT_270h, eHGT_271h, eHGT_272h, eHGT_273h, eHGT_274h , eHGT_275h, eHGT_276h, eHGT_315h, eHGT_316h, eHGT_391m, eHGT_398m, eHGT_402m, eHGT_40 9m, eHGT_396m, eHGT_393m, eHGT_399m, eHGT_400m, eHGT_405m, eHGT_406m, eHGT_410m, 3xcore eHGT_410m, eHGT_397m, eHGT_401m, eHGT_403m, eHGT_407m, eHGT_408m, eHGT_392h, eHGT_39 3h, eHGT_394h, eHGT_395h, eHGT_396h, eHGT_397h, eHGT_398h, eHGT_399h, eHGT_400h, eHGT _402h, eHGT_404h, eHGT_405h, eHGT_406h, eHGT_407h, eHGT_641m, eHGT_408h, eHGT413m, eH GT_414m, eHGT_415m, eHGT_416m, eHGT_417m, eHGT_418m, eHGT_419m, eHGT_420m, eHGT_421m,eHGT_423m, eHGT_428m, eHGT_429m, eHGT_430m, eHGT_411m, eHGT_412m, eHGT_422m, eHGT_424m, eHGT_425m, eHGT_4 26m, eHGT_427m, eHGT_411h, eHGT_412h, eHGT_413h, eHGT_414h, eHGT_417h, eHGT_418h, eHGT_419h, eHGT_420h, eHG T_423h, eHGT_424h, eHGT_425h, eHGT_426h, eHGT_427h, eHGT_428h, eHGT_429h, eHGT_430h, mscRE1023, mscRE1024 , mscRE1025, mscRE1026, mscRE1027, mscRE1028, mscRE1029, mscRE1030, mscRE1031, mscRE1032, mscRE1033, mscRE1 034, mscRE1035, mscRE1036, mscRE1037, mscRE1038, mscRE1039, mscRE1040, mscRE1041, mscRE1042, mscRE1043, ms cRE1044, mscRE1045, mscRE1046, mscRE1047, mscRE1048, mscRE1049, mscRE1050, mscRE1051, mscRE1052, mscRE1008 (ii) an enhancer selected from mscRE1009, mscRE1010, mscRE1011, mscRE1012, mscRE1013, mscRE1014, mscRE1015, mscRE1016, mscRE1017, mscRE1018, mscRE1019, mscRE1020, mscRE1021, and mscRE1022; (ii) a promoter; and (iii) a heterologous coding sequence. 2. The artificial expression construct of embodiment 1, wherein the heterologous coding sequence encodes an effector element or an expressible element. 3. The artificial expression construct of embodiment 2, wherein the effector element comprises a reporter protein or functional molecule. 4. The artificial expression construct of embodiment 3, wherein the reporter protein comprises a fluorescent protein. 5. The artificial expression construct of embodiment 3 or 4, wherein said functional molecule comprises a functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cellular transport protein, signaling molecule, neurotransmitter, calcium reporter, channelrhodopsin, CRISPR / CAS molecule, editase, guide RNA molecule, microRNA, homologous recombination donor cassette, or designer receptor activated exclusively by designer drugs (DREADD). 6. The artificial expression construct of embodiment 2, wherein the expressible element comprises a non-functional molecule. 7. The artificial expression construct of embodiment 6, wherein said non-functional molecule comprises a non-functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cellular transport protein, signaling molecule, neurotransmitter, calcium reporter, channelrhodopsin, CRISPR / CAS molecule, editase, guide RNA molecule, microRNA, homologous recombination donor cassette, or DREADD. 8. The artificial expression construct of any one of embodiments 1 to 7, wherein the artificial expression construct is associated with a capsid that crosses the blood-brain barrier. 9. The artificial expression construct of embodiment 8, wherein the capsid comprises PHP.eB, AAV-BR1, AAV-PHP.S, AAV-PHP.B, or AAV-PPS. 10. An artificial expression construct according to any one of embodiments 1 to 9, wherein said artificial expression construct comprises or encodes a skipping element. 11. The artificial expression construct of embodiment 10, wherein the skipping element comprises a 2A peptide and / or an internal ribosome entry site (IRES). 12. The artificial expression construct of embodiment 11, wherein said 2A peptide is selected from T2A, P2A, E2A, or F2A. 13. The artificial expression construct according to any one of embodiments 1 to 12, wherein the artificial expression construct comprises a minimal promoter, post-regulatory elements, and / or insulators (e.g., SP10 or 3xSP10). 14. The artificial expression construct is selected from the group consisting of eHGT_373m, 3xcore eHGT_373m, eHGT_375m, eHGT_379m, eHGT_372m, eHGT_384m, eHGT_386m, eHGT_390m, 3xcore eHGT_390m, eHGT_371m, eHGT_383m, eHGT_374m, eHGT_381m, eHGT_382m, eHGT_387m, eHGT_388m, eHGT_376m, eHGT_380m, eHGT_385m, eHGT_371h, eHGT_372h, eHGT_375h, eHGT _376h, eHGT_377h, eHGT_381h, eHGT_382h, eHGT_383h, eHGT_384h, eHGT_387h, eHGT_3 88h, eHGT_389h, eHGT_390h, eHGT_357h, eHGT_495m, eHGT_497m, mscRE1001, mscRE100 2, mscRE1003, mscRE1004, mscRE1005, mscRE1006, mscRE1007, eHGT_267h, eHGT_268h, eHGT_269h, eHGT_270h, eHGT_271h, eHGT_272h, eHGT_273h, eHGT_274h, eHGT_275h, eH GT_276h, eHGT_315h, eHGT_316h, eHGT_391m, eHGT_398m, eHGT_402m, eHGT_409m, eHGT _396m, eHGT_393m, eHGT_399m, eHGT_400m, eHGT_405m, eHGT_406m, eHGT_410m, 3xcore eHGT_410m, eHGT_397m, eHGT_401m, eHGT_403m, eHGT_407m, eHGT_408m, eHGT_392h, eHGT_39 3h, eHGT_394h, eHGT_395h, eHGT_396h, eHGT_397h, eHGT_398h, eHGT_399h, eHGT_400h, eHGT _402h, eHGT_404h, eHGT_405h, eHGT_406h, eHGT_407h, eHGT_641m, eHGT_408h, eHGT413m, eH GT_414m, eHGT_415m, eHGT_416m, eHGT_417m, eHGT_418m, eHGT_419m, eHGT_420m, eHGT_421m,eHGT_423m, eHGT_428m, eHGT_429m, eHGT_430m, eHGT_411m, eHGT_412m, eHGT_422m, eHGT_424m, eHGT_425m, eHGT_4 26m, eHGT_427m, eHGT_411h, eHGT_412h, eHGT_413h, eHGT_414h, eHGT_417h, eHGT_418h, eHGT_419h, eHGT_420h, eH GT_423h, eHGT_424h, eHGT_425h, eHGT_426h, eHGT_427h, eHGT_428h, eHGT_429h, eHGT_430h, mscRE1023, mscRE102 4, mscRE1025, mscRE1026, mscRE1027, mscRE1028, mscRE1029, mscRE1030, mscRE1031, mscRE1032, mscRE1033, mscRE 1034, mscRE1035, mscRE1036, mscRE1037, mscRE1038, mscRE1039, mscRE1040, mscRE1041, mscRE1042, mscRE1043, m scRE1044, mscRE1045, mscRE1046, mscRE1047, mscRE1048, mscRE1049, mscRE1050, mscRE1051, mscRE1052, mscRE100 8, mscRE1009, mscRE1010, mscRE1011, mscRE1012, mscRE1013, mscRE1014, mscRE1015, mscRE1016, mscRE1017, mscR E1018, mscRE1019, mscRE1020, mscRE1021, mscRE1022, AAV, scAAV, rAAv, minBglobin, CMV, minCMV, minRho, minRho, * 14. The artificial expression construct of any one of embodiments 1 to 13, comprising or encoding a set of features selected from: a fluorescent protein (e.g., EGFP, SYFP, GFP), Cre, iCre, dgCre, FlpO, tTA2, SP10, 3XSP10, WPRE, and / or BGHpA. 15. The artificial expression construct is: eHGT_373m-minBglobin-SYFP2-WPRE3-BGHpA; 3xCore_eHGT_373m-minBglobin-SYFP2-WPRE3-BGHpA; eHGT_375m-minBglobin-SYFP2-WPRE3-BGHpA; eHGT_379m-minBglobin-SYFP2-WPRE3-BGHpA; eHGT_372m-minBglobin-SYFP2-WPRE3-BGHpA; eHGT_384m-minBglobin-SYFP 2-WPRE3-BGHpA;eHGT_386m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_390m-minBglobin-SYFP2-WPRE3-BGHpA;3xCore2_eHGT_390m-minBglobin -SYFP2-WPRE3-BGHpA;eHGT_371m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_391m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_398m-minBglobin-SYF P2-WPRE3-BGHpA;eHGT_402m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_409m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_413m-minBglobin-SYFP2- WPRE3-BGHpA;eHGT_414m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_415m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_383m-minBglobin-SYFP2-WPRE 3-BGHpA;eHGT_374m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_396m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_381m-minBglobin-SYFP2-WPRE3-BG HpA;eHGT_382m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_387m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_388m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_393m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_399m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_400m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_405m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_406m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_410m-minBglobin-SYFP2-WPRE3-BGHpA;3xCore_eHGT_410m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_416m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_417m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_418m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_419m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_420m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_421m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_423m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_428m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_429m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_430m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_376m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_380m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_385m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_397m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_401m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_403m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_407m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_408m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_411m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_412m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_422m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_424m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_425m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_426m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_427m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_371h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_372h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_375h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_376h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_377h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_381h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_382h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_383h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_384h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_387h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_388h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_389h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_390h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_392h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_393h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_394h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_395h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_396h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_397h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_398h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_399h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_400h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_402h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_404h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_405h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_406h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_407h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_641m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_408h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_411h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_412h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_413h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_414h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_417h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_418h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_419h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_420h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_423h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_424h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_425h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_426h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_427h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_428h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_429h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_430h-minBglobin-SYFP2-WPRE3-BGHpA;hsA2-eHGT_267h-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_268h-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_269h-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_270h-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_271h-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_272h-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_273h-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_274h-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_275h-minRho-SYFP2-WPRE3-BGHpA;hsA2-eHGT_276h-minRho-SYFP2-WPRE3-BGHpA;eHGT_315h-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_316h-minBglobin-SYFP2-WPRE3-BGHpA;3xSP10ins-eHGT_357h-minRho*-SYFP2-WPRE3-BGHpA;eHGT_495m-minBglobin-SYFP2-WPRE3-BGHpA;eHGT_497m-minBglobin-SYFP2-WPRE3-BGHpA;mscRE1001-minBGlobin-FlpO-WPRE-BGHpA;mscRE1002-minBGlobin-FlpO-WPRE-BGHpA;mscRE1003-minBGlobin-FlpO-WPRE-BGHpA;mscRE1004-minBGlobin-FlpO-WPRE-BGHpA;mscRE1005-minBGlobin-FlpO-WPRE-BGHpA;mscRE1006-minBGlobin-FlpO-WPRE-BGHpA;mscRE1007-minBGlobin-FlpO-WPRE-BGHpA;mscRE1008-minBGlobin-FlpO-WPRE-BGHpA;mscRE1009-minBGlobin-FlpO-WPRE-BGHpA;mscRE1010-minBGlobin-FlpO-WPRE-BGHpA;mscRE1011-minBGlobin-FlpO-WPRE-BGHpA;mscRE1012-minBGlobin-FlpO-WPRE-BGHpA;mscRE1013-minBGlobin-FlpO-WPRE-BGHpA;mscRE1014-minBGlobin-FlpO-WPRE-BGHpA;mscRE1015-minBGlobin-FlpO; -WPRE-BGHpA;mscRE1016-minBGlobin-FlpO-WPRE-BGHpA;mscRE1017-minBGlobin-FlpO-WPRE-BGHpA;mscRE1018-minBGlobin-FlpO-WPRE-BGHpA;mscRE1019-minBGlobin-FlpO-WPRE-BGHpA;mscRE1020-minBGlobin-FlpO-WPRE-BGHpA;mscRE1021-minBGlobin-FlpO-WPRE-BGHpA;mscRE1022-minBGlobin-FlpO-WPRE-BGHpA;mscRE1023-minBGlobin-FlpO-WPRE-BGHpA;mscRE1024-minBGlobin-FlpO-WPRE-BGHpA;mscRE1025-minBGlobin-FlpO-WPRE-BGHpA;mscRE1026-minBGlobin-FlpO-WPRE-BGHpA;mscRE1027-minBGlobin-FlpO-WPRE-BGHpA;mscRE1028-minBGlobin-FlpO-WPRE-BGHpA;mscRE1029-minBGlobin-FlpO-WPRE-BGHpA;mscRE1030-minBGlobin-FlpO-WPRE-BGHpA;mscRE1031-minBGlobin-FlpO-WPRE-BGHpA;mscRE1032-minBGlobin-FlpO-WPRE-BGHpA;mscRE1033-minBGlobin-FlpO-WPRE-BGHpA;mscRE1034-minBGlobin-FlpO-WPRE-BGHpA;mscRE1035-minBGlobin-FlpO-WPRE-BGHpA;mscRE1036-minBGlobin-FlpO-WPRE-BGHpA;mscRE1037-minBGlobin-FlpO-WPRE-BGHpA;mscRE1038-minBGlobin-FlpO-WPRE-BGHpA;mscRE1039-minBGlobin-FlpO-WPRE-BGHpA;mscRE1040-minBGlobin-FlpO-WPRE-BGHpA;mscRE1041-minBGlobin-FlpO-WPRE-BGHpA;mscRE1042-minBGlobin-FlpO-WPRE-BGHpA;mscRE1043-minBGlobin-FlpO-WPRE-BGHpA;mscRE1044-minBGlobin-FlpO-WPRE-BGHpA;mscRE 1045-minBGlobin-FlpO-WPRE-BGHpA;mscRE1046-minBGlobin-FlpO-WPRE-BGHpA;mscRE1047-minBGlobin-FlpO-WPRE-BGHpA;mscRE1048- 15. An artificial expression construct according to any one of embodiments 1 to 14, comprising or encoding a set of features selected from: minBGlobin-FlpO-WPRE-BGHpA; mscRE1049-minBGlobin-FlpO-WPRE-BGHpA; mscRE1050-minBGlobin-FlpO-WPRE-BGHpA; mscRE1051-minBGlobin-FlpO-WPRE-BGHpA, or mscRE1052-minBGlobin-FlpO-WPRE-BGHpA; 16. The enhancer-promoter combination of embodiment 15 driving expression of a heterologous coding sequence. 17. The vector of embodiment 15, wherein the vector is a viral vector. 18. The vector of embodiment 17, wherein the viral vector is a recombinant adeno-associated viral (AAV) vector. 19. An adeno-associated virus (AAV) vector comprising at least one heterologous coding sequence, wherein the heterologous coding sequence is selected from the group consisting of eHGT_373m, 3xcore eHGT_373m, eHGT_375m, eHGT_379m, eHGT_372m, eHGT_384m, eHGT_386m, eHGT_390m, 3xcore eHGT_390m, eHGT_371m, eHGT_383m, eHGT_374m, eHGT_381m, eHGT_382m, eHGT_387m, eHGT_388m, eHGT_376m, eHGT_380m, eHGT_385m, eHGT_371h, eHGT_372h, eHGT_375h, eHGT _376h, eHGT_377h, eHGT_381h, eHGT_382h, eHGT_383h, eHGT_384h, eHGT_387h, eHGT_3 88h, eHGT_389h, eHGT_390h, eHGT_357h, eHGT_495m, eHGT_497m, mscRE1001, mscRE100 2, mscRE1003, mscRE1004, mscRE1005, mscRE1006, mscRE1007, eHGT_267h, eHGT_268h, eHGT_269h, eHGT_270h, eHGT_271h, eHGT_272h, eHGT_273h, eHGT_274h, eHGT_275h, eH GT_276h, eHGT_315h, eHGT_316h, eHGT_391m, eHGT_398m, eHGT_402m, eHGT_409m, eHGT _396m, eHGT_393m, eHGT_399m, eHGT_400m, eHGT_405m, eHGT_406m, eHGT_410m, 3xcore eHGT_410m, eHGT_397m, eHGT_401m, eHGT_403m, eHGT_407m, eHGT_408m, eHGT_39 2h, eHGT_393h, eHGT_394h, eHGT_395h, eHGT_396h, eHGT_397h, eHGT_398h, eHGT _399h, eHGT_400h, eHGT_402h, eHGT_404h, eHGT_405h, eHGT_406h, eHGT_407h, e HGT_641m, eHGT_408h, eHGT413m, eHGT_414m, eHGT_415m, eHGT_416m, eHGT_417m,eHGT_418m, eHGT_419m, eHGT_420m, eHGT_421m, eHGT_423m, eHGT_428m, eHGT_429m, eHGT_430m, eHGT_411m, eHGT_412m , eHGT_422m, eHGT_424m, eHGT_425m, eHGT_426m, eHGT_427m, eHGT_411h, eHGT_412h, eHGT_413h, eHGT_414h, eHGT_417h , eHGT_418h, eHGT_419h, eHGT_420h, eHGT_423h, eHGT_424h, eHGT_425h, eHGT_426h, eHGT_427h, eHGT_428h, eHGT_429 h, eHGT_430h, mscRE1023, mscRE1024, mscRE1025, mscRE1026, mscRE1027, mscRE1028, mscRE1029, mscRE1030, mscRE103 1, mscRE1032, mscRE1033, mscRE1034, mscRE1035, mscRE1036, mscRE1037, mscRE1038, mscRE1039, mscRE1040, mscRE10 41, mscRE1042, mscRE1043, mscRE1044, mscRE1045, mscRE1046, mscRE1047, mscRE1048, mscRE1049, mscRE1050, mscRE10 The vector is under the control of a promoter and enhancer selected from mscRE1051, mscRE1052, mscRE1008, mscRE1009, mscRE1010, mscRE1011, mscRE1012, mscRE1013, mscRE1014, mscRE1015, mscRE1016, mscRE1017, mscRE1018, mscRE1019, mscRE1020, mscRE1021, and mscRE1022. 20. A transgenic cell comprising an artificial expression construct or vector according to any one of the preceding embodiments. 21. The transgenic cell of embodiment 20, wherein the transgenic cell is an astrocyte, an oligodendrocyte, a microglial cell, a pericyte, an SMC, or an endothelial cell. 22. The transgenic cell of embodiment 20, wherein the transgenic cell is an L1 interlaminar astrocyte. 23. A non-human transgenic animal comprising an artificial expression construct, vector, or transgenic cell according to any one of the preceding embodiments. 24. The non-human transgenic animal of embodiment 23, wherein said non-human transgenic animal is a mouse or a non-human primate. 25. An administrable composition comprising an artificial expression construct, vector, or transgenic cell according to any one of the preceding embodiments. 26. A kit comprising an artificial expression construct, vector, transgenic cell, transgenic animal, and / or administrable composition according to any one of the preceding embodiments. 27. A method for selectively expressing a heterologous gene in a cell population in vivo or in vitro, comprising providing to a sample or subject comprising a population of non-neuronal cells an administrable composition of embodiment 25 in a sufficient dosage and for a sufficient period of time, thereby selectively expressing said gene in the population of non-neuronal cells. 28. The method of embodiment 27, wherein the heterologous gene encodes an effector element or an expressible element. 29. The method of embodiment 28, wherein the effector element comprises a reporter protein or a functional molecule. 30. The method of embodiment 29, wherein the reporter protein comprises a fluorescent protein. 31. The method of embodiment 29 or 30, wherein the functional molecule comprises a functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cellular transport protein, signaling molecule, neurotransmitter, calcium reporter, channelrhodopsin, CRISPR / CAS molecule, editase, guide RNA molecule, microRNA, homologous recombination donor cassette, or DREADD. 32. The method of embodiment 28, wherein the expressible element comprises a non-functional molecule. 33. The method of embodiment 32, wherein the non-functional molecule comprises a non-functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cellular transport protein, signaling molecule, neurotransmitter, calcium reporter, channelrhodopsin, CRISPR / CAS molecule, editase, guide RNA molecule, microRNA, homologous recombination donor cassette, or DREADD. 34. The method of any one of embodiments 27 to 33, wherein said providing comprises pipetting. 35. The method of embodiment 34, wherein the pipetting is performed on a brain slice. 36. The method of embodiment 35, wherein the brain slice comprises astrocytes, oligodendrocytes, microglial cells, pericytes, SMCs, and / or endothelial cells. 37. The method of embodiment 35, wherein the brain slice comprises L1 interlaminar astrocytes. 38. The method of any one of embodiments 35 to 37, wherein the brain slice is mouse, human, or non-human primate. 39. The method of any one of embodiments 27 to 33, wherein said providing comprises administering to a living organism. 40. The method of embodiment 39, wherein the organism is a human, a non-human primate, or a mouse. 41. The method according to any one of embodiments 39 or 40, wherein administration to a living organism is by injection. 42. The method of embodiment 41, wherein said injection comprises intravenous injection, intraparenchymal injection into brain tissue, intracerebroventricular (ICV) injection, intracisternal (ICM) injection, or intrathecal injection. 43.CN1781、CN1782、CN1783、CN1784、CN1785、CN1786、CN1787、CN1788、CN1789、CN1790、CN2044、CN2082、CN2083、CN2084、CN2560、CN2085、CN2086、CN2087、CN2088、CN2089、CN2558、CN2090、CN2091、CN2092、CN2093、CN2094、CN2095、CN2096、CN2097、CN2098、CN2099、CN2100、CN2101、CN2102、CN2103、CN2104、CN2105、CN2106、CN2107、CN2108、CN2109、CN2556、CN2110、CN2111、CN2112、CN2113、CN2114、CN2115、CN2116、CN2117、CN2118、CN2119、CN2120、CN2121、CN2122、CN2123、CN2124、CN2125、CN2126、CN2127、CN2128、CN2129、CN2130、CN2131、CN2132、CN2133、CN2134、CN2141、CN2142、CN2143、CN2144、CN2145、CN2146、CN2147、CN2148、CN2149、CN2150、CN2151、CN2152、CN2153、CN2154、CN2155、CN2156、CN2157、CN2158、CN2159、CN2160、CN2161、CN2162、CN2163、CN2164、CN2165、CN2166、CN2167、CN2845、CN2168、CN2169、CN2170、CN2171、CN2172、CN2173、CN2174、CN2175、CN2176、CN2177、CN2178、CN2179、CN2180、CN2181、CN2182、CN2183、CN2184、CN2243、CN2268、CN2345、CN2346、3001、3002、3003、3004、3005、3006、3007、3008、3009、3010、3011、3012、3013、3014、3015、3016、3017、3018、3019、3020、3021、3022、3023、3024、3025、3026、3027、3028、3029、3030、3031、3032、3033、3034、3035、3036、3037、An artificial expression construct comprising 3038, 3039, 3040, 3041, 3042, 3043, 3044, 3045, 3046, 3047, 3048, 3049, 3050, 3051, or 3052. 44. The embodiment of any one of the preceding embodiments, wherein the concatemer comprises an enriched core of the referenced enhancer, or a core thereof, and the concatemer comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the referenced enhancer or core thereof (representative examples include 3xcore eHGT_410m, 3xcore eHGT_390m, and 3xcore eHGT_373m, which are specifically disclosed herein, and the teachings thereof are applicable to the remainder of the disclosed enhancer sequences).

[0130] (viii) Experimental Methods. Cloning Enhancers. Enhancers were selected from the Open Chromatin Database for cloning by the following criteria: 1) subclass-specific ATAC-seq peaks (with -region flags) identified in Homer for both human and mouse (conserved) or human only (divergent), 2) subclass-specific DMRs in both human and mouse (conserved) or human only (divergent), 3) ranking by human ATAC-seq read count within the region, and 4) manual confirmation by visualization of read accumulation. For the rAAV (ssAAV) vector, a plasmid backbone from Addgene was used (plasmid number 51084 (AAV-hSyn1-GCaMP6s-P2A-nls-dTomato). This construct itself was originally derived from pAAV-GFP [Cell Biolabs catalog # VPK-410]). The enhancer was inserted upstream of a minimal beta-globin promoter and the reporter SYFP2, a bright EGFP surrogate that is well tolerated in neurons, using standard Gibson assembly techniques. NEB stable cells (New England Biolabs # C3040I) or Stbl3 cells (Thermo Fisher # C7373-03) were used for transformation and cultured at 32°C. No left-side ITR recombination was observed with the rAAV plasmids.

[0131] Virus production. The enhancer-AAV plasmid was maxiprepped and transfected into 15 cm plates of AAV-293 cells (Cell Biolabs catalog # AAV-100) using PEI Max 40K (Polysciences Inc, catalog # 24765-1) along with the helper plasmid pHelper (Cell Biolabs) and the PHP.eB rep / cap packaging plasmid (Chan et al., Nat Neurosci. 2017 Aug;20(8):1172-1179. doi:10.1038 / nn.4593. Epub 2017 Jun 26. PMID:28671695; PMCID:PMC5529245). The total mass was 150 μg PEI Max 40K, 30 μg pHelper, 15 μg rep / cap plasmid, and 15 μg enhancer-AAV vector. The next day, the medium was changed to 1% FBS. Five days later, cells and supernatants were harvested and AAV particles were released by three freeze-thaw cycles. The lysates were treated with benzonase to degrade free DNA (2 μL of benzonase at 37°C for 30 min, MilliporeSigma catalog no. E8263-25KU), followed by a low-speed spin (1500 g for 10 min) to remove cellular debris. The virus-containing supernatant was concentrated to a final volume of 150 μL via a 100 kDa molecular weight cutoff Centricon column (MilliporeSigma catalog no. Z648043). For highly purified large-scale preps, this protocol was modified to transfect 10 plates, harvest them together 3 days post-transfection, and then purify the crude virus by iodixanol gradient centrifugation.

[0132] Mouse virus testing. Mice were inoculated with 10 μL (2–3 × 10) of crude virus preparation diluted in 100 μL of PBS. 11Mice were injected retroorbitally with 1000 copies of 4% PFA in PBS at P42-P70 and subsequently sacrificed 21-28 days post-infection. Mouse brain hemispheres were immersion fixed in 4% PFA in PBS for 4-6 hours at 4°C and then sliced ​​into 350µm sagittal slices on a Leica VT1000S vibratome. Fluorescence detection across the entire sagittal section was performed using a 10x montage on an Olympus FV3000 confocal microscope, and stitched images were analyzed using ImageJ. Single-cell RNA-seq from mouse visual cortex was performed as previously described (Tasic et al., Curr. Opin. Neurobiol. 50, 242-249 (2018)).

[0133] Multiplexed FISH (mFISH) by hybridization chain reaction was performed on fixed primate brain slices by immersing them in 4% PFA in PBS at 4°C for 4–6 hours. After fixation, the hemispheres were rinsed in PBS and stored in PBS at 4°C for up to one month. To slice, the hemispheres were embedded in 1% low-melt agarose in PBS and cut into 50 μm sagittal slices in cold PBS buffer with a Leica VT1000S vibratome. The sections were fixed in 4% PFA in PBS for 2 hours, rinsed in PBS at room temperature, and then dehydrated in 70% ethanol at 4°C. The sections can then be stored at 4°C for up to one month. For staining, sections were cleared with 8% SDS in PBS for 2 hours at room temperature, then washed three times with 2x SSC for 1 hour each, then transferred to a new well with Hybridization Buffer (Molecular Instruments) and hybridized overnight at 37°C with the HCR probe in Hybridization Buffer. The next day, samples were washed with 30% Probe Wash Buffer for 1 hour at 37°C and then rinsed with 2x SSC. While the probe was being washed, the fluorescently labeled HCR hairpin was denatured at 95°C for 90 seconds and then snap-cooled in an aluminum block tube holder at room temperature for 30 minutes. The denatured hairpins were added to Amplification Buffer and applied to tissue slices for 2 hours at room temperature in the dark. Then, the slices were washed with 2x SSC containing DAPI, washed again with 2x SSC, and finally mounted on SuperFrost Plus slides in Prolong Glass Mounting medium (Thermo Fisher Scientific #P 36980). These HCR stains were imaged on an Olympus FV3000 confocal microscope using the manufacturer's software. Molecular Instruments generated HCR probes for SLC17A7, GAD1, FGFR3, and SOX10 transcripts.

[0134] In vivo non-human primate AAV vector testing. All procedures used in macaque monkeys conformed to guidelines provided by the US National Institutes of Health and were approved by the University of Washington Animal Care and Use Committee. Animals were injected with a single AAV vector at up to 10 injection sites during a single surgery. AAV was purified for this procedure by iodixanol gradient ultracentrifugation. After craniotomy, a total of 5 μL of AAV vector was injected into each site using a pneumatic picopump (World Precision Instruments), and 500 nL was expelled at 10 equally spaced sites at depths ranging from 2 mm to 200 μm below the pial surface. These sites were spaced 1 cm apart in each area with multiple injection sites. Animals were sacrificed 51 to 113 days after injection. The brain surface was examined, and tissue blocks (2 × 2 × 2 cm) were cut around each visible fluorescent spot. Each block was fixed in 4% PFA in PBS at 4°C for 24 hours. After fixation with PFA, the blocks were embedded in 2% agarose in PBS and cut into 350 μm sections, each of which was then examined for fluorescent cells. Appropriate recovery of the region was confirmed by PCR of DNA from the cut slices (collected using the QIAamp DNA FFPE Tissue Kit, Qiagen catalog # 56404) using primers common to all vectors: F5'-ACTCCATCACTAGGGGTTCCTG (SEQ ID NO: 366) and R5'-GGACACGCTGAACTTGTGGGGGGGGG (SEQ ID NO: 367), followed by Sanger sequencing using the nested reverse primer 5'-ACGTCGCCGTCCAGCTC (SEQ ID NO: 368). Slices were co-stained with DAPI and / or propidium iodide and imaged on a Nikon Tie inverted fluorescence microscope. 350 μm slices were used to assess precise expression using mFISH.

[0135] (ix) Closing paragraph Also included are variants of the sequences disclosed and referenced herein. Computer programs well known in the art, such as DNASTAR™ (Madison, Wisconsin) software, can be used to guide determining which amino acid residues can be substituted, inserted, or deleted without disrupting biological activity. Preferably, the amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve the substitution of one member of a family of amino acids that are related in their side chains.

[0136] Suitable conservative substitutions of amino acids in a peptide or protein are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art will recognize that single amino acid substitutions in non-essential regions of a polypeptide generally do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally classified into conservative substitution families as follows: Group 1: alanine (Ala), glycine (Gly), serine (Ser), and threonine (Thr); Group 2: (acidic): aspartic acid (Asp) and glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): asparagine (Asn), glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; also classified as polar, positively charged residues): arginine (Arg), lysine (Lys), and histidine (His); Group 6 (large aliphatic, nonpolar residues): isoleucine (I Group 11 (aliphatic): Gly, Ala, Val, Leu, and Ile; Group 10 (minor aliphatic, nonpolar, or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0137] In making such changes, the hydrophilicity index of amino acids may be taken into consideration. The importance of the hydrophilic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values ​​are as follows: 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), glutamate (-3.5), Gln (-3.5), aspartate (-3.5), Asn (-3.5), Lys (-3.9), and Arg (-4.5).

[0138] It is known in the art that certain amino acids may be substituted with other amino acids having a similar hydrophilicity index or score and still result in a protein with similar biological activity, i.e., still obtain a biologically functionally equivalent protein. In making such changes, substitutions of amino acids with hydrophilicity indices within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that substitutions of similar amino acids can be made effectively based on hydrophilicity.

[0139] As described in detail in U.S. Pat. No. 4,554,101, the following hydrophilicity values ​​have been assigned to amino acid residues: 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). It is understood that an amino acid can be substituted for another amino acid having a similar hydrophilicity value and still obtain a biologically valuable, particularly an immunologically valuable, protein. In such changes, substitutions of amino acids whose hydrophilicity values ​​are within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0140] As discussed above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.

[0141] As noted elsewhere, variants of a gene sequence can include codon-optimized variants, sequence polymorphisms, splice variants, and / or mutations that do not affect the function of the encoded product to a statistically significant extent.

[0142] Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences having at least 70% sequence identity, 80% sequence identity, 85% sequence identity, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to a protein, nucleic acid, or gene sequence disclosed herein.

[0143] "Percent sequence identity" refers to the relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences, as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated using known methods, see, for example, Computational Molecular Biology (Lesk, A. M. ed.), Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. Wed.), Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G. ed.), 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. ed.), Oxford University Press, NY (1992). Preferred methods for determining identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignment and percent identity calculations may be performed using the Megalign program in the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY = 10, GAP LENGTH PENALTY = 10).Related programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin), 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 Within the context of this disclosure, when sequence analysis software is used for the analysis, it will be understood that the results of the analysis will be based on the "default values" of the referenced program. As used herein, "default values" refers to any set of values ​​or parameters that are initially loaded into the software when first initialized.

[0144] Variants also include nucleic acid molecules that hybridize to the sequences disclosed herein under stringent hybridization conditions and provide the same function as the reference sequences. Exemplary stringent hybridization conditions include overnight incubation at 43°C in a solution containing 50% formamide, 5xSSC (750mM NaCl, 75mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5xDenhard's solution, 10% dextran sulfate, and 20µg / ml denatured, sheared salmon sperm DNA, followed by washing the filter in 0.1xSSC at 50°C. Varying the stringency of hybridization and signal detection is primarily achieved by manipulating formamide concentration (lower percentages of formamide result in lower stringency), salt conditions, or temperature. For example, moderately stringent conditions include overnight incubation at 37°C in a solution containing 6x SSPE (20x SSPE = 3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, and 100 μg / ml salmon sperm blocking DNA, followed by a wash at 50°C in 1x SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed after stringent hybridization can be performed at higher salt concentrations (e.g., 5x SSC). Modifications to the above conditions can be achieved through the inclusion and / or substitution of alternative blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of certain blocking reagents may require modification of the hybridization conditions described above due to compatibility issues.

[0145] As will be understood by those skilled in the art, each embodiment disclosed herein can comprise, consist essentially of, or consist of its specifically described element, step, ingredient, or component. Accordingly, the terms "include" or "including" should be interpreted as describing "comprising, consisting of, or consisting essentially of." The transitional terms "comprise" or "comprises" mean to allow for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts, without limitation. The transitional phrase "consisting of" excludes any unspecified element, step, ingredient, or component. The transitional phrase "consisting essentially of" limits the scope of the embodiment to the specified element, step, ingredient, or component, and those that do not have a substantial effect on the embodiment. A substantial effect would cause a statistically significant decrease in selective expression in the target cell population as determined by scRNA-Seq and the selected artificial expression construct / target cell population pairing.

[0146] Astrocytes: eHGT_373m, 3xcore eHGT_373m, eHGT_375m, eHGT_379m, eHGT_372m, eHGT_384m, eHGT_386m, eHGT_390m, 3xcore eHGT_390m, eHGT_371m, eHGT_383m, eHGT_374m, eHGT_381m, eHGT_382m, eHGT_387m, eHGT_388m, eHGT_ 376m, eHGT_380m, eHGT_385m, eHGT_371h, eHGT_372h, eHGT_375h, eHGT_376h, eHGT_377h, eHGT_381h, e HGT_382h, eHGT_383h, eHGT_384h, eHGT_387h, eHGT_388h, eHGT_389h, eHGT_390h, eHGT_357h, eHGT_495m, eHGT_497m, mscRE1001, mscRE1002, mscRE1003, mscRE1004, mscRE1005, mscRE1006, and mscRE1007;

[0147] L1 interlaminar astrocytes: eHGT_267h, eHGT_268h, eHGT_269h, eHGT_270h, eHGT_271h, eHGT_272h, eHGT_273h, eHGT_274h, eHGT_275h, eHGT_276h, eHGT_315h, and eHGT_316h;

[0148] Oligodendrocytes: eHGT_391m, eHGT_398m, eHGT_402m, eHGT_409m, eHGT_396m, eHGT_393m, eHGT_399m, eHGT_400m, eHGT_405m, eHGT_406m, eHGT_410m, 3xcore eHGT_410m, eHGT_397m, eHGT_401m, eHGT_403m, eHGT_407m, eHGT_408m, eHGT_392h, eHGT_393h, eHGT_394h, eHGT_395h, eHGT_396h, eHGT_397h, eHGT_398h, eHGT_399h, eHGT_400h, eHGT_402h, eHGT_404h, eHGT_405h, eHGT_406h, eHGT_407h, eHGT_641m, and eHGT_408h;

[0149] Microglia: eHGT413m, eHGT_414m, eHGT_415m, eHGT_416m, eHGT_417m, eHGT_418m, eHGT_419m, eHGT_420m, eHGT_4 21m, eHGT_423m, eHGT_428m, eHGT_429m, eHGT_430m, eHGT_411m, eHGT_412m, eHGT_422m, eHGT_424m, eHGT_425 m, eHGT_426m, eHGT_427m, eHGT_411h, eHGT_412h, eHGT_413h, eHGT_414h, eHGT_417h, eHGT_418h, eHGT_419h, eHGT_420h, eHGT_423h, eHGT_424h, eHGT_425h, eHGT_426h, eHGT_427h, eHGT_428h, eHGT_429h, and eHGT_430h;

[0150] Pericytes: mscRE1023, mscRE1024, mscRE1025, mscRE1026, mscRE1027, mscRE1028, mscRE1029, mscRE1030, mscRE1031, mscRE1032, mscRE1033, mscRE1034, mscRE1035, mscRE1036, and mscRE1037;

[0151] SMC: mscRE1038, mscRE1039, mscRE1040, mscRE1041, mscRE1042, mscRE1043, mscRE1044, mscRE1045, mscRE1046, mscRE1047, mscRE1048, mscRE1049, mscRE1050, mscRE1051, and mscRE1052; and

[0152] Endothelial cells: mscRE1008, mscRE1009, mscRE1010, mscRE1011, mscRE1012, mscRE1013, mscRE1014, mscRE1015, mscRE1016, mscRE1017, mscRE1018, mscRE1019, mscRE1020, mscRE1021, and mscRE1022.

[0153] Artificial means means not occurring in nature.

[0154] Unless otherwise indicated, all numbers used in the specification and claims expressing properties such as quantities of ingredients, molecular weights, reaction conditions, and the like, are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties to be obtained by the present invention. At the very least, and not as an attempt to limit the application of equivalence principles to the scope of the claims, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Where further clarity is needed, the term "about" has the meaning reasonably assigned by a person of ordinary skill in the art to a stated value or range, i.e., ±20% of the stated value, ±19% of the stated value, ±18% of the stated value, ±17% of the stated value, ±16% of the stated value, ±15% of the stated value, ±14% of the stated value, ±13% of the stated value, ±12% of the stated value, ±11% of the stated value, ±10% of the stated value, ±9% of the stated value, ±8% of the stated value, ±7% of the stated value, ±6% of the stated value, ±5% of the stated value, ±4% of the stated value, ±3% of the stated value, ±2% of the stated value, or ±1% of the stated value.

[0155] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0156] As used in the context of describing the present invention (particularly in the context of the claims below), "a," "an," "the," and similar referents should be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0157] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When such inclusion or deletion occurs, the specification is deemed to contain the modified group, thereby satisfying the description of all recited Markush groups used in the appended claims.

[0158] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect skilled artisans to adopt such variations as necessary, and the inventors intend the invention to be practiced other than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

[0159] Additionally, throughout this specification, numerous references are made to patents, printed publications, journal articles, and other written texts (materials referenced herein), each of which is individually incorporated herein by reference in its entirety for the purposes of its teaching.

[0160] Finally, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Accordingly, the invention is not limited to that precisely as shown and described.

[0161] The details set forth herein are presented by way of example only for illustrative purposes of discussion of preferred embodiments of the present invention, and to provide what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the present invention. In this regard, no attempt is made to show structural details of the present invention, but rather an attempt is made to provide a basic understanding of the present invention and, taken together with the drawings and / or examples, to make clear to those skilled in the art how some forms of the present invention may be embodied in practice.

[0162] The definitions and explanations used in this disclosure are meant and intended to control in any future interpretations unless clearly and unambiguously modified in the examples below, or unless application of the meaning would make any interpretation meaningless or essentially meaningless. If interpretation of a term would make it meaningless or essentially meaningless, the definition should be taken 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. 1. An artificial expression construct comprising: (i) 3xcore eHGT_390m, eHGT_390m, eHGT_373m, 3xcore eHGT_373m, eHGT_375m, eHGT_379m, eHGT_372m, eHGT_384m, eHGT_386m, eHGT_371m, eHGT_383m, eHGT_374m, eHGT_381m, eHGT_382m, eHGT_387m, eHGT_388m, eHGT_376m, eHGT_380m, eHGT_3 85m, eHGT_371h, eHGT_372h, eHGT_375h, eHGT_376h, eHGT_377h, eHGT_381h, eHGT_382h, eHGT _383h, eHGT_384h, eHGT_387h, eHGT_388h, eHGT_389h, eHGT_390h, eHGT_357h, eHGT_495m, eHG T_497m, mscRE1001, mscRE1002, mscRE1003, mscRE1004, mscRE1005, mscRE1006, mscRE1007, e HGT_267h, eHGT_268h, eHGT_269h, eHGT_270h, eHGT_271h, eHGT_272h, eHGT_273h, eHGT_274h , eHGT_275h, eHGT_276h, eHGT_315h, eHGT_316h, eHGT_391m, eHGT_398m, eHGT_402m, eHGT_40 9m, eHGT_396m, eHGT_393m, eHGT_399m, eHGT_400m, eHGT_405m, eHGT_406m, eHGT_410m, 3xcore eHGT_410m, eHGT_397m, eHGT_401m, eHGT_403m, eHGT_407m, eHGT_408m, eHGT_392h, eHGT_39 3h, eHGT_394h, eHGT_395h, eHGT_396h, eHGT_397h, eHGT_398h, eHGT_399h, eHGT_400h, eHGT _402h, eHGT_404h, eHGT_405h, eHGT_406h, eHGT_407h, eHGT_641m, eHGT_408h, eHGT413m, eH GT_414m, eHGT_415m, eHGT_416m, eHGT_417m, eHGT_418m, eHGT_419m, eHGT_420m, eHGT_421m,eHGT_423m, eHGT_428m, eHGT_429m, eHGT_430m, eHGT_411m, eHGT_412m, eHGT_422m, eHGT_424m, eHGT_425m, eHGT_4 26m, eHGT_427m, eHGT_411h, eHGT_412h, eHGT_413h, eHGT_414h, eHGT_417h, eHGT_418h, eHGT_419h, eHGT_420h, eHG T_423h, eHGT_424h, eHGT_425h, eHGT_426h, eHGT_427h, eHGT_428h, eHGT_429h, eHGT_430h, mscRE1023, mscRE1024 , mscRE1025, mscRE1026, mscRE1027, mscRE1028, mscRE1029, mscRE1030, mscRE1031, mscRE1032, mscRE1033, mscRE1 034, mscRE1035, mscRE1036, mscRE1037, mscRE1038, mscRE1039, mscRE1040, mscRE1041, mscRE1042, mscRE1043, ms cRE1044, mscRE1045, mscRE1046, mscRE1047, mscRE1048, mscRE1049, mscRE1050, mscRE1051, mscRE1052, mscRE1008 , mscRE1009, mscRE1010, mscRE1011, mscRE1012, mscRE1013, mscRE1014, mscRE1015, mscRE1016, mscRE1017, mscRE1018, mscRE1019, mscRE1020, mscRE1021, and mscRE1022; (ii) a promoter; and (iii) a heterologous coding sequence.

2. 2. The artificial expression construct of claim 1, wherein the heterologous coding sequence encodes an effector element or an expressible element.

3. 3. The artificial expression construct of claim 2, wherein the effector element comprises a reporter protein or a functional molecule.

4. 4. The artificial expression construct of claim 3, wherein the reporter protein comprises a fluorescent protein.

5. 4. The artificial expression construct of claim 3, wherein the functional molecule comprises a functional ion transporter, an enzyme, a transcription factor, a receptor, a membrane protein, a cellular transport protein, a signaling molecule, a neurotransmitter, a calcium reporter, a channelrhodopsin, a CRISPR / CAS molecule, an editase, a guide RNA molecule, a microRNA, a homologous recombination donor cassette, or a designer receptor that is exclusively activated by a designer drug (DREADD).

6. 3. The artificial expression construct of claim 2, wherein the expressible element comprises a non-functional molecule.

7. 7. The artificial expression construct of claim 6, wherein the non-functional molecule comprises a non-functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cellular transport protein, signaling molecule, neurotransmitter, calcium reporter, channelrhodopsin, CRISPR / CAS molecule, editase, guide RNA molecule, microRNA, homologous recombination donor cassette, or DREADD.

8. 2. The artificial expression construct of claim 1, wherein the artificial expression construct is associated with a capsid that crosses the blood-brain barrier.

9. 9. The artificial expression construct of claim 8, wherein the capsid comprises PHP.eB, AAV-BR1, AAV-PHP.S, AAV-PHP.B, or AAV-PPS.

10. 2. The artificial expression construct of claim 1, wherein the artificial expression construct comprises or encodes a skipping element.

11. 11. The artificial expression construct of claim 10, wherein the skipping element comprises a 2A peptide and / or an internal ribosome entry site (IRES).

12. 12. The artificial expression construct of claim 11, wherein the 2A peptide is selected from T2A, P2A, E2A, or F2A.

13. The artificial expression constructs are: AAV, scAAV, rAAv, minBglobin, CMV, minCMV, minRho, minRho ＊ , a fluorescent protein, Cre, iCre, dgCre, FlpO, tTA2, SP10, WPRE, and / or BGHpA.

14. A vector comprising the artificial expression construct of claim 1.

15. The vector of claim 14, wherein the vector is a viral vector.

16. The vector of claim 15, wherein the viral vector is a recombinant adeno-associated viral (AAV) vector.

17. 1. An adeno-associated virus (AAV) vector comprising at least one heterologous coding sequence, wherein the heterologous coding sequence is selected from the group consisting of 3xcore eHGT_390m, eHGT_390m, eHGT_373m, 3xcore eHGT_373m, eHGT_375m, eHGT_379m, eHGT_372m, eHGT_384m, eHGT_386m, eHGT_371m, eHGT_383m, eHGT_374m, eHGT_381m, eHGT_382m, eHGT_387m, eHGT_388m, eHGT_376m, eHGT_380m, eHGT_3 85m, eHGT_371h, eHGT_372h, eHGT_375h, eHGT_376h, eHGT_377h, eHGT_381h, eHGT_382h, eHGT _383h, eHGT_384h, eHGT_387h, eHGT_388h, eHGT_389h, eHGT_390h, eHGT_357h, eHGT_495m, eHG T_497m, mscRE1001, mscRE1002, mscRE1003, mscRE1004, mscRE1005, mscRE1006, mscRE1007, e HGT_267h, eHGT_268h, eHGT_269h, eHGT_270h, eHGT_271h, eHGT_272h, eHGT_273h, eHGT_274h , eHGT_275h, eHGT_276h, eHGT_315h, eHGT_316h, eHGT_391m, eHGT_398m, eHGT_402m, eHGT_40 9m, eHGT_396m, eHGT_393m, eHGT_399m, eHGT_400m, eHGT_405m, eHGT_406m, eHGT_410m, 3xcore eHGT_410m, eHGT_397m, eHGT_401m, eHGT_403m, eHGT_407m, eHGT_408m, eHGT_39 2h, eHGT_393h, eHGT_394h, eHGT_395h, eHGT_396h, eHGT_397h, eHGT_398h, eHGT _399h, eHGT_400h, eHGT_402h, eHGT_404h, eHGT_405h, eHGT_406h, eHGT_407h, e HGT_641m, eHGT_408h, eHGT413m, eHGT_414m, eHGT_415m, eHGT_416m, eHGT_417m,eHGT_418m, eHGT_419m, eHGT_420m, eHGT_421m, eHGT_423m, eHGT_428m, eHGT_429m, eHGT_430m, eHGT_411m, eHGT_412m , eHGT_422m, eHGT_424m, eHGT_425m, eHGT_426m, eHGT_427m, eHGT_411h, eHGT_412h, eHGT_413h, eHGT_414h, eHGT_417h , eHGT_418h, eHGT_419h, eHGT_420h, eHGT_423h, eHGT_424h, eHGT_425h, eHGT_426h, eHGT_427h, eHGT_428h, eHGT_429 h, eHGT_430h, mscRE1023, mscRE1024, mscRE1025, mscRE1026, mscRE1027, mscRE1028, mscRE1029, mscRE1030, mscRE103 1, mscRE1032, mscRE1033, mscRE1034, mscRE1035, mscRE1036, mscRE1037, mscRE1038, mscRE1039, mscRE1040, mscRE10 41, mscRE1042, mscRE1043, mscRE1044, mscRE1045, mscRE1046, mscRE1047, mscRE1048, mscRE1049, mscRE1050, mscRE10 The vector is under the control of a promoter and enhancer selected from mscRE1051, mscRE1052, mscRE1008, mscRE1009, mscRE1010, mscRE1011, mscRE1012, mscRE1013, mscRE1014, mscRE1015, mscRE1016, mscRE1017, mscRE1018, mscRE1019, mscRE1020, mscRE1021, and mscRE1022.

18. A transgenic cell comprising the artificial expression construct of claim 1.

19. 19. The transgenic cell of claim 18, wherein the transgenic cell is an astrocyte, an oligodendrocyte, a microglial cell, a pericyte, an SMC, or an endothelial cell.

20. 19. The transgenic cell of claim 18, wherein the transgenic cell is an L1 interlaminar astrocyte.

21. A non-human transgenic animal comprising the artificial expression construct of claim 1.

22. 22. The non-human transgenic animal of claim 21, wherein the non-human transgenic animal is a mouse or a non-human primate.

23. 10. An administrable composition comprising the artificial expression construct of claim 1.

24. A kit comprising the artificial expression construct of claim 1.

25. 24. A method for selectively expressing a heterologous gene in a cell population in vivo or in vitro, comprising providing to a sample or subject containing the cell population an administrable composition of claim 23 in a sufficient dosage and for a sufficient period of time, thereby selectively expressing said gene in the cell population.

26. 26. The method of claim 25, wherein the heterologous gene encodes an effector element or an expressible element.

27. 27. The method of claim 26, wherein the effector element comprises a reporter protein or a functional molecule.

28. 28. The method of claim 27, wherein the reporter protein comprises a fluorescent protein.

29. 28. The method of claim 27, wherein the functional molecule comprises a functional ion transporter, an enzyme, a transcription factor, a receptor, a membrane protein, a cellular transport protein, a signaling molecule, a neurotransmitter, a calcium reporter, a channelrhodopsin, a CRISPR / CAS molecule, an editase, a guide RNA molecule, a microRNA, a homologous recombination donor cassette, or a DREADD.

30. 27. The method of claim 26, wherein the expressible element comprises a non-functional molecule.

31. 31. The method of claim 30, wherein the non-functional molecule comprises a non-functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cellular transport protein, signaling molecule, neurotransmitter, calcium reporter, channelrhodopsin, CRISPR / CAS molecule, editase, guide RNA molecule, microRNA, homologous recombination donor cassette, or DREADD.

32. 26. The method of claim 25, wherein said providing comprises pipetting.

33. 33. The method of claim 32, wherein the pipetting is performed on a brain slice.

34. 34. The method of claim 33, wherein the brain slice comprises astrocytes, oligodendrocytes, microglial cells, pericytes, SMCs, and / or endothelial cells.

35. 34. The method of claim 33, wherein the brain slice comprises L1 interlaminar astrocytes.

36. 34. The method of claim 33, wherein the brain slice is from a mouse, human, or non-human primate.

37. 26. The method of claim 25, wherein said providing comprises administering to a living organism.

38. 38. The method of claim 37, wherein the organism is a human, a non-human primate, or a mouse.

39. The method according to claim 37, wherein the administration to a living body is by injection.

40. 40. The method of claim 39, wherein the injection comprises intravenous injection, intraparenchymal injection into brain tissue, intracerebroventricular (ICV) injection, intracisternal (ICM) injection, or intrathecal injection.

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