Artificial expression constructs for regulating gene expression in the cerebellum and second cell types

Abstract

We describe an artificial expression construct for regulating gene expression in a specific targeted type of central nervous system cell. The artificial expression construct of the invention can be used to express synthetic genes or regulate gene expression in cells of the cerebellum and a second type of cell.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 252,520, filed October 5, 2021, the contents of which are incorporated by reference in their entirety as if set forth herein.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant Nos. MH114126 and MH120095 awarded by the National Institutes of Health (NIH). The U.S. Government has certain rights in this invention.

[0003] Sequence Listing Reference The sequence listing accompanying this application is provided in XML format rather than in hard copy, and is incorporated herein by reference. The XML file containing this sequence listing is named A166-0031PCT.xml. The size of this XML file is 291KB, was created on October 4, 2022, and was submitted electronically via the Patent Center.

[0004] The present disclosure provides an artificial expression construct for regulating gene expression in the cerebellum and a second type of cell. The gene expression in the cerebellum can be regulated in Purkinje cells, granule cells, Bergmann glia, deep cerebellar nuclei (DCN) cells, molecular layer interneurons (MLI), mossy fiber (MF) cells, foliate white matter (WM) or oligodendrocytes. The gene expression in the second type of cell can be regulated in astrocytes, oligodendrocytes or neurons (whole brain); glutamatergic neurons in the thalamus; GABAergic neurons, glutamatergic neurons, astrocytes, pericytes or specialized smooth muscle cells (SMC) in the neocortex; cholinergic interneurons or medium spiny neurons (MSN) in the striatum; or spinal motor neurons. [Background technology]

[0005] To fully understand the biology of the brain, it is necessary to distinguish between different cell types, define them, and investigate them in detail, as well as to identify artificial expression constructs that can label and perturb those cells. In mice, driver lines expressing recombinases have been used successfully to label cell populations that share marker gene expression. However, the creation, maintenance, and use of such lines that can label specific cell types with high specificity is costly and often requires crossing transgenic animals between three species, which only results in low frequency of obtaining the desired experimental animals. Moreover, these tools cannot be applied to humans because of the need for germline transgenic animals.

[0006] Previous reports have described artificial expression constructs that induce gene expression in astrocytes or oligodendrocytes (throughout the brain), glutamatergic neurons of the thalamus or neocortex, GABAergic neurons of the neocortex, or cholinergic interneurons of the striatum. Summary of the Invention [Means for solving the problem]

[0007] The present disclosure provides an artificial expression construct for inducing gene expression in the cerebellum and a second type of cell. Gene expression in the cerebellum can be regulated in Purkinje cells, granule cells, Bergmann glia, deep cerebellar nuclei (DCN) cells, molecular layer interneurons (MLI), mossy fiber (MF) cells, foliate white matter (WM) or oligodendrocytes. Gene expression in the second type of cell can be modulated in astrocytes, oligodendrocytes or neurons (throughout the brain); glutamatergic neurons of the thalamus; GABAergic neurons of the neocortex (e.g., Pvalb neurons (e.g., chandelier cells), SST neurons (e.g., Chodl neurons), LAMP5 neurons, VIP neurons, medial ganglionic eminence (MGE) cells), glutamatergic neurons (e.g., L5 ET neurons, L5 NP neurons, L5 IT neurons), astrocytes, pericytes, or specialized smooth muscle cells (SMCs); cholinergic interneurons or medium spiny neurons (MSNs) of the striatum; or spinal motor neurons.

[0008] In certain embodiments of the artificial expression constructs of the invention, the following enhancers are utilized to drive gene expression in cells of the cerebellum and in a second type of cell, the combinations of which with cells of the cerebellum and with second type of cell are shown below in the order of enhancer / cells of the cerebellum and second type of cell. eHGT_023h v1 / Purkinje cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_023m / Purkinje cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_082h / DCN cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_087h / Purkinje cells in the cerebellum and Sst interneurons in the neocortex; eHGT_128h / DCN cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_181h / MLI cells in the cerebellum and Lamp5 interneurons in the neocortex; eHGT_260h / Purkinje cells of the cerebellum and whole glutamatergic neurons of the neocortex; eHGT_023h v2 / Purkinje and MLI cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_356h / DCN cells in the cerebellum and Vip interneurons in the neocortex; eHGT_359h / Purkinje cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_375m / Bergmann glia in the cerebellum and astrocytes throughout the brain; eHGT_387m / Bergmann glia in the cerebellum and astrocytes throughout the brain; eHGT_395h / oligodendrocytes, DCN cells, and lobar white matter (WM) in the cerebellum and oligodendrocytes throughout the brain; eHGT_453m / DCN cells in the cerebellum and L5 ET neurons in the neocortex; eHGT_470m / DCN cells in the cerebellum and Sst / Chodl neurons in the neocortex; eHGT_479m / Purkinje and MLI cells in the cerebellum and chandelier cells in the neocortex; eHGT_494m / Purkinje cells in the cerebellum and Vip and chandelier cells in the neocortex; eHGT_467m / Purkinje cells of the cerebellum and Sst / Chodl neurons of the neocortex; eHGT_483m / granule cells and mossy fiber (MF) cells in the cerebellum and Vip interneurons in the neocortex; eHGT_606h / Purkinje cells of the cerebellum and glutamatergic neurons of the thalamus; eHGT_738m / MLI cells in the cerebellum and cholinergic interneurons in the striatum; eHGT_796h / total cerebellar Purkinje cells and neocortical GABAergic neurons; eHGT_710m / MLI cells in the cerebellum and chandelier cells in the neocortex; eHGT_588m / Purkinje and MLI cells in the cerebellum and MGE cells in the neocortex; eHGT_007m / MLI cells in the cerebellum and Vip interneurons in the neocortex; eHGT_703m / MLI cells in the cerebellum and chandelier cells in the neocortex; eHGT_589m / DCN cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_086h / MLI (basket) cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_963m / MLI and DCN cells in the cerebellum and L5 NP neurons in the neocortex; eHGT_534h / Purkinje cells in the cerebellum and whole neurons in the whole brain; eHGT_830h / MLI cells in the cerebellum and glutamatergic neurons in the thalamus; eHGT_540h / Purkinje cells in the cerebellum and whole neurons in the whole brain; eHGT_882m / DCN cells in the cerebellum and MSN cells in the striatum; eHGT_1137m / DCN cells of the cerebellum and spinal motor neurons; eHGT_381h / Bergmann glia in the cerebellum and astrocytes in the neocortex; MGT_E118 / DCN cells in the cerebellum and astrocytes in the neocortex; MGT_E122 / Bergmann glia in the cerebellum and astrocytes in the neocortex; MGT_E146 / MLI cells in the cerebellum and pericytes in the neocortex; MGT_E16 / MLI cells in the cerebellum and L5 IT neurons in the neocortex; MGT_E150 / Purkinje cells of the cerebellum and specialized smooth muscle cells (SMCs) of the neocortex; eHGT_1032h / MLI cells in the cerebellum and total GABAergic neurons throughout the brain; eHGT_1027h / MLI cells in the cerebellum and total GABAergic neurons throughout the brain; eHGT_1027m / MLI cells in the cerebellum and total GABAergic neurons throughout the brain; Concatemerized multi-copy enhancer or concatemerized multi-copy enhancer core containing 3xCore-eHGT_023h v3 (3xPVALBCore) / Purkinje and MLI cells in the cerebellum and Pvalb interneurons in the neocortex; 3x(CoreB)eHGT_121h / MLI cells in the cerebellum and Pvalb interneurons in the neocortex; 3xcore3_eHGT_453m / DCN cells of the cerebellum; 3xcore2_eHGT_387m / Bergmann glia in the cerebellum and astrocytes in the neocortex; 3xcore2_eHGT_475m / Purkinje cells in the cerebellum and chandelier cells in the neocortex; 3xcore2_eHGT_351h / total DCN cells in the cerebellum and MSN cells in the striatum; and 3xcore_MGT_E116 / DCN cells in the cerebellum and L5 NP neurons in the neocortex.

[0009] In a particular embodiment, the artificial enhancer element of the invention comprises an enhancer core or a concatemerized enhancer core. An example is a concatemerized core comprising the cores of eHGT_023h v3, eHGT_121h, eHGT_453m, eHGT_387m, eHGT_475m, eHGT_351h and / or MGT_E116. By using such an artificial enhancer element, it is possible to rapidly express the transgene and obtain high expression, compared to the case where the full length of the original (natural) enhancer is used alone.

[0010] In certain embodiments, the enhancer core comprises a sequence as set forth in any of SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:112, and SEQ ID NO:121. In certain embodiments, these enhancer cores are concatemerized and have 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the core sequence. In certain embodiments, SEQ ID NO:26 provides a concatemer consisting of 3 copies of core-eHGT_023h v3 core. In certain embodiments, SEQ ID NO:28 provides a concatemer consisting of 3 copies of (coreB)eHGT_121h. In certain embodiments, SEQ ID NO:104 provides a concatemer consisting of 3 copies of core3_eHGT_453m. In certain embodiments, SEQ ID NO:106 provides a concatemer consisting of 3 copies of core2_eHGT_387m. In certain embodiments, SEQ ID NO: 109 provides a concatemer consisting of 3 copies of core2_eHGT_475m. In certain embodiments, SEQ ID NO: 113 provides a concatemer consisting of 3 copies of core2_eHGT_351h. In certain embodiments, SEQ ID NO: 122 provides a concatemer consisting of 3 copies of core_MGT_E116. In certain embodiments, the enhancer comprises an enhancer core and additional sequences.

[0011] In certain embodiments, there is provided an artificial expression construct comprising the features described herein and / or the use of the vectors described herein, including the vectors described herein, such as CN1259, CN1280, CN1521, CN1528, CN1533, CN1621, CN1674, CN1778, CN1932, CN2043, CN2045, CN2085, CN2102, CN2157, CN2216, CN2251, CN2257, CN2258, CN2267, CN2316, CN2339, and the like. , CN2431, CN2436, CN2643, CN2663, CN2717, CN2838, CN1415, CN2710, CN2839, CN2674, CN3301, CN3019, CN3569, CN2374, CN3584, CN3003, CN2379, CN3566, CN3453, HCT1, CN2146, AiP1347, AiP1351, AiP1375, AiP1307, AiP1530, AiP1379, CN3916, CN3869 and / or CN3870. [Brief description of the drawings]

[0012] Some of the drawings submitted in this application may be more easily understood in color, and applicants hereby contemplate color versions of these drawings as part of the original application and reserve the right to submit color images of such drawings in subsequent proceedings.

[0013] [Figure 1A-1C] CN2251 vector induces reporter transgene expression in primate deep cerebellar nucleus neurons. (1A) Shows epifluorescence microscopy image of rhesus monkey cerebellum stained with anti-NeuN antibody showing structural details and neurons. (1B) Shows epifluorescence microscopy image of anti-GFP signal in deep cerebellar nucleus neurons targeted by stereotaxic intracerebral viral injection of AAV vector CN2251 containing enhancer eHGT_453m packaged with serotype PHP.eB. (1C) Shows magnified image of box highlighting anti-GFP signal in deep cerebellar nucleus neurons.

[0014] [Fig. 2A-2I] Vector: CN2045, Enhancer: eHGT_359h. (2A-2E) Animal: Mouse 200910-08. (2A) Fluorescent montage images of native SYFP2 in sagittal sections of whole mouse brain and (2B) sagittal sections of mouse cerebellum and (2C) high magnification images of mouse cerebellum showing targeted SYFP2 expression in cells with Purkinje cell morphology. (2D) Native SYFP2 fluorescent signal in sagittal section of cerebellum showing labeling of Purkinje cells and (2E) native SYFP2 fluorescent signal overlaid with Pvalb mRNA expression (arrow). Adult mice were administered virus by intravascular (IV) injection (retro-orbital) of CN2045 virus packaged with PHP.eB capsid. (2F, 2G) Animal: Rat 585761. (2F) Fluorescence montage of native SYFP2 in sagittal sections of whole rat brain and (2G) a magnified image of the cerebellum of this rat showing targeted SYFP2 expression in cells with Purkinje cell morphology. Neonatal rats were administered virus by intracerebroventricular (ICV) injection of CN2045 virus packaged with PHP.eB capsids one day after birth. (2H, 2I) Animal: Monkey Q21.26.022. (2H) Fluorescence montage of native SYFP2 in sagittal sections of rhesus monkey cerebellum and (2I) a magnified image of the rhesus monkey cerebellum showing targeted SYFP2 expression in cells with Purkinje cell morphology. Virus was administered by intraparenchymal injection of CN2045 virus packaged with PHP.eB capsids.

[0015] [Figures 3A-3I]Vector: CN1259, Enhancer: eHGT_023h v1. (3A-3C) Animal: Mouse 190329-06. (3A) Fluorescent montage images of native SYFP2 in sagittal sections of whole mouse brain and (3B) sagittal sections of mouse cerebellum and (3C) high magnification of mouse cerebellum showing targeted SYFP2 expression in cells with Purkinje cell morphology. Adult mice were administered CN1259 virus packaged with PHP.eB capsids by intravascular (IV) injection (retro-orbital). (3D-3I) Cerebellum of mouse 190329-06 transduced with CN1259 administered to adult mice by intravascular (IV) injection (retro-orbital). (3D) Merge of whole cerebellum and (3E) Purkinje cells at high magnification. (3F-3I) Individual channels containing (3F) SYFP2 fluorescence, (3G) DAPI staining, (3H) Gad1 mFISH, and (3I) Pvalb mFISH are shown. Each image is a montage. Note that all SYFP2 cells overlap with Pvalb mRNA and Gad1 mRNA, consistent with their identity as Purkinje cells.

[0016] [Figure 4A-4I]Vector: CN1528, Enhancer: eHGT_082h, and Animal: Mouse 190425-01. (4A) Fluorescent montage images of native SYFP2 in sagittal sections of whole mouse brain and (4B) sagittal sections of mouse cerebellum and (4C) high magnification images of mouse cerebellum showing targeted SYFP2 expression in cells of the deep cerebellar nuclei. Adult mice were administered CN1528 virus packaged with PHP.eB capsids by intravascular (IV) injection (retro-orbital). (4D-4I) Mouse cerebellum transduced with CN1528 administered to adult mice by intravascular (IV) injection (retro-orbital). (4D) Merge images of deep cerebellar nuclei and (4E) high magnification images of deep cerebellar nuclei cells are shown. (4F-4I) Individual channels including (4F) SYFP2 fluorescence, (4G) DAPI staining, (4H) Gad1 mFISH, and (4I) Pvalb mFISH are shown. Each image is a montage. Note that all SYFP2 cells overlap with Pvalb, but only a portion of them express Gad1.

[0017] [Figure 5A-5C] Vector: CN2102, Enhancer: eHGT_387m, and Animal: Mouse: 200113-05 (5A), 200910-05 (5B-5C). (5A) Fluorescence montage images of native SYFP2 in sagittal sections of whole mouse brain and (5B) sagittal sections of mouse cerebellum and (5C) high magnification images of mouse cerebellum are shown, demonstrating that targeted SYFP2 is expressed in cells with Bergmann glia morphology. Adult mice were administered virus by intravascular (IV) injection (retro-orbital) of CN2102 virus packaged with PHP.eB capsids.

[0018] [Figure 6A-6B]Vector: CN2717, Enhancer: eHGT_710m, and Animal: Mouse C57BL6J-560070. (6A) Fluorescence montage images of native SYFP2 in sagittal sections of mouse cerebellum and (6B) magnified images of mouse cerebellum showing targeted SYFP2 expression in cells with small interneuron morphology in the molecular layer. Adult mice were administered virus by intravascular (IV) injection (retro-orbital) of CN2717 virus packaged with PHP.eB capsids.

[0019] [Figure 7A-7B] Vector: CN2251, Enhancer: eHGT_453m, and Animal: Mouse. (7A) Fluorescence montage images of native SYFP2 in a coronal section of a mouse cerebellum and (7B) a magnified image of a mouse cerebellum are shown, demonstrating targeted SYFP2 expression in cells of the deep cerebellar nuclei. Adult mice were administered virus by intravascular (IV) injection (retro-orbital) of CN2251 virus packaged with PHP.eB capsids.

[0020] [Figure 8]The sequences supporting the present disclosure are shown below. These sequences include: eHGT_023h v1 (SEQ ID NO:1); eHGT_023m (SEQ ID NO:2); eHGT_359h (SEQ ID NO:3); eHGT_023h v2 (SEQ ID NO:4); eHGT_082h (SEQ ID NO:5); eHGT_703m (SEQ ID NO:6); eHGT_087h (SEQ ID NO:7); eHGT_128h (SEQ ID NO:98); eHGT_181h (SEQ ID NO:8); eHGT_260h (SEQ ID NO:9); eHGT_356h (SEQ ID NO:10); eHGT_375m (SEQ ID NO:11); eHGT_387m (SEQ ID NO:12); eHGT_395h (SEQ ID NO:99); eHGT_007m (SEQ ID NO:13); eHGT _453m (SEQ ID NO:14);eHGT_470m (SEQ ID NO:15);eHGT_479m (SEQ ID NO:16);eHGT_494m (SEQ ID NO:17);eHGT_467m (SEQ ID NO:18);eHGT_483m (SEQ ID NO:19);eHGT_606h (SEQ ID NO:20);eHGT_738m (SEQ ID NO:21);eHGT_796h (SEQ ID NO:22);eHGT_710m (SEQ ID NO:23);eHGT_588m (SEQ ID NO:24);Core-eHGT_023h v3 (SEQ ID NO:25);3xCore-eHGT_023h v3 (SEQ ID NO: 26); (CoreB) eHGT_121h (SEQ ID NO: 27); 3x (CoreB) eHGT_121h (SEQ ID NO: 28); eHGT_589m (SEQ ID NO: 100); eHGT_086h (SEQ ID NO: 101); eHGT_963m (SEQ ID NO: 102); core3_eHGT_453m (SEQ ID NO: 103); 3xcore3_eHGT_453m (SEQ ID NO: 104); core2_eHGT_387m (SEQ ID NO: 105); 3xcore2_eHGT_387m (SEQ ID NO: 106); eHGT_5 34h (SEQ ID NO: 107); core2_eHGT_475m (SEQ ID NO: 108); 3xcore2_eHGT_475m (SEQ ID NO: 109); eHGT_830h (SEQ ID NO: 110); eHGT_540h (SEQ ID NO: 111); core2_eHGT_351h (SEQ ID NO: 112); 3xcore2_eHGT_351h (SEQ ID NO: 113); eHGT_882m (SEQ ID NO: 114); eHGT_1137m (SEQ ID NO: 115); eHGT_381h (SEQ ID NO: 116); MGT_E118 (SEQ ID NO: 117);MGT_E122 (SEQ ID NO: 118); MGT_E146 (SEQ ID NO: 119); MGT_E16 (SEQ ID NO: 120); core_MGT_E116 (SEQ ID NO: 121); 3xcore_MGT_E116 (SEQ ID NO: 122); MGT_E150 (SEQ ID NO: 123); eHGT_1032h (SEQ ID NO: 124); eHGT_1027h (SEQ ID NO: 125); eHGT_1027m (SEQ ID NO: 126); β-globin minimal promoter (SEQ ID NO: 29); minCMV promoter (SEQ ID NO: 30); mutated minCMV promoter (SEQ ID NO: 31); minRho promoter (SEQ ID NO: 32); minRho* promoter (SEQ ID NO: 33); Hsp68 minimal promoter (SEQ ID NO: 34); SYFP2 (SEQ ID NO: 35); EGFP (SEQ ID NO: 36); optimized Flp recombinase (SEQ ID NO: 37); 10aa (SEQ ID NO: 38); H2Bmod( SEQ ID NO:39; hsA2 (SEQ ID NO:40); Improved Cre recombinase (SEQ ID NO:41); SP10 insulator (SEQ ID NO:42); 3xSP10ins (SEQ ID NO:43); Flag coding sequence (SEQ ID NO:163); 3xFLAG (SEQ ID NO:164); H2B (SEQ ID NO:165); H2B* (SEQ ID NO:166); WPRE3 (SEQ ID NO:44); WPRE (SEQ ID NO:45); BGHpA (SEQ ID NO:46); HGHpA (SEQ ID NO:47); P2A (SEQ ID NO:48); T2A (SEQ ID NO:49); E2A (SEQ ID NO:50); F2A (SEQ ID NO:51); Exemplary plasmid backbone 1 - left ITR (SEQ ID NO:52); Exemplary plasmid backbone 1 - right ITR (SEQ ID NO:53); Exemplary plasmid backbone 2 - left ITR (SEQ ID NO:54); Exemplary plasmid backbone 2 - right ITR (SEQ ID NO:55); PHP.eB capsid (SEQ ID NO:56); AAV9 VP1 capsid protein (SEQ ID NO: 57); tet transactivator version 2 (SEQ ID NO: 58); GTPase HRas [Homo sapiens] (SEQ ID NO: 59); Substance P [Homo sapiens] consisting of residues 58 to 68 of Protachykinin-1 (SEQ ID NO: 60); Oxytocin [Homo sapiens] consisting of residues 20 to 28 of oxytocin-neurophysin 1 (SEQ ID NO: 61); GCaMP6m (SEQ ID NO: 62);GCaMP6s (SEQ ID NO: 63); GCaMP6f (SEQ ID NO: 64); CN1259 (SEQ ID NO: 65); CN1621 (SEQ ID NO: 127); CN2157 (SEQ ID NO: 128); CN2339 (SEQ ID NO: 66); CN1280 (SEQ ID NO: 67); CN2045 (SEQ ID NO: 68); CN1932 (SEQ ID NO: 69); CN1528 (SEQ ID NO: 70); CN2710 (SEQ ID NO: 71); CN1533 (SEQ ID NO: 72); CN1674 (SEQ ID NO: 73); CN1778 (SEQ ID NO: 74); No. 74);CN2043 (SEQ ID NO: 75);CN2085 (SEQ ID NO: 76);CN2102 (SEQ ID NO: 77);CN1415 (SEQ ID NO: 78);CN2251 (SEQ ID NO: 79);CN2257 (SEQ ID NO: 80);CN2258 (SEQ ID NO: 81);CN2267 (SEQ ID NO: 82);CN2316 (SEQ ID NO: 83);CN2431 (SEQ ID NO: 84);CN2436 (SEQ ID NO: 85);CN2643 (SEQ ID NO: 86);CN2663 (SEQ ID NO: 87);CN2717 ( SEQ ID NO:88; CN2838 (SEQ ID NO:89); CN1521 (SEQ ID NO:90); CN2216 (SEQ ID NO:91); CN2839 (SEQ ID NO:129); CN2674 (SEQ ID NO:130); CN3301 (SEQ ID NO:131); CN3019 (SEQ ID NO:132); CN3569 (SEQ ID NO:133); CN2374 (SEQ ID NO:134); CN3584 (SEQ ID NO:135); CN3003 (SEQ ID NO:136); CN2379 (SEQ ID NO:137); CN3566 (SEQ ID NO: 138); CN3453 (SEQ ID NO: 139); HCT1 (SEQ ID NO: 140); CN2146 (SEQ ID NO: 141); AiP1347 (SEQ ID NO: 142); AiP1351 (SEQ ID NO: 143); AiP1375 (SEQ ID NO: 144); AiP1307 (SEQ ID NO: 145); AiP1530 ​​(SEQ ID NO: 146); AiP1379 (SEQ ID NO: 147); CN3916 (SEQ ID NO: 148); CN3869 (SEQ ID NO: 149); and CN3870 (SEQ ID NO: 150). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] To fully understand the biology of the brain, we need to distinguish, define, and dissect different cell types, as well as identify artificial expression constructs that can label and perturb them (Tasic, Curr. Opin. Neurobiol. 50, 242-249 (2018); Zeng & Sanes, Nat. Rev. Neurosci. 18, 530-546 (2017)). In mice, driver lines expressing recombinases have been used successfully 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 creation, maintenance and use of such lines capable of labeling specific cell types with high specificity is costly and often requires crossing of transgenic animals between three species, resulting in low frequency of obtaining the desired experimental animals. Moreover, these tools cannot be applied to humans because of the need for germline transgenic animals.

[0022] The present disclosure provides an artificial expression construct for inducing gene expression in the cerebellum and a second type of cell. Gene expression in the cerebellum can be regulated in Purkinje cells, granule cells, Bergmann glia, deep cerebellar nuclei (DCN) cells, molecular layer interneurons (MLI), mossy fiber (MF) cells, foliate white matter (WM) or oligodendrocytes. Gene expression in the second type of cell can be modulated in astrocytes, oligodendrocytes or neurons (throughout the brain); glutamatergic neurons of the thalamus; GABAergic neurons of the neocortex (e.g., Pvalb neurons (e.g., chandelier cells), SST neurons (e.g., Chodl neurons), LAMP5 neurons, VIP neurons, medial ganglionic eminence (MGE) cells), glutamatergic neurons (e.g., L5 ET neurons, L5 NP neurons, L5 IT neurons), astrocytes, pericytes or specialized smooth muscle cells (SMCs); cholinergic interneurons or medium spiny neurons (MSNs) of the striatum; or spinal motor neurons.

[0023] The present disclosure provides enhancers and associated artificial expression constructs capable of inducing gene expression in cells of the cerebellum and a second type of cell, which are shown below. eHGT_023h v1 / Purkinje cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_023m / Purkinje cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_082h / DCN cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_087h / Purkinje cells in the cerebellum and Sst interneurons in the neocortex; eHGT_128h / DCN cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_181h / MLI cells in the cerebellum and Lamp5 interneurons in the neocortex; eHGT_260h / Purkinje cells of the cerebellum and whole glutamatergic neurons of the neocortex; eHGT_023h v2 / Purkinje and MLI cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_356h / DCN cells in the cerebellum and Vip interneurons in the neocortex; eHGT_359h / Purkinje cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_375m / Bergmann glia in the cerebellum and astrocytes throughout the brain; eHGT_387m / Bergmann glia in the cerebellum and astrocytes throughout the brain; eHGT_395h / oligodendrocytes, DCN cells, and lobar white matter (WM) in the cerebellum and oligodendrocytes throughout the brain; eHGT_453m / DCN cells in the cerebellum and L5 ET neurons in the neocortex; eHGT_470m / DCN cells in the cerebellum and Sst / Chodl neurons in the neocortex; eHGT_479m / Purkinje and MLI cells in the cerebellum and chandelier cells in the neocortex; eHGT_494m / Purkinje cells in the cerebellum and Vip and chandelier cells in the neocortex; eHGT_467m / Purkinje cells of the cerebellum and Sst / Chodl neurons of the neocortex; eHGT_483m / granule cells and mossy fiber (MF) cells in the cerebellum and Vip interneurons in the neocortex; eHGT_606h / Purkinje cells of the cerebellum and glutamatergic neurons of the thalamus; eHGT_738m / MLI cells in the cerebellum and cholinergic interneurons in the striatum; eHGT_796h / total cerebellar Purkinje cells and neocortical GABAergic neurons; eHGT_710m / MLI cells in the cerebellum and chandelier cells in the neocortex; eHGT_588m / Purkinje and MLI cells in the cerebellum and MGE cells in the neocortex; eHGT_007m / MLI cells in the cerebellum and Vip interneurons in the neocortex; eHGT_703m / MLI cells in the cerebellum and chandelier cells in the neocortex; eHGT_589m / DCN cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_086h / MLI (basket) cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_963m / MLI and DCN cells in the cerebellum and L5 NP neurons in the neocortex; eHGT_534h / Purkinje cells in the cerebellum and whole neurons in the whole brain; eHGT_830h / MLI cells in the cerebellum and glutamatergic neurons in the thalamus; eHGT_540h / Purkinje cells in the cerebellum and whole neurons in the whole brain; eHGT_882m / DCN cells in the cerebellum and MSN cells in the striatum; eHGT_1137m / DCN cells of the cerebellum and spinal motor neurons; eHGT_381h / Bergmann glia in the cerebellum and astrocytes in the neocortex; MGT_E118 / DCN cells in the cerebellum and astrocytes in the neocortex; MGT_E122 / Bergmann glia in the cerebellum and astrocytes in the neocortex; MGT_E146 / MLI cells in the cerebellum and pericytes in the neocortex; MGT_E16 / MLI cells in the cerebellum and L5 IT neurons in the neocortex; MGT_E150 / Purkinje cells of the cerebellum and specialized smooth muscle cells (SMCs) of the neocortex; eHGT_1032h / MLI cells in the cerebellum and total GABAergic neurons throughout the brain; eHGT_1027h / MLI cells in the cerebellum and total GABAergic neurons throughout the brain; eHGT_1027m / MLI cells in the cerebellum and total GABAergic neurons throughout the brain; 3xCore-eHGT_023h v3 / Purkinje and MLI cells in the cerebellum and Pvalb interneurons in the neocortex; 3x(CoreB)eHGT_121h / MLI cells in the cerebellum and Pvalb interneurons in the neocortex; 3xcore3_eHGT_453m / DCN cells of the cerebellum; 3xcore2_eHGT_387m / Bergmann glia in the cerebellum and astrocytes in the neocortex; 3xcore2_eHGT_475m / Purkinje cells in the cerebellum and chandelier cells in the neocortex; 3xcore2_eHGT_351h / total DCN cells in the cerebellum and MSN cells in the striatum; and 3xcore_MGT_E116 / DCN cells in the cerebellum and L5 NP neurons in the neocortex.

[0024] In a particular embodiment, the artificial enhancer element of the invention comprises an enhancer core or a concatemerized enhancer core. An example is a concatemerized core comprising the cores of eHGT_023h v3, eHGT_121h, eHGT_453m, eHGT_387m, eHGT_475m, eHGT_351h and / or MGT_E116. By using such an artificial enhancer element, it is possible to rapidly express the transgene and obtain high expression, compared to the case where the full length of the original (natural) enhancer is used alone.

[0025] In certain embodiments, the enhancer core comprises a sequence as set forth in any of SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:112, and SEQ ID NO:121. In certain embodiments, these enhancer cores are concatemerized and have 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the core sequence. In certain embodiments, SEQ ID NO:26 provides a concatemer consisting of 3 copies of Core-eHGT_023h v3 core. In certain embodiments, SEQ ID NO:28 provides a concatemer consisting of 3 copies of (coreB)eHGT_121h. In certain embodiments, SEQ ID NO:104 provides a concatemer consisting of 3 copies of core3_eHGT_453m. In certain embodiments, SEQ ID NO:106 provides a concatemer consisting of 3 copies of core2_eHGT_387m. In certain embodiments, SEQ ID NO: 109 provides a concatemer consisting of 3 copies of core2_eHGT_475m. In certain embodiments, SEQ ID NO: 113 provides a concatemer consisting of 3 copies of core2_eHGT_351h. In certain embodiments, SEQ ID NO: 122 provides a concatemer consisting of 3 copies of core_MGT_E116. In certain embodiments, the enhancer comprises an enhancer core and additional sequences.

[0026] In certain embodiments, there is provided an artificial expression construct comprising the features described herein and / or the use of the vectors described herein, including the vectors described herein, such as CN1259, CN1280, CN1521, CN1528, CN1533, CN1621, CN1674, CN1778, CN1932, CN2043, CN2045, CN2085, CN2102, CN2157, CN2216, CN2251, CN2257, CN2258, CN2267, CN2316, CN2339, and the like. , CN2431, CN2436, CN2643, CN2663, CN2717, CN2838, CN1415, CN2710, CN2839, CN2674, CN3301, CN3019, CN3569, CN2374, CN3584, CN3003, CN2379, CN3566, CN3453, HCT1, CN2146, AiP1347, AiP1351, AiP1375, AiP1307, AiP1530, AiP1379, CN3916, CN3869 and / or CN3870. These vectors can be used to induce gene expression in cells of the cerebellum and in cells of the second type. Combinations of vectors, cerebellar cells, and second type of cells used in the present invention are shown below in the order of vector / cerebellar cells and second type of cells. CN1259 / Purkinje cells in the cerebellum and Pvalb interneurons in the neocortex; CN1280 / Purkinje cells in the cerebellum and Pvalb interneurons in the neocortex; CN1521 / Purkinje and MLI cells in the cerebellum and Pvalb interneurons in the neocortex; CN1528 / DCN cells in the cerebellum and Pvalb interneurons in the neocortex; CN1533 / Purkinje cells in the cerebellum and Sst interneurons in the neocortex; CN1621 / DCN cells in the cerebellum and Pvalb interneurons in the neocortex; CN1674 / MLI cells in the cerebellum and Lamp5 interneurons in the neocortex; CN1778 / Purkinje cells of the cerebellum and whole glutamatergic neurons of the neocortex; CN1932 / Purkinje and MLI cells in the cerebellum and Pvalb interneurons in the neocortex; CN2043 / DCN cells in the cerebellum and Vip interneurons in the neocortex; CN2045 / Purkinje cells in the cerebellum and Pvalb interneurons in the neocortex; CN2085 / Bergmann glia in the cerebellum and astrocytes throughout the brain; CN2102 / Bergmann glia in the cerebellum and astrocytes throughout the brain; CN2157 / cerebellar oligodendrocytes, DCN cells, and lobar white matter (WM) and oligodendrocytes throughout the brain; CN2216 / MLI cells in the cerebellum and Pvalb interneurons in the neocortex; CN2251 / DCN cells in the cerebellum and L5 ET neurons in the neocortex; CN2257 / DCN cells in the cerebellum and Sst / Chodl neurons in the neocortex; CN2258 / Purkinje and MLI cells in the cerebellum and chandelier cells in the neocortex; CN2267 / Purkinje cells in the cerebellum and Vip and chandelier cells in the neocortex; CN2316 / Purkinje cells in the cerebellum and Sst / Chodl neurons in the neocortex; CN2339 / Purkinje cells in the cerebellum and Pvalb interneurons in the neocortex; CN2431 / granule cells and mossy fiber (MF) cells in the cerebellum and Vip interneurons in the neocortex; CN2436 / Purkinje cells of the cerebellum and glutamatergic neurons of the thalamus; CN2643 / MLI cells in the cerebellum and cholinergic interneurons in the striatum; CN2663 / total cerebellar Purkinje cells and neocortical GABAergic neurons; CN2717 / MLI cells in the cerebellum and chandelier cells in the neocortex; CN2838 / Purkinje and MLI cells in the cerebellum and MGE cells in the neocortex; CN1415 / MLI cells in the cerebellum and Vip interneurons in the neocortex; CN2710 / MLI cells in the cerebellum and chandelier cells in the neocortex; CN2839 / DCN cells in the cerebellum and Pvalb interneurons in the neocortex; CN2674 / MLI (basket) cells in the cerebellum and Pvalb interneurons in the neocortex; CN3301 / MLI and DCN cells in the cerebellum and L5 NP neurons in the neocortex; CN3019 / cerebellar DCN cells; CN3569 / Bergmann glia in the cerebellum and astrocytes in the neocortex; CN2374 / Purkinje cells in the cerebellum and whole neurons throughout the brain; CN3584 / Purkinje cells of the cerebellum and chandelier cells of the neocortex; CN3003 / MLI cells in the cerebellum and glutamatergic neurons in the thalamus; CN2379 / Purkinje cells in the cerebellum and whole neurons throughout the brain; CN3566 / whole DCN cells in the cerebellum and MSN cells in the striatum; CN3453 / DCN cells in the cerebellum and MSN cells in the striatum; HCT1 / cerebellar DCN cells and spinal motor neurons; CN2146 / Bergmann glia in the cerebellum and astrocytes in the neocortex; AiP1347 / DCN cells in the cerebellum and astrocytes in the neocortex; AiP1351 / Bergmann glia in the cerebellum and astrocytes in the neocortex; AiP1375 / MLI cells in the cerebellum and pericytes in the neocortex; AiP1307 / MLI cells in the cerebellum and L5 IT neurons in the neocortex; AiP1530 ​​(3xcore_E116) / DCN cells in the cerebellum and L5 NP neurons in the neocortex; AiP1379 / Purkinje cells of the cerebellum and specialized smooth muscle cells (SMCs) of the neocortex; CN3916 / MLI cells in the cerebellum and total GABAergic neurons throughout the brain; CN3869 / MLI cells in the cerebellum and total GABAergic neurons throughout the brain; and CN3870 / MLI cells in the cerebellum and total GABAergic neurons throughout the brain.

[0027] Various aspects of the disclosure are described in more detail below with further options: (i) artificial expression constructs and vectors for targeted expression of genes in target cells; (ii) compositions for administration; (iii) cell lines containing the artificial expression constructs; (iv) transgenic animals; (v) methods of use; (vi) kits and commercial packages; (vii) representative embodiments; and (viii) concluding remarks.

[0028] (i) Artificial expression constructs and vectors for targeted expression of genes in target cells The artificial expression constructs disclosed herein comprise (i) an enhancer sequence that induces targeted expression of a coding sequence in a targeted central nervous system cell, (ii) a coding sequence to be expressed, and (iii) a promoter. The artificial expression constructs of the invention may further comprise other regulatory elements as needed or beneficial.

[0029] In certain embodiments, an "enhancer" or "enhancer element" is a cis-acting sequence that increases the amount of transcription associated with a promoter, can function in either the forward or reverse direction relative to the promoter and the coding sequence to be transcribed, and can be located upstream or downstream relative to the promoter or the coding sequence to be transcribed. Various methods or techniques are known in the art for measuring the function of enhancer element sequences. Specific examples of enhancer sequences used in the artificial expression constructs disclosed herein include eHGT_023h v1, eHGT_023m, 3xCore-eHGT_023h v3, eHGT_082h, eHGT_087h, eHGT_128h, eHGT_181h, eHGT_260h, eHGT_023h v2, eHGT_023h v3, eHGT_023h v4, eHGT_023h v5, eHGT_023h v6, eHGT_023h v7, eHGT_023h v8, eHGT_023h v9, eHGT_023h v10, eHGT_023h v12, eHGT_023m, 3xCore-eHGT_023h v3, eHGT_082h, eHGT_087h, eHGT_128h, eHGT_181h, eHGT_260h, eHGT_023h v13, eHGT_023h v24, eHGT_023h v35, eHGT_023h v16, eHGT_023h v28, eHGT_023h v39, eHGT_023h v38, eHGT_023h v39, eHGT_023h v39, eHGT_023h v39, eHGT_023h v3 v2, eHGT_356h, eHGT_359h, eHGT_375m, eHGT_387m, eHGT_395h, 3x(CoreB)eHGT_121h, eHGT_453m, eHGT_470m, eHGT_479m, eHGT_494m, eHGT_ 467m, eHGT_483m, eHGT_606h, eHGT_738m, eHGT_796h, eHGT_710m, eHGT_588m, eHGT_007m, eHGT_703m, eHGT_589m, eHGT_086h, eHGT_963m, 3xc ore3_eHGT_453m, 3xcore2_eHGT_387m, eHGT_534h, 3xcore2_eHGT_475m, eHGT_830h, eHGT_540h, 3xcore2_eHGT_351h, eHGT_882m, eHGT_1137m, eHGT_381h, MGT_E118, MGT_E122, MGT_E146, MGT_E16, 3xcore_MGT_E116, MGT_E150, eHGT_1032h, eHGT_1027h and / or eHGT_1027m.

[0030] In certain embodiments, the enhancer used in targeting central nervous system cells is the enhancer that is only utilized in targeting central nervous system cells, or is the enhancer that is mainly utilized in targeting central nervous system cells.The enhancer used in targeting central nervous system cells is the enhancer that enhances the expression of genes in targeting central nervous system.In certain embodiments, the enhancer used in targeting central nervous system cells enhances the expression of genes in targeting central nervous system, but does not substantially induce the expression of genes in other non-targeting cells, and therefore is also the targeting central nervous system enhancer that has cell type specific transcription activity.

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

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

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

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

[0035] Subclassification of GABAergic neurons in the neocortex: · Overall: expresses the GABA synthesis genes Gad1 / GAD1 and / or Gad2 / GAD2. · Lamp5-, Sncg-, Serpinf1- and Vip-positive GABAergic neurons: neurons that arise from neural precursor cells derived from the caudal ganglia primordium (CGE) or preoptic area (POA) during development. · Sst and Pvalb positive GABAergic neurons: neurons that arise from neural precursor cells derived from the medial ganglia primordium (MGE) during development. Lamp5-positive GABAergic neurons are found in numerous neocortical layers, especially in the upper layers (L1-L2 / 3), and mainly have neurogliaform and single bouquet cell morphologies. · Lamp5_Lhx6 positive GABAergic neurons: a subset of Lamp5 positive GABAergic neurons that co-express Lamp5 and Lhx6. Sncg-positive GABAergic neurons are found in many neocortical layers and contain molecules common to Lamp5-positive and Vip-positive cells, but the expression of Lamp5 and Vip is inconsistent, whereas the expression of Sncg is consistent. Serpinf1-positive GABAergic neurons are found in many neocortical layers and share molecules common to Sncg-positive and Vip-positive cells, but the expression of Sncg and Vip is inconsistent, whereas the expression of Serpinf1 is consistent. Vip-positive GABAergic neurons: Found in many neocortical layers, but more frequently in the upper layers (L1-L4), they highly express the neurotransmitter vasoactive intestinal peptide (Vip). · Sst positive GABAergic neurons: found in many neocortical layers, but especially frequent in the lower layers (L5-L6). They highly express the neurotransmitter somatostatin (Sst) and frequently block dendritic inputs to postsynaptic neurons. This subclass includes sleep-active Sst Chodl neurons (which additionally express Nos1 and Tacr1), which are significantly different from other Sst neurons, but express some shared marker genes, including Sst. Expression of the SST gene in humans is frequently detected in a subtype of LAMP5+ GABAergic neurons in layer 1. Pvalb-positive GABAergic neurons are found in many neocortical layers, but are especially prevalent in the lower layers (L5-L6). These neurons highly express the calcium-binding protein parvalbumin (Pvalb) and the neuropeptide Tac1, which often dampen the output of postsynaptic neurons. Most GABAergic neurons with fast firing properties highly express Pvalb. This subclass includes chandelier cells, which have a characteristic chandelier-like morphology and express the markers Cpne5 and Vipr2 in mice and NOG and UNC5B in humans. Meis2: A distinct subclass defined by one type of cell, neocortical GABAergic neurons, that express the Meis2 gene but do not express several other genes expressed by other neocortical GABAergic neurons (e.g. Thy1 and Scn2b). These cells are found in L6b and subcortical white matter. · White matter (WM)-interneurons: a subgroup of interstitial neurons that express GABA and other neuronal markers.

[0036] Subclassification of neocortical glutamatergic neurons: Overall: These neurons express the glutamatergic transmitters Slc17a6 and / or Slc17a7. Both neurons express Snap25 and lack Gad1 / Gad2 expression. · L2 / 3 IT glutamatergic neurons: located mainly in layers 2 and 3, with mainly intratelencephalic (intercortical) projections. · L4 IT glutamatergic neurons: located mainly in layer 4, with mainly local or intratelencephalic (intercortical) projections. L5 IT glutamatergic neurons: mainly found in layer 5, with mainly intratelencephalic (intercortical) projections. Also called L5a. L5 PT glutamatergic neurons: mainly found in layer 5, mainly with cortico-subcortical (pyramidal or corticofugal) projections. Also called L5b or L5 CF (corticofugal) or L5 ET (extratelencephalic). This subclassification includes cells present in the primary motor cortex and adjacent areas, which are corticospinal projection neurons associated with motor neuron / movement disorders (e.g. ALS). This subclassification also includes thick tufted pyramidal neurons, which include specialized subtypes found only in certain areas, e.g. Betz, Meynert, and von Economo cells. · L5 NP glutamatergic neurons: mainly located in layer 5 and project mainly to nearby areas. · L6 CT glutamatergic neurons: mainly found in layer 6, mainly projecting to the corticothalamus. · L6 IT glutamatergic neurons: located mainly in layer 6 and project primarily intratelencephalic (intercortical) L6 IT Car3 glutamatergic neurons: Most densely present in the claustrum and endopyriform nucleus, but sparsely present throughout L6 in many cortical areas, including the primary visual cortex. These neurons project primarily intratelencephalic (intercortical). Additional marker genes for claustrum-enriched neurons include Gnb4 and Ntng2. · L6b glutamatergic neurons: mainly present in the neocortical subplate (L6b), project locally (near the cell body), and some also project cortically from the VISp to the anterior cingulate bundle and cortico-subcortically to the thalamus. CR neurons: a distinctive subclass defined by a single type found in L1. Cajal-Retzius cells express the characteristic molecular markers Lhx5 and Trp73.

[0037] Classification and subclassification of striatal cells: Medium spiny neurons (total): These account for 95% of striatal neurons and are known to express the GABA synthesis genes Gad1 / GAD1 and Gad2 / GAD2, as well as Ppp1r1b / PPP1R1B. In this specification, medium spiny neurons that express Drd3 are referred to as Drd3+ medium spiny neurons. Medium spiny neurons, direct pathway projection type: Accounting for nearly 50% of striatal neurons, they are enriched in Drd1 / DRD1, Pdyn / PDYN and Slc35d3 / SLC35D3. The main axonal projection destinations from direct pathway type medium spiny neurons are the substantia nigra pars reticulata (SNr) or the globus pallidus internal segment (GPi). Medium spiny neurons, indirect pathway projection type: Accounting for nearly 50% of striatal neurons, they are enriched in Drd2 / DRD2, Adora2a / ADORA2A, Gpr6 / GPR6 and Penk / PENK. The main axonal projection destination from indirect pathway medium spiny neurons is the external segment of the globus pallidus (GPe). Striatal interneurons - cholinergic: A rare interneuron population that comprises 1% of striatal neurons. These local interneurons have large cell bodies, few dendritic spines, express Chat / CHAT, and are known to release the neurotransmitter acetylcholine.

[0038] Subdivision of the cerebellum: Purkinje cells: large GABAergic neurons that are the only projection neurons from the cerebellum. The cell bodies of cerebellar Purkinje cells form a single layer, the so-called "Purkinje cell layer," and express parvalbumin. Other known marker genes include Gad1, Pcp2, and Calb1. Deep cerebellar nucleus (DCN) neurons: neurons found in the deep cerebellar nucleus structures. DCN neurons include excitatory and inhibitory cells, some of which express the Pvalb gene. Excitatory DCN cells can be identified by the lack of Gad1 expression. Glutamatergic excitatory cells express Slc17a6, and glycinergic excitatory cells express Slc6a5. Inhibitory DCN cells express Gad1, and glycinergic inhibitory cells additionally express Slc6a5. Molecular layer interneurons (MLIs): They control Purkinje cell activity through inhibitory synaptic transmission. Molecular layer interneurons include stellate cells and basket cells. Typical marker genes include Gad1, Pvalb, Kit and Sorcs3. Stellate cells and basket cells are GABAergic interneurons characterized by the expression of Pvalb in the molecular layer of the cerebellum. Bergmann glial cells: glutamate uptake and extracellular potassium ions (K + ) homeostasis. Typical marker genes include Gpr37l1, Gdf10, Id4, and Npy. Granule cells: Granule cells in the cerebellar cortex give rise to parallel fibers, specialized axons that ascend to the molecular layer of the cerebellar cortex. Exemplary marker genes include Slc17a7, Calb2, Gabra6 and Fat2.

[0039] Striatal cholinergic interneurons are the major source of acetylcholine in the striatum and are important for basal ganglia physiology and pathophysiology.

[0040] Spinal motor neurons: Motor neurons are specialized neurons located in the spinal cord and brain that integrate signals from the central nervous system and sensory systems to control voluntary and involuntary movements. Spinal motor neurons receive input from intracortical neurons and relay that information to control muscles throughout the body.

[0041] Subclassification of non-neuronal cells: Astrocytes: Glial cells derived from the neuroectoderm that express the marker Aqp4 and often also GFAP, but not the neuronal marker SNAP25. Astrocytes can have a characteristic star-shaped morphology and are involved in the metabolic support of other cells in the brain. Many types of astrocyte morphology are observed in mice and humans. Oligodendrocytes: Neuroectoderm-derived glial cells that express the Sox10 marker. This category includes oligodendrocyte precursor cells (OPCs). Oligodendrocytes are a subcategory primarily responsible for myelination of neurons. · VLMC: Vascular leptomeningeal cells (VLMCs), which are part of the meninges surrounding the outer layer of the cortex, 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. Smooth muscle cells (SMCs): Specialized smooth muscle cells, vascular associated cells that express the Acta2 marker gene. Smooth muscle cells line arterioles in the brain and are involved in blood-brain barrier crossing. · Endothelial cells: These are the cells that line the blood vessels in the brain. Endothelial cells express the Tek and PDGF-B markers. Microglia: immune cells derived from hematopoietic cells, macrophages localized in brain tissue, perivascular macrophages (PVM) that can migrate from the blood and associate with brain tissue, and can be seen as a by-product of brain dissection procedures. Microglia are known to express Cx3cr1, Tmem119, and PTPRC (CD45).

[0042] In certain embodiments, the coding sequence is a heterologous coding sequence that codes for an effector element.Effector element is the sequence that is expressed to obtain an intended effect, and the intended effect is actually achieved by this effector element.Examples of effector elements include reporter genes / proteins and functional genes / proteins.

[0043] Representative reporter genes / proteins include those expressed by Addgene ID No. 83894 (pAAV-hDlx-Flex-dTomato-Fishell_7), ID No. 83895 (pAAV-hDlx-Flex-GFP-Fishell_6), ID No. 83896 (pAAV-hDlx-GiDREADD-dTomato-Fishell-5), ID No. 83898 (pAAV-mDlx-ChR2-mCherry-Fishell-3), ID No. 83899 (pAAV-mDlx-GCaMP6f-Fishell-2), ID No. 83900 (pAAV-mDlx-GFP-Fishell-1), and ID No. 89897 (pcDNA3-FLAG-mTET2(N500)). Representative reporter genes include, inter alia, expressible fluorescent proteins or expressible biotin; blue fluorescent proteins (e.g., eBFP, eBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire); cyan fluorescent proteins (e.g., eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan, mTurquoise); green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green (mAzamigreen), CopGFP, AceGFP, avGFP, ZsGreen1, Oregon Green, etc.) TM (Thermo Fisher Scientific); luciferase; orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato, dTomato); red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRuby, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred, Texas Red) TM(Thermo Fisher Scientific)); far-red fluorescent proteins (e.g., mPlum and mNeptune); yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, SYFP2, Venus, YPet, PhiYFP, ZsYellow1); or reporter genes encoding tandemly linked complexes.

[0044] GFP is composed of 238 amino acids (26.9 kDa) and was first isolated from the jellyfish Aequorea victoria / Aequorea aequorea / Aequorea forskalea, which fluoresces green when exposed to blue light. GFP isolated from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor excitation peak at 475 nm. Its emission peak is at 509 nm, which is in the low wavelength range of green light in the visible spectrum. GFP from the sea pansy (Renilla reniformis) has one major excitation peak at 498 nm. Due to its wide range of applications and the demand from researchers for further improvements, various GFP variants have been created. The first major improvement was a single point mutation (S65T) reported in Nature by Roger Tsien in 1995. This mutation dramatically improved the spectral properties of GFP, increasing its fluorescence and photostability, and shifting the major excitation peak to 488 nm while maintaining the emission peak at 509 nm. Enhanced GFP (EGFP) was obtained by adding a point mutation (F64L) to GFP that improves folding efficiency at 37 °C. EGFP has an extinction coefficient (denoted ε) of 55,000 L / mol cm, which is 9.13 × 10 per molecule. -21 m 2 Also known as the optical cross section of GFP, Superfolder GFP was reported in 2006 as a series of GFP mutants that can rapidly fold and mature even when fused to poorly folded peptides.

[0045] "Yellow fluorescent protein" (YFP) is a genetic variant of the green fluorescent protein derived from Aequorea victoria. Its excitation peak is at 514 nm and its emission peak is at 527 nm.

[0046] Representative functional molecules include ion transporters, cell transport proteins, enzymes, transcription factors, neurotransmitters, calcium reporters, channelrhodopsins, guide RNAs, nucleases, microRNAs, or designer receptors activated only by designer drugs (DREADDs), each of which has a function.

[0047] Ion transporters are transmembrane proteins responsible for the transport of ions across cell membranes. Ion transporters are found in the majority of cells and are important in regulating cellular excitability and homeostasis. Ion transporters are involved in numerous cellular processes, including action potentials, synaptic transmission, hormone secretion, and muscle contraction. Many biological processes important to living cells involve the transport of calcium ions (Ca) through ion channels. 2+ ), potassium ion (K + ), sodium ion (Na + In certain embodiments, ion transporters include voltage-gated sodium channels (e.g., SCN1A), potassium channels (e.g., KCNQ2), and calcium channels (e.g., CACNA1C).

[0048] Representative enzymes, transcription factors, receptors, membrane proteins, cellular transport proteins, signaling molecules and neurotransmitters include enzymes such as lactase, lipase, helicase, α-glucosidase, amylase; transcription factors such as SP1, AP-1, heat shock factor protein 1, C / EBP (CCAAT / enhancer binding protein), Oct-1; receptors such as transforming growth factor receptor β1, platelet-derived growth factor receptor, epidermal growth factor receptor, vascular endothelial growth factor receptor, interleukin-8 receptor α; membrane proteins and cellular transport proteins such as clathrin, dynamin, caveolin, Rab4A, Rab-11A; signaling molecules such as nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGFβ), epidermal growth factor (EGF), GTPase, HRas; and neurotransmitters such as cocaine- and amphetamine-regulated transcript, substance P, oxytocin, somatostatin.

[0049] In certain embodiments, the functional molecules include reporters that indicate cell function and state, such as calcium reporters. Intracellular calcium concentration is an important predictor of many cellular activities, such as neuronal activation, muscle cell contraction, and second messenger signaling. A sensitive and simple technique for monitoring intracellular calcium concentration is the use of genetically encoded calcium indicators (GECIs). Among GECIs, a green fluorescent protein (GFP)-based calcium sensor, named GCaMP, is highly efficient and widely used. GCaMP is formed by fusing M13 and calmodulin proteins to the N- and C-termini of circularly permuted GFP. Some types of GCaMP exhibit characteristic fluorescence emission spectra (Zhao et al., Science, 2011, 333(6051): 1888-1891). Representative GECIs that exhibit green fluorescence include GCaMP3, GCaMP5G, GCaMP6s, GCaMP6m, GCaMP6f, jGCaMP7s, jGCaMP7c, jGCaMP7b, jGCaMP7f, jGCaMP8s, jGCaMP8m, and jGCaMP8f. In addition, GECIs that exhibit red fluorescence include jRGECO1a and jRGECO1b. AAV products containing GECIs are commercially available.For example, AAV8-CAG-GCaMP3 (catalog no. BS4-CX3AAV8), AAV8-Syn-FLEX-GCaMP6s-WPRE (catalog no. BS1-NXSAAV8), AAV8-Syn-FLEX-GCaMP6s-WPRE (catalog no. BS1-NXSAAV8), AAV9-CAG-FLEX-GCaMP6m-WPRE (catalog no. BS2-CXMAAV9), AAV9-Syn-FLEX-jGCaMP7s-WPRE ( AAV products available include AAV9-CAG-FLEX-jGCaMP7f-WPRE (Catalog No. BS12-CXFAAV9), AAV9-Syn-FLEX-jGCaMP7b-WPRE (Catalog No. BS12-NXBAAV9), AAV9-Syn-FLEX-jGCaMP7c-WPRE (Catalog No. BS12-NXCAAV9), AAV9-Syn-FLEX-NES-jRGECO1a-WPRE (Catalog No. BS8-NXAAAV9), and AAV8-Syn-FLEX-NES-jRCaMP1b-WPRE (Catalog No. BS7-NXBAAV8).

[0050] In certain embodiments, the calcium reporter includes the genetically encoded calcium indicator (GECI) NTnC; a myosin light chain kinase-GFP-calmodulin chimera; the calcium indicator TN-XXL; a BRET-based auto-luminescent calcium indicator; and / or the calcium indicator protein OeNL(Ca2+)-18μ.

[0051] In certain embodiments, the functional molecules include modulators of neuron-active channelrhodopsins (e.g., channelrhodopsin 1, channelrhodopsin 2, and variants thereof). Channelrhodopsins are a subfamily of retinylidene 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) describe the creation of transmembrane domain chimeras of ChR1 and ChR2 using site-directed mutagenesis. Zhang et al. (Nat Neurosci, 2008, 11(6): 631-3) describe a red-light shifted channelrhodopsin variant, VChR1. VChR1 has reduced photosensitivity and reduced membrane trafficking and expression. Other known channelrhodopsin variants include the ChR2 variant 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), all of which are activated by blue light (470 nm) but are insensitive to orange / red light. Other variants are described in Lin, Experimental Physiology, 2010, 96.1: 19-25; Knopfel et al., The Journal of Neuroscience, 2010, 30(45): 14998-15004; and Mardinly et al., Nat Neurosci. 2018, 21(6):881-893.

[0052] In certain embodiments, the functional molecule includes DNA and RNA editing tools, such as CRISPR / Cas (e.g., guide RNA and nuclease such as Cas, Cas9, cpf1).Furthermore, the functional molecule includes recombinant Cpf1 as described in US Patent Publication No. 2018 / 0030425, US Patent Publication No. 2016 / 0208243, WO / 2017 / 184768 and Zetsche et al. (2015) Cell 163: 759-771; single-stranded gRNA (see, for example, Jinek et al. (2012) Science 337:816-821; Jinek et al. (2013) eLife 2:e00471; Segal (2013) eLife 2:e00563), editase, guide RNA molecule, microRNA, or homologous recombination donor cassette.

[0053] In certain embodiments, the functional molecule includes a localization cassette. In certain embodiments, the localization cassette is used to localize a molecule (e.g., a vector, a protein, a sensor) to a specific subcellular compartment, such as the cell body, axon, or dendrite of a neuron. In certain embodiments, the localization cassette includes a somatic tag (e.g., soma (EE-RR)) for localization to the cell body; an axon tag (e.g., derived from GAP43) or synaptophysin (sy) for localization to the axon; a hydrophobic tail for localization to the cell membrane; and a hydrophobic or alkyl chain for localization to the endoplasmic reticulum. In certain embodiments, the localization cassette is fused to a sensor molecule, such as a GECI. In certain embodiments, the fusion protein of the localization cassette and the GECI includes soma-jGCaMP8s, axon-jRGECO1a, syGCaMP5G, and soma-jGCaMP7s.

[0054] In certain embodiments, the functional molecule includes a tag cassette. The tag cassettes include His tag (HHHHHH; SEQ ID NO: 151), Flag tag (DYKDDDDK; SEQ ID NO: 152), Xpress tag (DLYDDDDK; SEQ ID NO: 153), Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO: 154), calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 155), polyglutamic acid tag, HA tag (YPYDVPDYA; SEQ ID NO: 156), Myc tag (EQKLISEEDL; SEQ ID NO: 157), Strep tag (meaning the original STREP (registered trademark) tag) (WRHPQFGG; SEQ ID NO: 158), STREP tag II (WSHPQFEK; SEQ ID NO: 159; (Institut fur Bioanalytik (IBA) GmbH, Germany; see, e.g., U.S. Patent Publication No. 7,981,632), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 160), Softag In certain embodiments, the tag cassette includes a fusion tag cassette such as 3XFLAG. In certain embodiments, the 3XFLAG includes the sequence shown in SEQ ID NO: 164. In certain embodiments, the artificial expression construct of the present invention includes 10aa (SEQ ID NO: 38).

[0055] The sequences of the aforementioned functional molecules have been published, for example, lactase (e.g., GenBank: EAX11622.1), lipase (e.g., GenBank: AAA60129.1), helicase (e.g., GenBank: AMD82207.1), amylase (e.g., GenBank: AAA51724.1), α-glucosidase (e.g., GenBank: ABI53718.1), transcription factor SP1 (e.g., UniProtKB / Swiss-Prot: P08047.3), transcription factor AP-1 (e.g., NP_002219.1), heat shock factor protein 1 (e.g., UniProtK B / 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), TGF-β (e.g. GenBank:CAF02096.2), glial cell line-derived neurotrophic factor (GDNF) (e.g. NP_001177397.1), platelet-derived growth factor receptor (e.g. GenBank:AAA60049.1), epidermal growth factor receptor (e.g. GenBank:CAA25 240.1), vascular endothelial growth factor receptor (e.g., GenBank: AAC16449.2), interleukin-8 receptor α (e.g., GenBank: AAB59436.1), caveolin (e.g., GenBank: CAA79476.1), dynamin (e.g., GenBank: AAA88025.1), clathrin heavy chain 1 isoform 1 (e.g., NP_004850.1), clathrin heavy chain 2 isoform 1 (e.g., NP_009029.3), clathrin light chain A isoform a (e.g., NP_001824.1), clathrin light chain B isoform a (e.g. NP_001825.1), ras-related protein Rab-4A isoform 1 (e.g. NP_004569.2), ras-related protein Rab-11A (e.g. UniProtKB / Swiss-Prot:P62491.3), platelet-derived growth factor (e.g. GenBank:AAA60552.1), transforming growth factor beta 3 (e.g. GenBank:AAA61161.1), nerve growth factor (e.g. GenBank:CAA37703.1), EGF (e.g. GenBank:CAA34902.2), cocaine-amphetamine regulated transcript (A chain) (e.g. PDB:1HY9_A), protachykinin-1 (e.g. UniProtKB-P20366), oxytocin neurophysin 1 (e.g. UniProtKB-P01178), somatostatin (e.g. GenBank:AAH32625.1), genetically encoded green calcium indicator NTnC (A chain) [synthetic construct] (e.g. PDB:5MWC_A), calcium indicator TN-XXL [synthetic construct] (e.g. GenBank:ACF93133.1), BRET-based auto-luminescent calcium indicator [synthetic construct] (e.g., GenBank: ADF42668.1), calcium indicator protein OeNL(Ca2+)-18μ [synthetic construct] (e.g., GenBank: BBB18812.1), myosin light chain kinase, green fluorescent protein, calmodulin chimera (A chain) [synthetic construct] (e.g., PDB: 3EKJ_A), channelopsin 1 (e.g., UniProtKB-F8UVI5), channelopsin 1 (e.g., GenBank: AER58217.1), channelrhodopsin 2 (e.g., UniProtKB-B4Y105), channelrhodopsin 2 [synthetic construct] (e.g., GenBank: ABO64386.1), CRISPR-associated proteins (Cas) (e.g., GenBank: AKG27598.1), Cas9 [synthetic construct] (e.g., GenBank: AST09977.1), CRISPR-associated endonuclease Cpf1 (e.g., UniProtKB / Swiss-Prot: U2UMQ6.1), ribonuclease Examples of such proteins include ribonuclease 4 or ribonuclease L (e.g. UniProtKB / Swiss-Prot:Q05823.2), deoxyribonuclease IIβ (e.g. GenBank:AAF76893.1), sodium channel protein type 1 subunit α (e.g. UniProtKB-P35498), member 2 of the voltage-gated potassium channel subfamily KQT (e.g. UniProtKB-O43526) and voltage-gated L-type calcium channel subunit α-1C (e.g. UniProtKB-Q13936).

[0056] Further effector elements include Cre, iCre, dgCre, FlpO and tTA2. iCre refers to codon-improved Cre. dgCre is a GFP / Cre recombinase fusion gene enhanced by the N-terminal fusion of the first 159 amino acids of the dihydrofolate reductase gene (DHFR or folA) of the E. coli K12 chromosome, which has a G67S mutation and a destabilization domain mutation R12Y / Y100I upon recombination. FlpO is a codon-optimized form of FLPe, which significantly improves protein expression and FRT recombination efficiency in mouse cells. The FLP / FRT system is widely used for gene expression, as is the Cre / LoxP system (the generation of conditional knockout mice using the FLP / FRT system is also widely practiced). tTA2 refers to tetracycline transactivator.

[0057] Representative expressible elements include expression products that do not include effector elements, such as non-functional or defective proteins. In certain embodiments, such expressible elements can be used to perform methods for testing the effect of their corresponding functional molecules. In certain embodiments, the expressible elements are non-functional or defective due to recombinant mutations that abolish their function. In these aspects, the non-expressible elements are as similar in structure as possible to their corresponding functional molecules.

[0058] A representative self-cleaving peptide is the 2A peptide, which allows two proteins to be produced from one mRNA. The 2A sequence is a short sequence (e.g., 20 amino acids long) and is often used in size-restricted 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 can initiate ribosome translation from a second internal site on the mRNA molecule, allowing two proteins to be produced from one mRNA.

[0059] The artificial expression construct may encode nuclear transport proteins such as histone H1, histone H2A, histone H2B, histone H3, histone H4, histone-like proteins HPhA, H2Bmod, and H2B*.

[0060] Coding sequences encoding the molecules (e.g., RNA and proteins) described herein can be obtained from publicly available databases and publications. The coding sequences may further contain various sequence polymorphisms, mutations and / or sequence variants, and such changes do not affect the function of the encoded molecule. "Encode" refers to the property of a nucleic acid sequence, such as a vector, plasmid, gene, cDNA, mRNA, etc., to function as a template for the synthesis of other molecules, such as proteins.

[0061] The term "gene" may include not only coding sequences, but also regulatory regions such as promoters, enhancers, insulators and / or post-transcriptional regulatory elements (e.g., termination regions). In addition, the term may include any introns and other DNA sequences spliced ​​from the mRNA transcript, as well as variants resulting from alternative splice sites. These sequences may further include sequences or degenerate codons of a reference sequence that may be introduced to confer codon preference in a particular type of organism or cell.

[0062] The promoter may be a general promoter, a tissue-specific promoter, a cell-specific promoter, and / or a cytoplasm-specific promoter. The promoter may be a strong promoter, a weak promoter, a constitutive expression promoter, and / or an inducible promoter. An inducible promoter induces expression in response to a specific condition, signal, or cellular event. For example, the promoter may be an inducible promoter that requires a specific ligand, small molecule, transcription factor, or hormone protein to induce transcription from the promoter. Specific examples of promoters include minBglobin (also called minBGprom), CMV, minCMV, minCMV* (minCMV* is minCMV with the SacI restriction site removed), minRho, minRho* (minRho* is minRho with the SacI restriction site removed), SV40 immediate early promoter, Hsp68 minimal promoter (proHSP68), and Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter. A minimal promoter does not have the activity of inducing gene expression by itself, but when linked to an enhancer element nearby, it is activated and can induce gene expression.

[0063] In certain embodiments, the expression construct is provided in a vector. A "vector" refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule, such as an expression construct. The transferred nucleic acid is usually linked to, e.g., inserted into, the nucleic acid molecule of the vector. The vector may contain a sequence that induces autonomous replication of the cell or may contain a sequence that allows integration into the DNA of the host cell. Useful vectors include, for example, plasmids (e.g., DNA and RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.

[0064] The term "viral vector" is used broadly to refer to a nucleic acid molecule that contains components derived from a virus that facilitate the transfer and expression of non-natural nucleic acid molecules in cells. An "adeno-associated viral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements or portions thereof that are primarily derived from AAV. A "retroviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements or portions thereof that are primarily derived from a retrovirus. A "lentiviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements or portions thereof that are primarily derived from a lentivirus or the like. A "hybrid vector" refers to a vector that contains structural and / or functional genetic elements that are primarily derived from more than one virus.

[0065] "Adenoviral vector" refers to a construct that contains sufficient adenoviral sequences (a) to facilitate packaging of an artificial expression construct, and (b) to express a coding sequence cloned in the sense or antisense orientation. Recombinant adenoviral vectors include genetically engineered forms of adenovirus. The genetic makeup of adenovirus is a 36 kb linear double-stranded DNA virus, allowing replacement of large portions of adenoviral DNA with up to 7 kb of foreign sequence. Adenoviral DNA can replicate episomally without causing genotoxicity, and thus, unlike retroviruses, is not integrated into chromosomes upon infection of a host cell with adenovirus. Furthermore, adenoviruses are structurally stable, and no genome rearrangements have been detected after extensive amplification.

[0066] Adenoviruses are particularly suitable for use as gene transfer vectors due to their moderate genome size, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the adenovirus genome contain inverted repeats (ITRs) of 100-200 base pairs in length, which are cis elements required for viral DNA replication and packaging. The early (E) and late (L) regions of the adenovirus genome contain various transcription units that are divided by the initiation of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for regulating the transcription of the adenoviral 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, expression of late genes and shut-off of host cell protein biosynthesis. Late gene products, including the majority of adenovirus capsid proteins, are expressed only after significant processing of a single primary transcript driven by the major late promoter (MLP). The MLP is particularly efficient at the late stages of infection, when all mRNAs driven by this promoter have a tripartite 5'-leader (TPL) sequence and are preferentially selected by the mRNA for translation.

[0067] Other than the requirement that the adenoviral vector be replication-deficient or at least conditionally-deficient, the characteristics of the adenoviral vector are not believed to be critical to the successful implementation of certain embodiments disclosed herein. The adenovirus may be of any of the 42 known serotypes or subgenuses A-F. In certain embodiments, adenovirus of serotype 5 of the C subgenus is preferred as the starting material for obtaining a conditionally replication-deficient adenoviral vector for use in certain embodiments, since type 5 adenovirus is a human adenovirus with a large amount of known biochemical and genetic information and has been used historically in the majority of constructions using adenoviruses as vectors.

[0068] As described herein, the vector is generally replication-defective and lacks the E1 region of adenovirus. Therefore, it is most convenient to introduce the polynucleotide encoding the gene of interest into the position where the coding sequence of the E1 region has been deleted. However, the insertion position of the construct within the adenovirus sequence is not critical. The polynucleotide encoding the gene of interest may be inserted into the deleted E3 region of the E3 replacement vector, or into the E4 region, and the defect in the E4 region is complemented by a helper cell line or a helper virus.

[0069] Adeno-associated virus (AAV) is a parvovirus found as a contaminant of adenovirus stocks. AAV is a ubiquitous virus (85% of the US population has anti-AAV antibodies) and does not cause disease. Furthermore, AAV is classified as a dependovirus because its replication depends on the presence of a helper virus (e.g., adenovirus). Various serotypes have been isolated, of which AAV-2 is the most extensively characterized. AAV has a single-stranded linear DNA that is packaged with the capsid proteins VP1, VP2, and VP3 to form icosahedral virions with a diameter of 20-24 nm.

[0070] The length of AAV DNA is 4.7 kilobases. AAV DNA contains two open reading frames, flanked by two ITRs. There are two main genes in the AAV genome: rep and cap. The rep gene codes for the proteins responsible for AAV viral replication, and the cap gene codes for the capsid proteins VP1-3. Each ITR forms a T-shaped hairpin structure. These terminal repeats are the only cis components of AAV required for chromosomal integration. Thus, AAV can be used as a vector in which all viral coding sequences can be removed and replaced with gene cassettes for delivery. Three AAV viral promoters have been identified and named p5, p19, and p40, respectively, based on their map location. Transcription from p5 and p19 results in the production of the rep protein, and transcription from p40 produces the capsid protein.

[0071] AAV is outstanding for use in the present disclosure because it has a good safety profile and can be expressed in target cell populations by modifying capsid and genome.scAAV refers to self-complementary AAV.pAAV refers to plasmid adeno-associated virus.rAAV refers to recombinant adeno-associated virus.

[0072] Other viral vectors may also be used, for example vectors derived from viruses such as vaccinia virus, poliovirus or herpes virus, which offer beneficial characteristics for a variety of mammalian cells.

[0073] Retroviruses are commonly used tools for gene delivery. A retrovirus is an RNA virus whose genomic RNA is reverse transcribed to produce a double-stranded linear DNA copy, which is then covalently integrated into the host genome. Once integrated into the host genome, the retrovirus is called a provirus. The provirus functions as a template for RNA polymerase II to induce the expression of RNA molecules that code for structural proteins and enzymes required for the production of new viral particles.

[0074] Examples of retroviruses suitable for use in certain embodiments include Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), Rous sarcoma virus (RSV), and lentiviruses.

[0075] "Lentivirus" refers to the complex retrovirus group (or complex retrovirus genus). Examples of lentiviruses include HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Visna-Maedi virus (VMV); Caprine arthritis-encephalomyelitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV). In certain embodiments, a vector backbone based on HIV (i.e., HIV cis-acting sequence elements) can be used.

[0076] In some types of vectors, safety can be improved by replacing the U3 region of the 5'LTR, which induces the transcription of the viral genome in the production of viral particles, with a heterologous promoter. Examples of heterologous promoters that can be used for this purpose include, for example, Simian Virus 40 (SV40) (e.g., early or late) promoter, Cytomegalovirus (CMV) (e.g., immediate early) promoter, Moloney Murine Leukemia Virus (MoMLV) promoter, Rous Sarcoma Virus (RSV) promoter, and Herpes Simplex Virus (HSV) (thymidine kinase) promoter. Conventional promoters can induce high levels of transcription independent of Tat. Replacement of the U3 region with a heterologous promoter removes the complete U3 sequence from the virus production system, reducing the possibility of recombination that produces a replicable virus. In certain embodiments, a heterologous promoter has the additional advantage of being able to control the way the viral genome is transcribed. For example, the heterologous promoter can be an inducible promoter, such that the entire viral genome or a portion thereof is transcribed only when an inducer is present. Inducers include one or more compounds or physiological conditions, such as the culture temperature or pH of the host cell.

[0077] In certain embodiments, the viral vector comprises a TAR element. "TAR" refers to the "transactivation response" gene element present in the R region of the LTR of lentivirus. This element interacts with the transactivator (tat) gene element of lentivirus to enhance viral replication. However, this element is not required in the embodiment that replaces the U3 region of 5'LTR with a heterologous promoter.

[0078] The "R region" refers to the region in the retroviral LTR from the start of the cap site (i.e., the transcription start site) to just before the start of the poly(A) tail. The R region is also defined as the region between the U3 and U5 regions. The R region plays a role in moving nascent DNA from one end of the genome to the other during reverse transcription.

[0079] In certain embodiments, the expression of heterologous sequences in viral vectors can be increased by incorporating post-transcriptional regulatory elements and efficient polyadenylation sites into the viral vector, and a transcription termination signal may also be incorporated into the viral vector. Various post-transcriptional regulatory elements can increase the expression of heterologous nucleic acids. Examples of post-transcriptional regulatory elements include the Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the Hepatitis B virus post-transcriptional regulatory element (HPRE) (Smith et al., Nucleic Acids Res. 26(21):4818-4827, 1998); and other post-transcriptional regulatory elements (Liu et al., 1995, Genes Dev., 9:1766). In certain embodiments, the vector includes a post-transcriptional regulatory element such as a WPRE or HPRE. In certain embodiments, the vector lacks or does not include a post-transcriptional regulatory element such as a WPRE or HPRE.

[0080] Expression of heterologous genes can be increased by elements capable of inducing efficient transcription termination and polyadenylation of heterologous nucleic acid transcripts. Transcription termination signals are usually found downstream of polyadenylation signals. In certain embodiments, vectors contain a polyadenylation signal at the 3' end of a polynucleotide encoding an expressed molecule (e.g., a protein). "Poly(A) site" or "poly(A) sequence" refers to a DNA sequence that induces both transcription termination and polyadenylation of a nascent RNA transcript transcribed by RNA polymerase II. Polyadenylation sequences can improve mRNA stability by adding a poly(A) tail to the 3' end of a coding sequence, thereby contributing to improved translation efficiency. In certain embodiments, BGHpA, hGHpA, or SV40pA may be utilized. In certain embodiments, a preferred embodiment of an expression construct includes a terminator element. Terminator elements can increase the amount of transcription and minimize transcription from the construct to another plasmid sequence by read-through.

[0081] In certain embodiments, the viral vector further comprises one or more insulator elements. The insulator elements may protect sequences expressed from the viral vector, such as effector elements and expressible elements, from integration site effects. Integration site effects occur through cis-acting elements in genomic DNA, meaning that the imported sequence is either expressed or not expressed (i.e., position effects; see, for example, Burgess-Beusse et al., PNAS., USA, 99:16433, 2002; and Zhan et al., Hum. Genet., 109:471, 2001). In certain embodiments, the viral import vector comprises one or more insulator elements in the 3'LTR, and upon provirus integration into the host genome, this insulator is integrated into both the 5'LTR and the 3'LTR during the replication of the 3'LTR. Insulators suitable for use in certain embodiments include the chicken β-globin insulator (see Chung et al., Cell 74:505, 1993; Chung et al., PNAS USA 94:575, 1997; and Bell et al., Cell 98:387, 1999), the SP10 insulator (Abhyankar et al., JBC 282:36143, 2007), or other small CTCF recognition sequences that function as enhancer-blocking insulators (Liu et al., Nature Biotechnology, 33:198, 2015).

[0082] In addition to the above, various types of suitable expression vectors are also known to those skilled in the art. These known expression vectors include commercially available expression vectors designed for general recombinant manipulation, such as plasmids that contain one or more reporter genes and the regulatory elements required for the reporter gene to be expressed in cells. Many vectors are commercially available, such as from Invitrogen, Stratagene, Clontech, etc., and are described in various accompanying guidebooks. In certain embodiments, suitable expression vectors include any plasmid, cosmid, or phage construct that can express the encoded gene in mammalian cells, such as the pUC plasmid system and the Bluescript plasmid system.

[0083] Particular embodiments of the vectors disclosed herein include those set forth in the table below. [Table 1] TIFF2024537165000003.tif38154

[0084] Subcomponent sequences within a larger vector sequence can be readily identified by one of skill in the art based on the disclosure herein (see FIG. 8). The nucleotides between the identifiable subcomponents listed in the table above are restriction enzyme recognition sites used in construct assembly (cloning) and, in some cases, additional nucleotides with no identifiable function. These segments of the complete vector sequence can be adjusted using different cloning techniques and / or different vectors. Short palindromic sequences of six bases usually represent vector construction artifacts that are not critical to the function of the vector.

[0085] In certain embodiments, a vector (e.g., AAV) is selected that has a capsid that can cross the blood-brain barrier (BBB). In certain embodiments, the vector is engineered to contain a capsid that crosses the blood-brain barrier. Examples of AAVs with viral capsids that can 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 capsid of PHP.eB differs from that of AAV9 in that the amino acid residues from position 586 onwards, S-AQ-A (SEQ ID NO: 92), are changed to S-DGTLAVPFK-A (SEQ ID NO: 93), when comparing AAV9 as a reference. In certain embodiments, PHP.eb refers to the sequence of SEQ ID NO: 56.

[0086] AAV9 is a naturally occurring AAV serotype that, unlike many other naturally occurring serotypes, is able to cross the blood-brain barrier (BBB) ​​upon intravenous injection. AAV9 transduces a wide area of ​​the central nervous system (CNS), allowing for minimally invasive therapeutic approaches (Naso et al., BioDrugs. 2017; 31(4): 317). Such cases have been reported, for example, in connection with AveXis' clinical trial for the treatment of spinal muscular atrophy (SMA) syndrome (AVXS-101, NCT03505099) and the clinical trial for the treatment of CLN3-associated neuronal ceroid lipofuscinosis (NCT03770572).

[0087] AAVrh.10 is an AAV originally isolated from rhesus macaques that has weak human seroreactivity compared to other common serotypes used in gene delivery applications (Selot et al., Front Pharmacol. 2017; 8: 441) and has been evaluated in several clinical trials (LYS-SAF302, LYSOGENE and NCT03612869).

[0088] AAV1R6 and AAV1R7 are two variants isolated from a library of chimeric AAV vectors in which the capsid domain of AAVrh.10 is replaced by that of AAV1, which retain the ability to cross the BBB and transduce the central nervous system but show significantly reduced transduction of liver and vascular endothelium.

[0089] rAAVrh.8, also an AAV isolated from rhesus macaques, showed widespread transduction of glial and neuronal cells in clinically relevant areas following peripheral administration, with reduced peripheral tissue tropism compared to other vectors.

[0090] AAV-BR1 is an AAV2 variant that displays the NRGTEWD ​​epitope (SEQ ID NO: 94) and was isolated during in vivo screening of a random AAV-display peptide library. AAV-BR1 exhibits high specificity with high transgene expression in the brain and minimal off-target affinity, including for the liver (Korbelin et al., EMBO Mol Med. 2016; 8(6): 609).

[0091] AAV-PHP.S (Addgene, Watertown, MA) is a variant of AAV9 generated by the CREATE method that encodes the 7-mer sequence QAVRTSL (sequence number 95) and transduces neurons of the enteric nervous system and potently transduces peripheral sensory afferents that project to the spinal cord and brainstem.

[0092] AAV-PHP.B (Addgene, Watertown, MA) is a variant of AAV9 generated by the CREATE method that encodes the 7-mer sequence TLAVPFK (SEQ ID NO: 96). AAV-PHP.B transfers genes throughout the CNS more efficiently than AAV9, transducing a large proportion of astrocytes and neurons in multiple CNS regions.

[0093] AAV-PPS is an AAV2 variant created by inserting the DSPAHPS epitope (SEQ ID NO: 97) into the capsid of AAV2, and exhibits dramatically improved brain tropism compared to AAV2.

[0094] For more information regarding capsids crossing the blood-brain barrier, see Chan et al., Nat. Neurosci. 2017 Aug: 20(8): 1172-1179.

[0095] (ii) Composition for Administration The artificial expression constructs and vectors (herein referred to as bioactive components) of the present disclosure can be formulated with carriers suitable for administration to cells, tissue slices, animals (e.g., mice and non-human primates), or humans. The bioactive components contained in the compositions described herein can be prepared in a neutral form, can be prepared as a free base, or can be prepared as a pharmacologically acceptable salt.

[0096] Pharmaceutically acceptable salts include the acid addition salts (formed from the free amino groups of the protein) which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, mandelic, etc. Also, salts formed with the free carboxyl groups can be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, or organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like.

[0097] Carriers for biologically active ingredients include solvents, dispersion media, vehicles, coating agents, diluents, isotonicity agents, absorption delaying agents, buffers, solutions, suspensions, colloids, etc. The use of such carriers for biologically active ingredients is well known in the art. Except insofar as a conventional media or agent is incompatible with the biologically active ingredients of the present invention, any conventional media or agent can be used in combination with the compositions described herein.

[0098] A "pharmacologically 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 retro-orbital plexus).

[0099] In certain embodiments, compositions of the invention can be formulated for intravenous, intraparenchymal, intraocular, intravitreal, parenteral, subcutaneous, intraventricular, intramuscular, intrathecal, intraspinal, intraperitoneal, oral or nasal inhalation, or for direct injection or administration into one or more cells, tissues or organs.

[0100] The compositions of the present invention may comprise liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres and / or nanoparticles.

[0101] The formation of liposomes and their use are widely known to those skilled in the art. Liposomes have been developed to improve serum stability and blood half-life (see, for example, U.S. Patent No. 5,741,516). In addition, various methods have been reported for using liposomes and liposome-like preparations as potential drug carriers (see, for example, U.S. Patent No. 5,567,434; U.S. Patent No. 5,552,157; ​​U.S. Patent No. 5,565,213; U.S. Patent No. 5,738,868; and U.S. Patent No. 5,795,587).

[0102] The present disclosure also provides pharma- ceutically acceptable nanocapsule formulations of the bioactive ingredients of the present invention. In general, nanocapsule formulations can encapsulate compounds in a stable and reproducible manner (Quintanar-Guerrero et al., Drug Dev Ind Pharm 24(12):1113-1128, 1998; Quintanar-Guerrero et al., Pharm Res. 15(7):1056-1062, 1998; Quintanar-Guerrero et al., J. Microencapsul. 15(1):107-119, 1998; Douglas et al., Crit Rev Ther Drug Carrier Syst 3(3):233-261, 1987). To avoid side effects caused by large amounts of macromolecules being taken up into cells, such ultrafine particles can be designed using polymers that can be degraded in vivo. Biodegradable polyalkyl cyanoacrylate nanoparticles that meet such requirements are also envisioned for use in the present disclosure.Such microparticles can be easily prepared, and are described, for example, in Couvreur et al., J Pharm Sci 69(2):199-202, 1980; Couvreur et al., Crit Rev Ther Drug Carrier Syst. 5(1)1-20, 1988; zur Muhlen et al., Eur J Pharm Biopharm, 45(2):149-155, 1998; Zambaux et al., J Control Release 50(1-3):31-40, 1998; and U.S. Patent No. 5,145,684.

[0103] Injectable compositions include sterile aqueous solutions or dispersions and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions (US Pat. No. 5,466,468). Injectable compositions delivered by injection are in the form of a sterile fluid to the extent that they can be delivered using a syringe. In certain embodiments, injectable compositions are usually stable during manufacturing and storage, and may contain one or more preservative compounds to prevent the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium, which may include, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof, and / or vegetable oils. To maintain proper fluidity, for example, a coating agent such as lecithin may be used, or in the case of dispersions, the particle size may be maintained to the required size, and / or a surfactant may be used. To prevent the action of microorganisms, various antibacterial and / or antifungal agents may be used, such as, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In various embodiments, the injectable composition contains an isotonic agent, such as, for example, sugars or sodium chloride. Prolonged absorption of the injectable composition can be achieved by incorporating an agent that delays absorption, such as, for example, aluminum monostearate or gelatin, into the injectable composition. If necessary, an appropriate buffer may be added to the injectable composition, and the diluted liquid is first made isotonic with sufficient saline or glucose.

[0104] Dispersions may also be prepared in glycerol, liquid polyethylene glycols or mixtures thereof, or oils. As described herein, under ordinary conditions of storage and use, such preparations may contain a preservative to prevent the growth of microorganisms.

[0105] Sterile compositions can be prepared by mixing the physiologically active ingredient with any other ingredients (e.g., those mentioned above) in an appropriate amount of solvent and sterilizing by filtration. Dispersions are usually prepared by dispersing various sterilized physiologically active ingredients in a sterile solvent containing a basic dispersion medium and other necessary ingredients (e.g., those mentioned above). In the case of sterile powders for preparing sterile injectable solutions, a preferred method is to sterilize a solution containing the physiologically active ingredient and other desired ingredients in advance by filtration, and then vacuum drying or freeze-drying the solution to prepare a powder containing the physiologically active ingredient and other desired ingredients.

[0106] Oral compositions may be in liquid form, such as solutions, syrups or suspensions, and may be provided as pharmaceutical products to be reconstituted with water or other suitable solvents before use. Such liquid preparations may be prepared by conventional methods using pharmaceutically acceptable additives, such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or gum arabic); non-aqueous solvents (e.g., almond oil, ester oils or fractionated vegetable oils); and preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate or sorbic acid). The composition of the present invention may be prepared, for example, in the form of tablets or capsules, by conventional methods using pharma- ceutically acceptable excipients, such as, for example, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods known in the art.

[0107] Compositions for inhalation can be delivered in the form of an aerosol spray from a pressurized pack or nebulizer using a suitable propellant, such as, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The compositions may be formulated into capsules or cartridges (e.g., gelatin capsules or cartridges) for use in an inhaler or nebulizer, which contain a powder mix of the compositions described herein and a suitable powder base, such as lactose or starch.

[0108] Additionally, compositions of the present invention 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).

[0109] Supplementary active ingredients can also be included in the compositions of the present invention.

[0110] Typically, the compositions of the present invention may contain at least 0.1% or more of the physiologically active ingredient, but it goes without saying that the percentage of the physiologically active ingredient may vary and may conveniently range from 1% or 2% to 70% or 80% or more, or may range from 0.5 to 99%, based on the total weight or volume of the composition of the present invention. Of course, the amount of physiologically beneficial physiologically active ingredient in each composition may be adjusted so that a given unit dose of said compound provides an appropriate dosage. Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, and other pharmacological considerations will be considered by those skilled in the art responsible for preparing such pharmaceutical formulations, and therefore various compositions and dosages may be desirable.

[0111] In certain embodiments, for human administration, compositions of the invention should meet sterility, pyrogenicity, general safety and purity standards as required by the U.S. Food and Drug Administration (FDA) or other responsible regulatory agency in other countries.

[0112] (iii) Cell lines containing the artificial expression constructs The present disclosure includes cells comprising the artificial expression constructs described herein. Cells transformed with the artificial expression constructs can be used for a variety of purposes, such as neuroanatomical studies, evaluation of functional and / or non-functional proteins, drug screening to evaluate the regulatory properties of enhancers, etc.

[0113] While a variety of host cell lines can be used, in certain embodiments, the host cell is a mammalian cell. In certain embodiments, the artificial expression constructs are selected from the group consisting of eHGT_023h v1, eHGT_023m, 3xCore-eHGT_023h v3, eHGT_082h, eHGT_087h, eHGT_128h, eHGT_181h, eHGT_260h, eHGT_023h v2, eHGT_356h, eHGT_359h, eHGT_375m, eHGT_387m, eHGT_395h, 3x(CoreB)eHGT_121h, eHGT_453m, eHGT_470m, eHGT_479m, eHGT_494m, eHGT_467m, eHGT_483m, eHGT_606h, eHGT_738m, eHGT_796h, eHGT_710m, eHGT_588m, eHGT_007m, eHGT_703m, eHG T_589m, eHGT_086h, eHGT_963m, 3xcore3_eHGT_453m, 3xcore2_eHGT_387m, eHGT_534h, 3xcore2_eHGT_475m, eHGT_830h, e HGT_540h, 3xcore2_eHGT_351h, eHGT_882m, eHGT_1137m, eHGT_381h, MGT_E118, MGT_E122, MGT_E146, MGT_E16, 3xcore_MGT _E116, MGT_E150, eHGT_1032h, eHGT_1027h and / or eHGT_1027m, and / or CN1259, CN1280, CN1521, CN1528, CN1533, CN1621, CN1674, CN1778, CN1932, CN2043, CN2045, CN2085, CN2102, CN2157, CN2216, CN2251, CN2257, CN2258, CN2267, CN2316, CN 2339, CN2431, CN2436, CN2643, CN2663, CN2717, CN2838, CN1415, CN2710, CN2839, CN2674, CN3301, CN3019, CN3569, CN2374 , CN3584, CN3003, CN2379, CN3566, CN3453, HCT1, CN2146, AiP1347, AiP1351, AiP1375, AiP1307, AiP1530, AiP1379, CN3916,The enhancer and / or vector sequence selected from CN3869 and / or CN3870, and the host cell line is a human cell, a primate cell, or a mouse cell. Additionally, cell lines that can be utilized for gene transfer in the present disclosure include primary cell lines derived from living tissues such as rat or mouse brain, and organotypic cell cultures such as brain slices from animals such as rat, mouse, non-human primate, or human neurosurgical tissue. The PC12 cell line (available from the American Type Culture Collection (ATCC), Manassas, VA) has been shown to express many neuronal marker proteins in response to nerve growth factor (NGF). The PC12 cell line is believed to be a neuronal cell line and is applicable for use in the present disclosure. JAR cells (available from the ATCC) are a platelet-derived cell line that express several neuronal genes, such as the serotonin transporter gene, and may be used in the embodiments described herein.

[0114] WO91 / 13150 describes various cell lines, including neuronal cell lines, and methods for their production. Similarly, WO97 / 39117 describes neuronal cell lines and methods for the production of such cell lines. The neuronal cell lines disclosed in these patent applications are applicable for use in the present disclosure.

[0115] In certain embodiments, the term "neuronal cell" is used to describe any neuronal cell, related to neuronal cells, or including neuronal cells. Neuronal cells are defined by the characteristic of having an axon and a dendrite. The term "neuron-specific" refers to something that is found in a neuronal cell or cells derived therefrom, but is not found or is substantially absent in non-neuronal cells (e.g., glial cells such as astrocytes and oligodendrocytes); or activity that occurs in a neuronal cell or cells derived therefrom, but is not found or is substantially absent in non-neuronal cells (e.g., glial cells such as astrocytes and oligodendrocytes).

[0116] In certain embodiments, non-neuronal cell lines such as mouse embryonic stem cells may be used. Cultured mouse embryonic stem cells can be transiently transfected with plasmid constructs to analyze the expression of gene constructs. Mouse embryonic stem cells are pluripotent undifferentiated cells. Mouse embryonic stem cells can be maintained in an undifferentiated state by leukemia inhibitory factor (LIF). Mouse embryonic stem cells can be induced to differentiate by removing LIF. Mouse embryonic stem cells form various types of differentiated cells in culture. Differentiation of mouse embryonic stem cells occurs through the expression of tissue-specific transcription factors, which allows the evaluation of the function of enhancer sequences (see, for example, Fiskerstrand et al., FEBS Lett 458: 171-174, 1999).

[0117] The method of differentiating stem cells into neural cells includes replacing the stem cell culture medium with a medium containing basic fibroblast growth factor (bFGF), heparin, N2 supplement (e.g., transferrin, insulin, progesterone, putrescine and selenite), laminin and polyornithine. The method of producing myelinating oligodendrocytes from stem cells is described in Hu, et al., 2009, Nat. Protoc. 4:1614-22. Bibel, et al., 2007, Nat. Protoc. 2:1034-43 describes a protocol for producing glutamatergic neurons from stem cells, and Chatzi, et al., 2009, Exp. Neurol. 217:407-16 describes a procedure for producing GABAergic neurons. This procedure includes exposing stem cells to all-trans retinoic acid for 3 days. GABAergic neurons, which account for 95% of all cells, are then obtained by culturing in a serum-free neuronal induction medium such as neurobasal medium supplemented with B27, bFGF and EGF.

[0118] US Patent Publication No. 2012 / 0329714 describes the use of prolactin to increase neural stem cell numbers, and US Patent Publication No. 2012 / 0308530 describes a culture surface with amino groups that promotes differentiation of neural cells into neurons, astrocytes, and oligodendrocytes. Thus, the fate of neural stem cells can be controlled by various extracellular factors. Commonly used extracellular factors include brain-derived 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. Pat. Nos. 5,948,428 and 6,001,654); isobutyl-3-methylxanthine; leukemia growth inhibitory factor (LIF; U.S. Pat. No. 6,103,530); somatostatin; amphiregulin; neurotrophins (e.g., cyclic adenosine monophosphate); epidermal growth factor (EGF); dexamethasone (a glucocorticoid hormone); forskolin; ligands for the GDNF family of receptors; potassium; retinoic acid (U.S. Pat. No. 6,395,546); tetanus toxoid; and transforming growth factors alpha and TGF-beta (U.S. Pat. Nos. 5,851,832 and 5,753,506).

[0119] In certain embodiments, the yeast one-hybrid system may be used to identify compounds that inhibit specific protein-DNA interactions, such as eHGT_023h v1, eHGT_023m, 3xCore-eHGT_023h v3, eHGT_082h, eHGT_087h, eHGT_128h, eHGT_181h, eHGT_260h, eHGT_023h v2, eHGT_356h, eHGT_359h, eHGT_375m, eHGT_387m, eHGT_395h, 3x(CoreB)eHGT_121h, eHGT_453m, eHGT_470m, eHGT_479m, eHGT_494m, eHGT_4 67m, eHGT_483m, eHGT_606h, eHGT_738m, eHGT_796h, eHGT_710m, eHGT_588m, eHGT_007m, eHGT_703m, eHGT_589m, eHGT_086h, eHGT_963m, 3xcor eHGT_453m, 3xcore2_eHGT_387m, eHGT_534h, 3xcore2_eHGT_475m, eHGT_830h, eHGT_540h, 3xcore2_eHGT_351h, eHGT_882m, eHGT_1137m, eHGT_381h, MGT_E118, MGT_E122, MGT_E146, MGT_E16, 3xcore_MGT_E116, MGT_E150, eHGT_1032h, eHGT_1027h and / or eHGT_1027m transcription factors.

[0120] Transgenic animals are described below. Cell lines may be derived from such transgenic animals. For example, cell lines having artificial expression constructs integrated into their genomes can be obtained from primary tissue cultures derived from transgenic mice (e.g., as described below) (see, e.g., MacKenzie & Quinn, Proc Natl Acad Sci USA 96: 15251-15255, 1999).

[0121] (iv) Transgenic animals Another aspect of the disclosure is to provide a method for the preparation of eHGT_023h v1, eHGT_023m, 3xCore-eHGT_023h v3, eHGT_082h, eHGT_087h, eHGT_128h, eHGT_181h, eHGT_260h, eHGT_023h v4, eHGT_082h, eHGT_087h, eHGT_128h, eHGT_181h, eHGT_260h, eHGT_023h v5, eHGT_082h, eHGT_087h, eHGT_128h, eHGT_181h, eHGT_260h, eHGT_023h v6, eHGT_082h, eHGT_087h, eHGT_128h, eHGT_181h,v2, eHGT_356h, eHGT_359h, eHGT_375m, eHGT_387m, eHGT_395h, 3x(CoreB)eHGT_121h, eHGT_453m, eHGT_470m, eHGT_479m, eHGT_494m, eHGT_467m, eH GT_483m, eHGT_606h, eHGT_738m, eHGT_796h, eHGT_710m, eHGT_588m, eHGT_007m, eHGT_703m, eHGT_589m, eHGT_086h, eHGT_963m, 3xcore3_eHGT_453m , 3xcore2_eHGT_387m, eHGT_534h, 3xcore2_eHGT_475m, eHGT_830h, eHGT_540h, 3xcore2_eHGT_351h, eHGT_882m, eHGT_1137m, eHGT_381h, MGT_E118, MGT_E122, MGT_E146, MGT_E16, 3xcore_MGT_E116, MGT_E150, eHGT_1032h, eHGT_1027h and / or eHGT_1027m. In certain embodiments, the genome of the transgenic animal is selected from the group consisting of CN1259, CN1280, CN1521, CN1528, CN1533, CN1621, CN1674, CN1778, CN1932, CN2043, CN2045, CN2085, CN2102, CN2157, CN2216, CN2251, CN2257, CN2258, CN2267, CN2316, CN2339, CN2431, CN2436, CN2643, CN 2663, CN2717, CN2838, CN1415, CN2710, CN2839, CN2674, CN3301, CN3019, CN3569, CN2374, CN3584, CN3003, CN2379, CN3566, CN3453, HCT1, CN2146, AiP1347, AiP1351, AiP1375, AiP1307, AiP1530, AiP1379, CN3916, CN3869 and / or CN3870. In certain embodiments, when a non-integrating vector is utilized, the transgenic animal may bev3, eHGT_082h, eHGT_087h, eHGT_128h, eHGT_181h, eHGT_260h, eHGT_023h v2, eHGT_356h, eHGT_359h, eHGT_375m, eHGT_387m, eHGT_395h, 3x(CoreB)eHGT_121h, eHGT_453m, eHGT_470m, eHGT_479m, eHGT_4 94m, eHGT_467m, eHGT_483m, eHGT_606h, eHGT_738m, eHGT_796h, eHGT_710m, eHGT_588m, eHGT_007m, eHGT_703m, eHGT_589m, eHGT _086h, eHGT_963m, 3xcore3_eHGT_453m, 3xcore2_eHGT_387m, eHGT_534h, 3xcore2_eHGT_475m, eHGT_830h, eHGT_540h, 3xcore2_ eHGT_351h, eHGT_882m, eHGT_1137m, eHGT_381h, MGT_E118, MGT_E122, MGT_E146, MGT_E16, 3xcore_MGT_E116, MGT_E150, eHGT_103 2h, eHGT_1027h and / or eHGT_1027m, and / or CN1259, CN1280, CN1521, CN1528, CN1533, CN1621, CN1674, CN1778, CN1932, CN2043, CN2045, CN2085, CN2102, CN2157, CN2216, CN2251, CN2257, CN2258, CN2267, CN2316, CN2339, CN2431, CN2436, CN2643, CN2663, C The one or more cells contain an artificial expression construct comprising N2717, CN2838, CN1415, CN2710, CN2839, CN2674, CN3301, CN3019, CN3569, CN2374, CN3584, CN3003, CN2379, CN3566, CN3453, HCT1, CN2146, AiP1347, AiP1351, AiP1375, AiP1307, AiP1530, AiP1379, CN3916, CN3869 and / or CN3870.

[0122] A detailed description of the methods for producing transgenic animals is provided in U.S. Patent No. 4,736,866. The transgenic animals may be of any non-human species, but are preferably non-human primates (NHPs), sheep, horses, cows, pigs, goats, dogs, cats, rabbits, chickens; or rodents, such as guinea pigs, hamsters, gerbils, rats, mice, ferrets, etc.

[0123] In certain embodiments, by producing transgenic animals, organisms are obtained in which recombinant constructs are introduced into the same genome integration site of every cell.Therefore, the cell lines derived from such transgenic animals have consistent characteristics in that they have recombinant constructs in the same genome integration site of every cell, and therefore all of these cells undergo the same variegated position effect.In contrast, when gene is introduced into cell lines or primary cell cultures, heterologous expression of constructs is obtained.This method has the disadvantage that the expression of introduced DNA is affected by the specific genetic background of host animal.

[0124] As previously described 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 constructs are introduced into cultured mouse embryonic stem cells. The transformed ES cells are then injected into blastocysts from a host mother, and the host embryo is reimplanted into the host mother. This procedure results in chimeric mice with tissues composed of cells derived from both embryonic stem cells present in the cultured cell line and embryonic stem cells present in the host embryo. Typically, mice are selected for isolating cultured ES cells used for gene transfer that have a different coat color than the host mouse into whose embryo the transformed cells are injected. Thus, the chimeric mice have a mixed coat color. If at least a portion of the germline tissue is derived from the genetically modified cells, the chimeric mice can then be crossed with an appropriate line to obtain offspring carrying the transgene.

[0125] In addition to the delivery methods described above, other methods of delivering artificial expression constructs to target cells or tissues or organs of animals, particularly cells, organs or tissues of mammalian vertebrates, are contemplated, including sonophoresis (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), as well as other delivery methods available and / or described elsewhere in this disclosure.

[0126] (v) How to use In certain embodiments, a composition comprising a bioactive ingredient described herein is administered to a subject to produce a physiological effect.

[0127] In certain embodiments, the present disclosure includes the use of the artificial expression constructs described herein to regulate the expression of a heterologous gene encoded in part or in its entirety downstream of an enhancer in a recombinant sequence. Accordingly, provided herein are methods of using the artificial expression constructs of the present disclosure in the research, study and future development of pharmaceuticals for the prevention, treatment or alleviation of symptoms of a disease, dysfunction or disorder.

[0128] Certain embodiments include a method of inducing expression of a gene in a target cell by administering to a subject an artificial expression construct, the artificial expression construct being selected from the group consisting of eHGT_023h v1, eHGT_023m, 3xCore-eHGT_023h v3, eHGT_082h, eHGT_087h, eHGT_128h, eHGT_181h, eHGT_260h, eHGT_023h v2, eHGT_023h v3, eHGT_023h v4, eHGT_023h v5, eHGT_023h v6, eHGT_023h v7, eHGT_023h v8, eHGT_023h v9, eHGT_023h v10, eHGT_023m, 3xCore-eHGT_023h v3, eHGT_082hv2, eHGT_356h, eHGT_359h, eHGT_375m, eHGT_387m, eHGT_395h, 3x(CoreB)eHGT_121h, eHGT_453m, eHGT_470m, eHGT_479m, eHGT_ 494m, eHGT_467m, eHGT_483m, eHGT_606h, eHGT_738m, eHGT_796h, eHGT_710m, eHGT_588m, eHGT_007m, eHGT_703m, eHGT_589m, eH GT_086h, eHGT_963m, 3xcore3_eHGT_453m, 3xcore2_eHGT_387m, eHGT_534h, 3xcore2_eHGT_475m, eHGT_830h, eHGT_540h, 3xcor e2_eHGT_351h, eHGT_882m, eHGT_1137m, eHGT_381h, MGT_E118, MGT_E122, MGT_E146, MGT_E16, 3xcore_MGT_E116, MGT_E150, eHG T_1032h, eHGT_1027h and / or eHGT_1027m, and / or CN1259, CN1280, CN1521, CN1528, CN1533, CN1621, CN1674, CN1778, CN1932, CN2043, CN2045, CN2085, CN2102, CN2157, CN2216, CN2251, CN2257, CN2258, CN2267, CN2316, CN2339, CN2431, CN2436, CN2643, C The subject may be an isolated cell, a network of cells, a tissue section, a laboratory animal, a veterinary animal, or a human.

[0129] As is well known in the medical arts, the dose administered to a subject will depend on a variety of factors, such as the subject's body size, surface area and age, the particular compound administered, sex, duration and route of administration, general health, other drugs being administered concomitantly, etc. Although doses of the compounds of the present disclosure may vary, in certain embodiments, doses of the artificial expression constructs of the present disclosure may be administered in doses of 10 to 20 mg / kg. 5 ~10 100 In certain embodiments, patients receiving intravenous, intraparenchymal, intraspinal, retroorbital or intrathecal administration may receive 10 copies of the 6 ~10 22 A copy of the artificial expression construct can be injected.

[0130] "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 amount disclosed herein is an amount that can produce a statistically significant effect in animal models, human studies, in vivo assays, or in vitro assays.

[0131] The dose of the expression construct and the duration of administration of such compositions are determined by those skilled in the art who have the benefit of the teachings of the present invention. However, it is contemplated that administration of an effective amount of the composition of the present disclosure may be performed by a single administration, for example, by a single injection of a sufficient number of infectious particles to confer an effect on the subject. Alternatively, in some circumstances, it may be desirable to administer multiple or sequential administrations of the artificial expression construct composition or other genetic constructs over a relatively short or long period of time, and the decision to administer such may be determined by the person overseeing the administration of such compositions. For example, the number of infectious particles administered to a mammal may be as little as 10 or more times as necessary to achieve the intended effect. 7 pieces / ml, 10 8 pieces / ml, 10 9 pieces / ml, 10 10 pieces / ml, 10 11 pieces / ml, 10 12 pieces / ml, 10 13This may be in the form of a single dose or two or more divided doses of cells / ml or more, and in certain embodiments, it may actually be desirable to administer two or more expression constructs in combination to achieve the desired effect.

[0132] In certain circumstances, it may be desirable to deliver the artificial expression construct in the form of an appropriately formulated composition as disclosed herein using a pipette or by retro-orbital injection, subcutaneous administration, intraocular administration, intravitreal administration, parenteral administration, subcutaneous administration, intravenous administration, intraparenchymal administration, intraventricular administration, intramuscular administration, intrathecal administration, intraspinal administration, intraperitoneal administration, oral administration, nasal inhalation, or direct administration or injection into one or more cells, tissues, or organs. Methods of administration may include those described in U.S. Patent No. 5,543,158; U.S. Patent No. 5,641,515, and U.S. Patent No. 5,399,363.

[0133] (vi) Kits and commercial packages The kits and commercial packages include an artificial expression construct as described herein. The artificial expression construct can be isolated. In certain embodiments, the components of the expression product can be separated from each other. In certain embodiments, the expression product is found within a vector, a viral vector, a cell, a tissue section or tissue sample, and / or a transgenic animal. Such kits may further include one or more reagents, restriction enzymes, peptides, therapeutic agents, pharmaceutical compounds, or a means for delivery of the compositions of the invention (e.g., a syringe, injection, etc.).

[0134] Embodiments of the kit or commercial package further include instructions for use of the components included in the kit or commercial package, e.g., in basic research, electrophysiological studies, neuroanatomical studies, and / or in the study and / or treatment of a disorder, disease or condition.

[0135] The following representative embodiments are set forth to illustrate specific embodiments of the present disclosure. Those skilled in the art having reference to this disclosure will appreciate that various modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit and scope of the present disclosure.

[0136] (vii) Representative Embodiments 1. Concatamerized cores including eHGT_023h v3 enhancer core, eHGT_121h enhancer core, eHGT_453m enhancer core, eHGT_387m enhancer core, eHGT_475m enhancer core, eHGT_351h enhancer core and / or MGT_E116 enhancer core. 2. The concatemerized core of embodiment 2, comprising a sequence as set forth in SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:112 and / or SEQ ID NO:121, or a sequence having at least 90% sequence identity to a sequence as set forth in SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:112 and / or SEQ ID NO:121. 3. A concatemerized core according to embodiment 2, comprising 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of eHGT_023h v3 core, eHGT_121h core, eHGT_453m core, eHGT_387m core, eHGT_475m core, eHGT_351h core and / or MGT_E116 core. 4. The concatemerized core according to embodiment 2 or 3, comprising 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of a sequence as set forth in SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:112 and / or SEQ ID NO:121, or a sequence having at least 90% sequence identity to a sequence as set forth in SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:112 and / or SEQ ID NO:121. 5. A concatemerized core according to any one of embodiments 2 to 4, having 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the sequence shown in SEQ ID NO: 25. 6. A concatemerized core according to any one of embodiments 2 to 5, having 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the sequence shown in SEQ ID NO: 27. 7. A concatemerized core according to any one of embodiments 2 to 6, having 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the sequence set forth in SEQ ID NO: 103. 8. A concatemerized core according to any one of embodiments 2 to 7, having 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the sequence set forth in SEQ ID NO: 105. 9. A concatemerized core according to any one of embodiments 2 to 8, having 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the sequence set forth in SEQ ID NO: 108. 10. A concatemerized core according to any one of embodiments 2 to 9, having 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the sequence set forth in SEQ ID NO: 112. 11. A concatemerized core according to any one of embodiments 2 to 10, having 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the sequence set forth in SEQ ID NO: 121. 12. A concatemerized core according to embodiment 5, having three copies of the sequence shown in SEQ ID NO:25. 13. The concatemerized core of embodiment 6, having three copies of the sequence shown in SEQ ID NO:27. 14. The concatemerized core of embodiment 7, having three copies of the sequence shown in SEQ ID NO: 103. 15. The concatemerized core of embodiment 8, having three copies of the sequence shown in SEQ ID NO: 105. 16. The concatemerized core of embodiment 9, having three copies of the sequence shown in SEQ ID NO: 108. 17. A concatemerized core described in embodiment 10, having three copies of the sequence shown in SEQ ID NO:112. 18. The concatemerized core of embodiment 11, having three copies of the sequence shown in SEQ ID NO: 121. 19. The concatemerized core of embodiment 12, having the sequence shown in SEQ ID NO:26. 20. The concatemerized core of embodiment 13, having the sequence shown in SEQ ID NO:28. 21. The concatemerized core of embodiment 14, having the sequence shown in SEQ ID NO: 104. 22. The concatemerized core of embodiment 15, having the sequence shown in SEQ ID NO: 106. 23. The concatemerized core of embodiment 16, having the sequence shown in SEQ ID NO: 109. 24. The concatemerized core of embodiment 17, having the sequence shown in SEQ ID NO: 113. 25. The concatemerized core of embodiment 18, having the sequence shown in SEQ ID NO: 122. 26. An artificial expression construct comprising: (i) eHGT_023h v1, eHGT_023m, 3xCore-eHGT_023h v3, eHGT_082h, eHGT_087h, eHGT_128h, eHGT_181h, eHGT_260h, eHGT_023h v2, eHGT_356h, eHGT_359h, eHGT_375m, eHGT_387m, eHGT_395h, 3x(CoreB)eHGT_121h, eHGT_453m, eHGT_470m, eHGT_479m, eHGT_494m, eHGT_467m, eHGT_48 3m, eHGT_606h, eHGT_738m, eHGT_796h, eHGT_710m, eHGT_588m, eHGT_007m, eHGT_703m, eHGT_589m, eHGT_086h, eHGT_963m, 3xcore3_eHGT_453m, 3xcore2_e 16. An artificial expression construct comprising: (i) an enhancer selected from among eHGT_387m, eHGT_534h, 3xcore2_eHGT_475m, eHGT_830h, eHGT_540h, 3xcore2_eHGT_351h, eHGT_882m, eHGT_1137m, eHGT_381h, MGT_E118, MGT_E122, MGT_E146, MGT_E16, 3xcore_MGT_E116, MGT_E150, eHGT_1032h, eHGT_1027h and / or eHGT_1027m; (ii) a promoter; and (iii) a heterologous coding sequence. 27. The artificial expression construct described in embodiment 26, wherein the heterologous coding sequence encodes an effector element or an expressible element. 28. The artificial expression construct of embodiment 27, wherein the effector element comprises a reporter protein or functional molecule. 29. The artificial expression construct described in embodiment 28, wherein the reporter protein is a fluorescent protein. 30. The artificial expression construct of embodiment 28, wherein the functional molecule is a functional ion transporter, a functional enzyme, a functional transcription factor, a functional receptor, a functional membrane protein, a functional cell transport protein, a functional signaling molecule, a functional neurotransmitter, a functional calcium reporter, a functional channelrhodopsin, a functional CRISPR / Cas molecule, a functional editase, a functional guide RNA molecule, a functional microRNA, a functional homologous recombination donor cassette, or a functional designer receptor activated only by designer drugs (DREADD). 31. The artificial expression construct of embodiment 27, wherein the expressible element comprises a non-functional molecule. 32. The artificial expression construct of embodiment 31, wherein the non-functional molecule is a non-functional ion transporter, a non-functional enzyme, a non-functional transcription factor, a non-functional receptor, a non-functional membrane protein, a non-functional cell transport protein, a non-functional signaling molecule, a non-functional neurotransmitter, a non-functional calcium reporter, a non-functional channelrhodopsin, a non-functional CRISPR / Cas molecule, a non-functional editase, a non-functional guide RNA molecule, a non-functional microRNA, a non-functional homologous recombination donor cassette, or a non-functional designer receptor activated only by designer drugs (DREADD). 33. An artificial expression construct described in any one of embodiments 26 to 32, which is associated with a capsid that crosses the blood-brain barrier. 34. The artificial expression construct of embodiment 33, wherein the capsid is PHP.eB, AAV-BR1, AAV-PHP.S, AAV-PHP.B or AAV-PPS. 35. An artificial expression construct according to any one of embodiments 26 to 34, comprising or encoding a skipping element. 36. The artificial expression construct of embodiment 35, wherein the skipping element is a 2A peptide or an internal ribosome entry site (IRES). 37. The artificial expression construct of embodiment 36, wherein the 2A peptide is T2A, P2A, E2A or F2A. 38. eHGT_023h v1, eHGT_023m, 3xCore-eHGT_023h v3, eHGT_082h, eHGT_087h, eHGT_128h, eHGT_181h, eHGT_260h, eHGT_023h v2, eHGT_356h, eHGT_359h, eHGT_375m, eHGT_387m, eHGT_395h, 3x(CoreB)eHGT_121h, eHGT_453m, eHGT_4 70m, eHGT_479m, eHGT_494m, eHGT_467m, eHGT_483m, eHGT_606h, eHGT_738m, eHGT_796h, eHGT_710m, eHGT_ 588m, eHGT_007m, eHGT_703m, eHGT_589m, eHGT_086h, eHGT_963m, 3xcore3_eHGT_453m, 3xcore2_eHGT_387 m, eHGT_534h, 3xcore2_eHGT_475m, eHGT_830h, eHGT_540h, 3xcore2_eHGT_351h, eHGT_882m, eHGT_1137m, 38. The artificial expression construct according to any one of embodiments 26 to 37, comprising or encoding a set of features selected from: eHGT_381h, MGT_E118, MGT_E122, MGT_E146, MGT_E16, 3xcore_MGT_E116, MGT_E150, eHGT_1032h, eHGT_1027h, eHGT_1027m, AAV, scAAV, rAAV, pAAV, minBglobin, CMV, minCMV, minCMV*, minRho, minRho*, fluorescent protein, 10aa, H2B, H2Bmod, H2B*, hsA2, Cre, iCre, dgCre, FlpO, tTA2, SP10, tag cassette, nuclear transport protein, self-cleaving peptide, WPRE, WPRE3, hGHpA and / or BGHpA. 39. eHGT_023h v1-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_023m-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; hsA2-3xCore-eHGT_023h v3-minRho-[heterologous coding sequence]-WPRE3-BGHpA; hsA2-eHGT_082h-minRho-[heterologous coding sequence]-WPRE3-BGHpA; hsA2-eHGT_087h-minRho-[heterologous coding sequence]-WPRE3-BGHpA; hsA2-eHGT_128h-minRho-[heterologous coding sequence]-WPRE3-BGHpA; hsA2-eHGT_181h-minRho-[heterologous coding sequence]-WPRE3-BGHpA; hsA2-eHGT_260h-minRho-[heterologous coding sequence]-WPRE3-BGHpA; hsA2-eHGT_023h v2-minRho-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_356h-minRho*-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_359h-minRho*-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_375m-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_387m-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_395h-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; 3x(coreB)eHGT121h-minCMV*-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_453m-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_470m-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_479m-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_494m-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_467m-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_023h v1-minRho*-[heterologous coding sequence]-10aa-H2Bmod-WPRE3-BGHpA; eHGT_023h v1-minRho*-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_483m-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_606h-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_738m-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_796h-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_710m-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_588m-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_007m-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_703m-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_589m-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_086h-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_963m-minBG-[heterologous coding sequence]-P2A-3XFLAG-10aa-H2B-WPRE3-BGHpA; eHGT_963m-minBG-[heterologous coding sequence]-WPRE3-BGHpA; 3xcore3_eHGT_453m-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; 3xcore2_eHGT_387m-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_534h-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; 3xcore2_eHGT_475m-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_830h-minBglobin-[heterologous coding sequence]-P2A-3XFLAG-10aa-H2B-WPRE3-BGHpA; eHGT_830h-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_540h-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; 3xcore2_eHGT_351h-minRho*-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_882m-minBglobin-[heterologous coding sequence]-P2A-3XFLAG-10aa-H2B-WPRE3-BGHpA; eHGT_882m-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_1137m-minBglobin-[heterologous coding sequence]-WPRE3-bGHpA; eHGT_381h-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; MGT_E118-minBglobin-[heterologous coding sequence]-WPRE3-bGHpA; MGT_E122-minBglobin-[heterologous coding sequence]-WPRE3-bGHpA; MGT_E146-minBglobin-[heterologous coding sequence]-WPRE3-bGHpA; MGT_E16-minBglobin-[heterologous coding sequence]-WPRE3-bGHpA; 3xCore_MGT_E116-minBglobin-[heterologous coding sequence]-WPRE3-bGHpA; MGT_E150-minBglobin-[heterologous coding sequence]-WPRE3-bGHpA; eHGT_1032h-minBglobin-[heterologous coding sequence]-P2A-3XFLAG-10aa-H2B-WPRE3-BGHpA; eHGT_1032h-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_1027h-minBglobin-[heterologous coding sequence]-P2A-3XFLAG-10aa-H2B*-WPRE3-BGHpA; eHGT_1027h-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_1027m-minBglobin-[heterologous coding sequence]-P2A-3XFLAG-10aa-H2B*-WPRE3-BGHpA; eHGT_1027m-minBglobin-[heterologous coding sequence]-WPRE3-BGHpA; eHGT_023h v1-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_023m-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; hsA2-3xCore-eHGT_023h v3-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; hsA2-eHGT_082h-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; hsA2-eHGT_087h-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; hsA2-eHGT_128h-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; hsA2-eHGT_181h-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; hsA2-eHGT_260h-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; hsA2-eHGT_023h v2-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_356h-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_359h-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_375m-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_387m-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_395h-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; 3x(coreB)eHGT121h-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_453m-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_470m-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_479m-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_494m-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_467m-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_023h v1-[minimal promoter]-[heterologous coding sequence]-10aa-H2Bmod-[post-transcriptional regulatory element]; eHGT_483m-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_606h-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_738m-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_796h-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_710m-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_588m-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_007m-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_703m-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_589m-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_086h-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_963m-minBG-[heterologous coding sequence]-P2A-3XFLAG-10aa-H2B-[post-transcriptional regulatory element]; eHGT_963m-minBG-[heterologous coding sequence]-[post-transcriptional regulatory element]; 3xcore3_eHGT_453m-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; 3xcore2_eHGT_387m-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_534h-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; 3xcore2_eHGT_475m-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_830h-[minimal promoter]-[heterologous coding sequence]-P2A-3XFLAG-10aa-H2B-[post-transcriptional regulatory element]; eHGT_830h-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_540h-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; 3xcore2_eHGT_351h-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_882m-[minimal promoter]-[heterologous coding sequence]-P2A-3XFLAG-10aa-H2B-[post-transcriptional regulatory element]; eHGT_882m-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_1137m-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_381h-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; MGT_E118-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; MGT_E122-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; MGT_E146-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; MGT_E16-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; 3xCore_MGT_E116-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; MGT_E150-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_1032h-[minimal promoter]-[heterologous coding sequence]-P2A-3XFLAG-10aa-H2B-[post-transcriptional regulatory element]; eHGT_1032h-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_1027h-[minimal promoter]-[heterologous coding sequence]-P2A-3XFLAG-10aa-H2B*-[post-transcriptional regulatory element]; eHGT_1027h-[minimal promoter]-[heterologous coding sequence]-[post-transcriptional regulatory element]; eHGT_1027m-[minimal promoter]-[heterologous coding sequence]-P2A-3XFLAG-10aa-H2B*-[post-transcriptional regulatory element]; and eHGT_1027m - [minimal promoter] - [heterologous coding sequence] - [post-transcriptional regulatory element] 39. The artificial expression construct according to any one of embodiments 26 to 38, comprising or encoding a set of features selected from: 40. A vector comprising an artificial expression construct described in any one of embodiments 26 to 39. 41. The vector described in embodiment 40, which is a viral vector. 42. The vector described in embodiment 41, wherein the viral vector is a recombinant adeno-associated viral (AAV) vector. 43. 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_023h v1, eHGT_023m, 3xCore-eHGT_023h v3, eHGT_082h, eHGT_087h, eHGT_128h, eHGT_181h, eHGT_260h, eHGT_023h v2, eHGT_356h, eHGT_359h, eHGT_375m, eHGT_387m, eHGT_395h, 3x(CoreB)eHGT_121h, eHGT_453m, eHGT_470m, eHGT_479m, eHGT_494m, eHGT_467m, eH GT_483m, eHGT_606h, eHGT_738m, eHGT_796h, eHGT_710m, eHGT_588m, eHGT_007m, eHGT_703m, eHGT_589m, eHGT_086h, eHGT_963m, 3xcore3_eHGT_453 m, 3xcore2_eHGT_387m, eHGT_534h, 3xcore2_eHGT_475m, eHGT_830h, eHGT_540h, 3xcore2_eHGT_351h, eHGT_882m, eHGT_1137m, eHGT_381h, MGT_E118, MGT_E122, MGT_E146, MGT_E16, 3xcore_MGT_E116, MGT_E150, eHGT_1032h, eHGT_1027h and eHGT_1027m. 44. The AAV vector of embodiment 43, wherein the heterologous coding sequence encodes an effector element or an expressible element. 45. The AAV vector of embodiment 44, wherein the effector element comprises a reporter protein or functional molecule. 46. ​​The AAV vector of embodiment 45, wherein the reporter protein is a fluorescent protein. 47. The AAV vector of embodiment 45, wherein the functional molecule is a functional ion transporter, a functional enzyme, a functional transcription factor, a functional receptor, a functional membrane protein, a functional cell transport protein, a functional signaling molecule, a functional neurotransmitter, a functional calcium reporter, a functional channelrhodopsin, a functional CRISPR / Cas molecule, a functional editase, a functional guide RNA molecule, a functional microRNA, a functional homologous recombination donor cassette, or a functional designer receptor activated only by designer drugs (DREADD). 48. The AAV vector of embodiment 44, wherein the expressible element comprises a non-functional molecule. 49. The AAV vector of embodiment 48, wherein the non-functional molecule is a non-functional ion transporter, a non-functional enzyme, a non-functional transcription factor, a non-functional receptor, a non-functional membrane protein, a non-functional cell transport protein, a non-functional signaling molecule, a non-functional neurotransmitter, a non-functional calcium reporter, a non-functional channelrhodopsin, a non-functional CRISPR / Cas molecule, a non-functional editase, a non-functional guide RNA molecule, a non-functional microRNA, a non-functional homologous recombination donor cassette, or a non-functional designer receptor activated only by designer drugs (DREADD). 50. A transgenic cell comprising an artificial expression construct or vector according to any one of the preceding embodiments. 51. The transgenic cell of embodiment 50, comprising a cell of the cerebellum, a GABAergic neuron, a glutamatergic neuron, a non-neuronal cell, a cholinergic interneuron, a medium spiny neuron (MSN) and / or a spinal motor neuron. 52. The transgenic cell of embodiment 51, wherein the cells of the cerebellum include Purkinje cells, molecular layer interneuron (MLI) cells, deep cerebellar nucleus (DCN) cells, Bergmann glia, granule cells, mossy fiber (MF) cells, lobar white matter (WM) cells and / or oligodendrocytes. 53. The transgenic cell of embodiment 51 or 52, wherein the GABAergic neurons include Pvalb neurons, Sst neurons, Lamp5 neurons, Vip neurons, medial ganglia eminence (MGE) (Sst / Pvalb) cells and / or whole GABAergic neurons. 54. The transgenic cell of embodiment 53, wherein the Pvalb neuron comprises a chandelier cell. 55. The transgenic cell of embodiment 53, wherein the Sst neuron comprises an Sst Chodl cell. 56. A transgenic cell according to any one of embodiments 51 to 55, wherein the glutamatergic neurons include whole glutamatergic neurons, layer 5 extraencephalic glutamatergic (L5 ET) neurons, layer 5 proximal projection (NP) neurons, layer 5 intratelencephalic projection (IT) neurons, and / or thalamic glutamatergic neurons. 57. A transgenic cell described in any one of embodiments 51 to 56, wherein the non-neuronal cells include astrocytes, oligodendrocytes, pericytes or specialized smooth muscle cells (SMCs). 58. A transgenic cell described in any one of embodiments 50 to 57, which is a mouse cell, a human cell or a non-human primate cell. 59. A non-human transgenic animal comprising an artificial expression construct, vector, or transgenic cell according to any one of the preceding embodiments. 60. A non-human transgenic animal described in embodiment 59, which is a mouse or a non-human primate. 61. An administrable composition comprising an artificial expression construct, vector and / or transgenic cell according to any one of the preceding embodiments. 62. A kit comprising an artificial expression construct, a vector, a transgenic cell, a non-human transgenic animal and / or an administrable composition according to any one of the preceding embodiments. 63. A method for expressing a gene in a population of cells of the cerebellum and a population of a second type of cell, in vivo or in vitro, comprising: 62. A method comprising the step of providing to a sample or subject comprising a population of cerebellar cells and a population of a second type of cell an administrable composition of embodiment 61 in a sufficient dose and for a sufficient period of time, thereby expressing a gene in the cell populations. 64. A method for expressing a heterologous gene in a population of cells of the cerebellum and a population of a second type of cells, in vivo or in vitro, comprising: providing an administrable composition to a sample or subject comprising a population of cells of the cerebellum and a population of a second type of cell in a sufficient dose and for a sufficient period of time, thereby expressing the heterologous gene in said population of cells; The composition comprises an artificial expression construct, the artificial expression construct being selected from the group consisting of: (i) eHGT_023h v1, eHGT_023m, 3xCore-eHGT_023h v3, eHGT_082h, eHGT_087h, eHGT_128h, eHGT_181h, eHGT_260h, eHGT_023h v2, eHGT_356h, eHGT_359h, eHGT_375m, eHGT_387m, eHGT_395h, 3x(CoreB)eHGT_121h, eHGT_453m, eHGT_470m, eHGT_479m, eHGT_494m, e HGT_467m, eHGT_483m, eHGT_606h, eHGT_738m, eHGT_796h, eHGT_710m, eHGT_588m, eHGT_007m, eHGT_703m, eHGT_589m, eHGT_086h, eHGT _963m, 3xcore3_eHGT_453m, 3xcore2_eHGT_387m, eHGT_534h, 3xcore2_eHGT_475m, eHGT_830h, eHGT_540h, 3xcore2_eHGT_351h, eHGT_882m, eHGT_1137m, eHGT_381h, MGT_E118, MGT_E122, MGT_E146, MGT_E16, 3xcore_MGT_E116, MGT_E150, eHGT_1032h, eHGT_1027h and / or eHGT_1027m; (ii) a promoter; and (iii) a heterologous coding sequence. 65. The method of embodiment 64, wherein the artificial expression construct is associated with a capsid that crosses the blood-brain barrier. 66. The method of embodiment 65, wherein the capsid is PHP.eB, AAV-BR1, AAV-PHP.S, AAV-PHP.B or AAV-PPS. 67. A method according to any one of embodiments 64 to 66, wherein the artificial expression construct comprises or encodes a skipping element. 68. The method of embodiment 67, wherein the skipping element is a 2A peptide and / or an internal ribosome entry site (IRES). 69. The method of embodiment 68, wherein the 2A peptide is T2A, P2A, E2A or F2A. 70. The artificial expression construct is selected from the group consisting of eHGT_023h v1, eHGT_023m, 3xCore-eHGT_023h v3, eHGT_082h, eHGT_087h, eHGT_128h, eHGT_181h, eHGT_260h, and eHGT_023h. v2, eHGT_356h, eHGT_359h, eHGT_375m, eHGT_387m, eHGT_395h, 3x(CoreB)eHGT_121h, eHGT_453m, eHGT_4 70m, eHGT_479m, eHGT_494m, eHGT_467m, eHGT_483m, eHGT_606h, eHGT_738m, eHGT_796h, eHGT_710m, eHGT _588m, eHGT_007m, eHGT_703m, eHGT_589m, eHGT_086h, eHGT_963m, 3xcore3_eHGT_453m, 3xcore2_eHGT_3 87m, eHGT_534h, 3xcore2_eHGT_475m, eHGT_830h, eHGT_540h, 3xcore2_eHGT_351h, eHGT_882m, eHGT_1137 70. The method of any one of embodiments 64-69, wherein the polypeptide comprises or encodes a set of features selected from: m, eHGT_381h, MGT_E118, MGT_E122, MGT_E146, MGT_E16, 3xcore_MGT_E116, MGT_E150, eHGT_1032h, eHGT_1027h, eHGT_1027m, hsA2, AAV, scAAV, rAAV, minBglobin, CMV, minCMV, minCMV*, minRho, minRho*, fluorescent proteins (e.g., EGFP, SYFP, GFP), Cre, iCre, dgCre, FlpO, tTA2, SP10 (e.g., 3xSP10), 10aa, H2B, H2Bmod, H2B*, WPRE, WPRE3, hGHpA and / or BGHpA. 71. A method according to any one of embodiments 64 to 70, wherein the gene encodes an effector element or an expressible element. 72. The method of embodiment 71, wherein the effector element comprises a reporter protein or functional molecule. 73. The method of embodiment 72, wherein the reporter protein is a fluorescent protein. 74. The method of embodiment 72, wherein the functional molecule is a functional ion transporter, a functional enzyme, a functional transcription factor, a functional receptor, a functional membrane protein, a functional cell transport protein, a functional signaling molecule, a functional neurotransmitter, a functional calcium reporter, a functional channelrhodopsin, a functional CRISPR / Cas molecule, a functional editase, a functional guide RNA molecule, a functional microRNA, a functional homologous recombination donor cassette, or a functional designer receptor activated only by designer drugs (DREADD). 75. The method of embodiment 71, wherein the expressible element comprises a non-functional molecule. 76. The method of embodiment 75, wherein the non-functional molecule is a non-functional ion transporter, a non-functional enzyme, a non-functional transcription factor, a non-functional receptor, a non-functional membrane protein, a non-functional cell transport protein, a non-functional signaling molecule, a non-functional neurotransmitter, a non-functional calcium reporter, a non-functional channelrhodopsin, a non-functional CRISPR / Cas molecule, a non-functional editase, a non-functional guide RNA molecule, a non-functional microRNA, a non-functional homologous recombination donor cassette, or a non-functional designer receptor activated only by designer drugs (DREADD). 77. The method of any one of embodiments 64 to 76, wherein the providing step comprises pipetting. 78. The method of embodiment 77, wherein the pipetting is performed on a brain slice. 79. The method of embodiment 78, wherein the brain slice comprises cells of the cerebellum and / or a second type of cells. 80. The method of embodiment 79, wherein the cells of the cerebellum include Purkinje cells, molecular layer interneuron (MLI) cells, deep cerebellar nucleus (DCN) cells, Bergmann glia, granule cells, mossy fiber (MF) cells, lobar white matter (WM) cells and / or oligodendrocytes. 81. The method of embodiment 79, wherein the second type of cell comprises a GABAergic neuron, a glutamatergic neuron, a non-neuronal cell, a cholinergic interneuron, a medium spiny neuron (MSN), or a spinal motor neuron. 82. The method of embodiment 81, wherein the GABAergic neurons include Pvalb neurons, Sst neurons, Lamp5 neurons, Vip neurons, medial ganglia eminence (MGE) (Sst / Pvalb) cells and / or whole GABAergic neurons. 83. The method of embodiment 82, wherein the Pvalb neurons comprise chandelier cells. 84. The method of embodiment 82, wherein the Sst neurons comprise Sst Chodl cells. 85. The method of embodiment 81, wherein the glutamatergic neurons include whole glutamatergic neurons, L5 ET neurons, L5 NP neurons, L5 IT neurons and / or thalamic glutamatergic neurons. 86. The method of embodiment 81, wherein the non-neuronal cells comprise astrocytes, oligodendrocytes, pericytes or specialized smooth muscle cells (SMCs). 87. The method of any one of embodiments 79 to 86, wherein the cells of the cerebellum comprise Purkinje cells and the enhancer is eHGT_023h v1, eHGT_023m, 3xCore-eHGT_023h v3, eHGT_087h, eHGT_260h, eHGT_023h v2, eHGT_359h, eHGT_479m, eHGT_494m, eHGT_467m, eHGT_606h, eHGT_796h, eHGT_588m, eHGT_534h, 3xcore2_eHGT_475m, eHGT_540h or MGT_E150. 88. The method of any one of embodiments 79 to 87, wherein the cells of the cerebellum comprise molecular layer interneuron (MLI) cells and the enhancer is 3xCore-eHGT_023h v3, eHGT_181h, eHGT_023h v2, 3x(CoreB)eHGT_121h, eHGT_479m, eHGT_738m, eHGT_710m, eHGT_588m, eHGT_007m, eHGT_703m, eHGT_086h, eHGT_963m, eHGT_830h, MGT_E146, MGT_E16, eHGT_1032h, eHGT_1027h or eHGT_1027m. 89. The method of embodiment 88, wherein the molecular layer interneuron (MLI) cell is a basket cell and the enhancer is eHGT_086h. 90. The method of any one of embodiments 79 to 89, wherein the cells of the cerebellum comprise deep cerebellar nucleus (DCN) cells and the enhancer is eHGT_082h, eHGT_128h, eHGT_356h, eHGT_395h, eHGT_453m, eHGT_470m, eHGT_589m, eHGT_963m, 3xcore3_eHGT_453m, 3xcore2_eHGT_351h, eHGT_882m, eHGT_1137m, MGT_E118 or 3xcore_MGT_E116. 91. The method of any one of embodiments 79 to 90, wherein the cells of the cerebellum comprise Bergmann glia and the enhancer is eHGT_375m, eHGT_387m, 3xcore2_eHGT_387m, eHGT_381h or MGT_E122. 92. The method of any one of embodiments 79 to 91, wherein the cells of the cerebellum include granule cells and mossy fiber (MF) cells, and the enhancer is eHGT_483m. 93. The method of any one of embodiments 79 to 92, wherein the cells of the cerebellum include oligodendrocytes, deep cerebellar nuclei (DCN) cells and / or lobar white matter (WM) cells, and the enhancer is eHGT_395h. 94. The method of any one of embodiments 79 to 93, wherein the second type of cell is a Pvalb interneuron of the neocortex and the enhancer is eHGT_023h v1, eHGT_023m, 3xCore-eHGT_023h v3, eHGT_082h, eHGT_128h, eHGT_023h v2, eHGT_359h, 3x(CoreB)eHGT_121h, eHGT_589m or eHGT_086h. 95. The method of any one of embodiments 79 to 94, wherein the second type of cells are medial ganglionic eminence (MGE) cells of the neocortex and the enhancer is eHGT_588m. 96. The method of any one of embodiments 79 to 95, wherein the second type of cells are neocortical Sst / Chold neurons and the enhancer is eHGT_470m or eHGT_467m. 97. The method of any one of embodiments 79 to 96, wherein the second type of cell is a Vip interneuron of the neocortex and the enhancer is eHGT_356h, eHGT_483m, eHGT_007m or eHGT_494m. 98. The method of any one of embodiments 79 to 97, wherein the second type of cell is a neocortical Sst interneuron and the enhancer is eHGT_087h. 99. The method of any one of embodiments 79 to 98, wherein the second type of cell is a neocortical Lamp5 interneuron and the enhancer is eHGT_181h. 100. The method of any one of embodiments 79 to 99, wherein the second type of cells are whole neocortical GABAergic neurons and the enhancer is eHGT_796h. 101. The method of any one of embodiments 79 to 100, wherein the second type of cells are L5 ET neurons of the neocortex and the enhancer is eHGT_453m. 102. The method of any one of embodiments 79 to 101, wherein the second type of cells are neocortical L5 NP neurons and the enhancer is 3xcore_MGT_E116. 103. The method of any one of embodiments 79 to 102, wherein the second type of cell is a neocortical L5 IT neuron and the enhancer is MGT_E16. 104. The method of any one of embodiments 79 to 103, wherein the second type of cells are whole neocortical glutamatergic neurons and the enhancer is eHGT_260h. 105. The method of any one of embodiments 79 to 104, wherein the second type of cells are neocortical chandelier cells and the enhancer is eHGT_479m, eHGT_494m, 3xcore2_eHGT_475m, eHGT_710m or eHGT_703m. 106. The method of any one of embodiments 79 to 105, wherein the second type of cells are neocortical astrocytes and the enhancer is 3xcore2_eHGT_387m, eHGT_381h, MGT_E118 or MGT_E122. 107. The method of any one of embodiments 79 to 106, wherein the second type of cells are neocortical pericytes and the enhancer is MGT_E146. 108. The method of any one of embodiments 79 to 107, wherein the second type of cell is a neocortical specialized smooth muscle cell (SMC) and the enhancer is MGT_E150. 109. The method of any one of embodiments 79 to 108, wherein the second type of cell is a striatal cholinergic interneuron and the enhancer is eHGT_738m. 110. The method of any one of embodiments 79 to 109, wherein the second type of cell is a striatal medium spiny neuron (MSN) and the enhancer is eHGT_882m. 111. The method of any one of embodiments 79 to 110, wherein the second type of cells are whole striatal medium spiny neuron (MSN) cells and the enhancer is 3xcore2_eHGT_351h. 112. The method of any one of embodiments 79 to 111, wherein the second type of cells are glutamatergic neurons of the thalamus and the enhancer is eHGT_606h or eHGT_830h. 113. The method of any one of embodiments 79 to 112, wherein the second type of cells are astrocytes throughout the brain and the enhancer is eHGT_375m or eHGT_387m. 114. The method of any one of embodiments 79 to 113, wherein the second type of cells are oligodendrocytes throughout the brain and the enhancer is eHGT_395h. 115. The method of any one of embodiments 79 to 114, wherein the second type of cells is GABAergic neurons throughout the brain and the enhancer is eHGT_1032h, eHGT_1027h or eHGT_1027m. 116. The method of any one of embodiments 79 to 115, wherein the second type of cells are entire neurons throughout the brain, and the enhancer is eHGT_534h or eHGT_540h. 117. The method of any one of embodiments 79 to 116, wherein the second type of cell is a spinal motor neuron and the enhancer is eHGT_1137m. 118. The method of any one of embodiments 78 to 117, wherein the brain slice is a mouse, human or non-human primate brain slice. 119. The method of any one of embodiments 77 to 118, wherein the providing step comprises administration to a living subject. 120. The method of embodiment 119, wherein the living subject is a human, a non-human primate, or a mouse. 121. The method of embodiment 119 or 120, wherein administration to the living subject is by injection. 122. The method of embodiment 121, wherein the injection is an intravenous injection, an intraparenchymal injection into brain tissue, an intracerebroventricular (ICV) injection, an intracisternal (ICM) injection or an intrathecal injection. 123. An artificial expression construct comprising: CN1259, CN1280, CN1521, CN1528, CN1533, CN1621, CN1674, CN1778, CN1932, CN2043, CN2045, CN2085, CN2102, CN2157, CN2216, CN2251, CN2257, CN2258, CN2267, CN2316, CN2339, CN2431, CN2436, CN2643, CN2663, CN2717 , CN2838, CN1415, CN2710, CN2839, CN2674, CN3301, CN3019, CN3569, CN2374, CN3584, CN3003, CN2379, CN3566, CN3453, HCT1, CN2146, AiP1347, AiP1351, AiP1375, AiP1307, AiP1530, AiP1379, CN3916, CN3869 or CN3870, or an artificial expression construct.

[0137] (viii) Conclusion Variants of the sequences disclosed and referenced herein are also included herein. Guidelines for determining which amino acid residues can be substituted, inserted or deleted without losing biological activity can be determined using computer programs well known in the art, such as DNASTAR. TM Software (Madison, WI, USA) can be used to find the amino acid changes in the protein variants disclosed herein. The amino acid changes are preferably conservative amino acid changes, i.e., substitutions of similarly charged amino acids with each other or of uncharged amino acids with each other. Conservative amino acid changes include substitutions with members of a family of amino acids whose side chains are related.

[0138] Suitable conservative substitutions of amino acids in peptides or proteins are known to those skilled in the art, and such conservative substitutions can be made without generally altering the biological activity of the resulting molecule.Those skilled in the art will be familiar with the fact that generally, a single amino acid substitution in a non-essential region of a polypeptide will not substantially alter the biological activity (see, for example, 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, specifically: Group 1: alanine (Ala), glycine (Gly), serine (Ser), and threonine (Thr); Group 2: (acidic): aspartic acid (Asp) and glutamic acid (Glu); Group 3: (acidic; 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 (Ile), leucine (Leu), and ketone (K). Group 7 (polar uncharged): tyrosine (Tyr), Gly, Asn, Gln, Cys, Ser and Thr; Group 8 (large aromatic residues): phenylalanine (Phe), tryptophan (Trp) and Tyr; Group 9 (non-polar): proline (Pro), Ala, Val, Leu, Ile, Phe, Met and Trp; Group 11 (aliphatic): Gly, Ala, Val, Leu and Ile; Group 10 (small aliphatic residues that are non-polar or slightly polar): Ala, Ser, Thr, Pro and Gly; and Group 12 (sulfur-containing residues): Met and Cys. Further information can be found in Creighton (1984) Proteins, WH Freeman and Company.

[0139] In making such changes, the hydropathic index of amino acids may be taken into consideration. The importance of the hydropathic index of amino acids in conferring interactive biological function on a protein is widely understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). The hydrophobicity index of each amino acid is Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); glutamic acid (-3.5); Gln (-3.5); aspartic acid (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[0140] It is well known in the art that substitution of a particular amino acid with another amino acid having a similar hydrophobicity index or hydrophobicity degree can also result in a protein with similar biological activity, i.e., a protein with biologically equivalent functionality. When making such changes, substitution of amino acids with hydrophobicity indices within ±2 is preferred, substitution of amino acids with hydrophobicity indices within ±1 is particularly preferred, and substitution of amino acids with hydrophobicity indices within ±0.5 is even more particularly preferred. Furthermore, it is well known in the art that substitution of similar amino acids can be effectively carried out based on hydrophilicity.

[0141] As detailed in U.S. Patent No. 4,554,101, each amino acid residue is assigned a hydrophilicity value, which is as follows: 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 well known that certain amino acids can be substituted with other amino acids having a similar hydrophilicity value, and that such substitutions will result in biologically equivalent proteins, and in particular immunologically equivalent proteins. When making such changes, substitutions between amino acids whose hydrophilicity values ​​are within the range of ±2 are preferred, substitutions between amino acids whose hydrophilicity values ​​are within the range of ±1 are particularly preferred, and substitutions between amino acids whose hydrophilicity values ​​are within the range of ±0.5 are even more particularly preferred.

[0142] As outlined above, amino acid substitutions may be made on the basis of the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.

[0143] As described elsewhere herein, variants of a gene sequence include codon-optimized variants, sequence polymorphisms, splice variants, and / or mutations that have no statistically significant effect on the function of the encoded product.

[0144] Variants of the protein, nucleic acid and gene sequences disclosed herein also include sequences having at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity or at least 99% sequence identity to the protein, nucleic acid or gene sequences disclosed herein.

[0145] "Percent sequence identity" refers to the relatedness of two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of relatedness between protein, nucleic acid or gene sequences, as determined by the matching between strings of protein, nucleic acid or gene sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Methods for determining identity are preferably designed to give the best match between the sequences tested. Methods for determining identity and similarity are codified in publicly available computer programs. Sequence alignment and identity calculations may be performed using the Megalign program (DNASTAR, Inc., Madison, Wis.) in the LASERGENE suite of bioinformatics computing software.Multiple alignment of sequences can also be performed using the Clustal format alignment method (Higgins and Sharp CABIOS, 5, 151-153 (1989) using default parameters (gap penalty=10, gap length penalty=10)). Related programs further include the GCG 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 York, NY). In this disclosure, when sequence analysis software is used for analysis, the analysis results are interpreted as being based on the "default values" that are the basis of the program. In this specification, "default values" refers to a set of numerical values ​​or parameters that are preregistered in the software at the time of initialization of the software.

[0146] Variants also include nucleic acid molecules that hybridize to the sequences disclosed herein under stringent hybridization conditions and have the same function as the reference sequences. Exemplary stringent hybridization conditions include overnight incubation at 42°C in a solution containing 50% formamide, 5xSSC (750mM NaCl, 75mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5xDenhardt's solution, 10% dextran sulfate, and 20μg / ml denatured salmon sperm DNA that has been fragmented, followed by washing the filter at 50°C with 0.1xSSC. Hybridization stringency and signal detection are altered primarily by adjusting the concentration of formamide (lower percentage of formamide results in lower stringency), salt conditions, or temperature. For example, moderately stringent conditions include overnight incubation at 37° C. in 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg / ml blocking salmon sperm DNA, followed by washing with 1×SSPE and 0.1% SDS at 50° C. Even lower stringency is achieved by performing stringent post-hybridization washes at high salt concentrations (e.g., 5×SSC). The above conditions can be varied by adding and / or substituting other blocking reagents used to reduce the background of hybridization experiments. Common blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary preparations. The addition of certain blocking reagents may require some modification of the hybridization conditions described above due to compatibility issues.

[0147] The term "concatemerize" is used in a broad sense and means to link in a chain or to link in a series. The term is used to describe the linking of multiple nucleotide sequences to obtain a single nucleotide sequence or the linking of multiple amino acid sequences to obtain a single amino acid sequence. Also, "concatemerize" is understood to refer to "concatenation."

[0148] As will be appreciated by those of skill in the art, each embodiment disclosed herein comprises, consists essentially of, or consists of the particular components, steps, materials, or ingredients described. Thus, the terms "comprise" or "comprising" should be interpreted to mean "comprise, consist essentially of, or consist of." The transitional phrase "comprise" means that an unrecited component, step, material, or ingredient is included, even if in large amounts. The transitional phrase "consisting of" excludes any unrecited component, step, material, or ingredient. The transitional phrase "consisting essentially of" limits the scope of the embodiment to the recited components, steps, materials, or ingredients and those components, steps, materials, or ingredients that do not materially affect the embodiment. A significant effect is an effect that results in a statistically significant decrease in targeted expression in the target cell population as measured by scRNA-Seq, as a combination of enhancer / target cell population and subclassification of cerebellar cells. eHGT_023h v1 / Purkinje cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_023m / Purkinje cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_082h / DCN cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_087h / Purkinje cells in the cerebellum and Sst interneurons in the neocortex; eHGT_128h / DCN cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_181h / MLI cells in the cerebellum and Lamp5 interneurons in the neocortex; eHGT_260h / Purkinje cells of the cerebellum and whole glutamatergic neurons of the neocortex; eHGT_023h v2 / Purkinje and MLI cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_356h / DCN cells in the cerebellum and Vip interneurons in the neocortex; eHGT_359h / Purkinje cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_375m / Bergmann glia in the cerebellum and astrocytes throughout the brain; eHGT_387m / Bergmann glia in the cerebellum and astrocytes throughout the brain; eHGT_395h / oligodendrocytes, DCN cells, and lobar white matter (WM) in the cerebellum and oligodendrocytes throughout the brain; eHGT_453m / DCN cells in the cerebellum and L5 ET neurons in the neocortex; eHGT_470m / DCN cells in the cerebellum and Sst / Chodl neurons in the neocortex; eHGT_479m / Purkinje and MLI cells in the cerebellum and chandelier cells in the neocortex; eHGT_494m / Purkinje cells in the cerebellum and Vip and chandelier cells in the neocortex; eHGT_467m / Purkinje cells of the cerebellum and Sst / Chodl neurons of the neocortex; eHGT_483m / granule cells and mossy fiber (MF) cells in the cerebellum and Vip interneurons in the neocortex; eHGT_606h / Purkinje cells of the cerebellum and glutamatergic neurons of the thalamus; eHGT_738m / MLI cells in the cerebellum and cholinergic interneurons in the striatum; eHGT_796h / total cerebellar Purkinje cells and neocortical GABAergic neurons; eHGT_710m / MLI cells in the cerebellum and chandelier cells in the neocortex; eHGT_588m / Purkinje and MLI cells in the cerebellum and MGE cells in the neocortex; eHGT_007m / MLI cells in the cerebellum and Vip interneurons in the neocortex; eHGT_703m / MLI cells in the cerebellum and chandelier cells in the neocortex; eHGT_589m / DCN cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_086h / MLI (basket) cells in the cerebellum and Pvalb interneurons in the neocortex; eHGT_963m / MLI and DCN cells in the cerebellum and L5 NP neurons in the neocortex; eHGT_534h / Purkinje cells in the cerebellum and whole neurons in the whole brain; eHGT_830h / MLI cells in the cerebellum and glutamatergic neurons in the thalamus; eHGT_540h / Purkinje cells in the cerebellum and whole neurons in the whole brain; eHGT_882m / DCN cells in the cerebellum and MSN cells in the striatum; eHGT_1137m / DCN cells of the cerebellum and spinal motor neurons; eHGT_381h / Bergmann glia in the cerebellum and astrocytes in the neocortex; MGT_E118 / DCN cells in the cerebellum and astrocytes in the neocortex; MGT_E122 / Bergmann glia in the cerebellum and astrocytes in the neocortex; MGT_E146 / MLI cells in the cerebellum and pericytes in the neocortex; MGT_E16 / MLI cells in the cerebellum and L5 IT neurons in the neocortex; MGT_E150 / Purkinje cells of the cerebellum and specialized smooth muscle cells (SMCs) of the neocortex; eHGT_1032h / MLI cells in the cerebellum and total GABAergic neurons throughout the brain; eHGT_1027h / MLI cells in the cerebellum and total GABAergic neurons throughout the brain; eHGT_1027m / MLI cells in the cerebellum and total GABAergic neurons throughout the brain; Concatamerized multi-copy enhancers or concatemerized multi-copy enhancer cores containing 3xCore-eHGT_023h v3 / Purkinje and MLI cells in the cerebellum and Pvalb interneurons in the neocortex; 3x(CoreB)eHGT_121h / MLI cells in the cerebellum and Pvalb interneurons in the neocortex; 3xcore3_eHGT_453m / DCN cells of the cerebellum; 3xcore2_eHGT_387m / Bergmann glia in the cerebellum and astrocytes in the neocortex; 3xcore2_eHGT_475m / Purkinje cells in the cerebellum and chandelier cells in the neocortex; 3xcore2_eHGT_351h / total DCN cells in the cerebellum and MSN cells in the striatum; and 3xcore_MGT_E116 / DCN cells in the cerebellum and L5 NP neurons in the neocortex Examples include:

[0149] In certain embodiments, "artificial" means not naturally occurring.

[0150] Unless otherwise indicated, all numerical values ​​expressing quantities or properties of materials, such as molecular weight and reaction conditions, in the specification and claims are to be construed in all instances as modified by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Without intending to limit the scope of the doctrine of equivalents to the scope of the claims, each numerical parameter should, at the very least, be construed in light of the number of reported significant digits and by applying ordinary rounding procedures. For clarity, the term "about," when used in conjunction with a stated value or range, has a meaning that would be reasonably interpreted by one of ordinary skill in the art, i.e., within ±20% of the stated value; within ±19% of the stated value; within ±18% of the stated value; within ±17% of the stated value; within ±16% of the stated value; within ±15% of the stated value; within ±14% of the stated value; within ±13% of the stated value; within ±12% of the stated value; within ±11% of the stated value; within ±10% of the stated value; within ±9% of the stated value; within ±8% of the stated value; within ±7% of the stated value; within ±6% of the stated value; within ±5% of the stated value; within ±4% of the stated value; within ±3% of the stated value; within ±2% of the stated value; or within ±1% of the stated value.

[0151] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations and approximate ranges, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain certain errors necessarily resulting from the standard deviation associated with their respective testing measurements.

[0152] In the description of the present invention (particularly in the description of the claims below), the terms "a", "an", "the" and similar modifiers are intended to include both the singular and the plural unless otherwise indicated or the context clearly indicates otherwise. Numerical ranges described herein are intended to be a shorthand way of referring to each numerical value falling within the range individually. Unless otherwise indicated, each numerical value is described herein as if it were individually described herein. Any method described herein can be performed in any suitable order unless otherwise indicated or the context clearly indicates otherwise. The use of any examples or language of examples (e.g., "etc.") provided herein is intended to be for the purpose of illustrating the invention only and does not limit the scope of the invention as described in the claims. No term described herein should be construed as indicating any non-claimed element essential to the practice of the invention.

[0153] Groupings of other elements of the invention disclosed herein or of various embodiments of the invention should not be construed as limiting the invention. Members of each group may be described herein or in the claims individually or in combination with other members of the group or other elements described herein. It is anticipated that for reasons of convenience and / or patentability, one or more members of a group may be added to another group, or one or more members may be deleted from a group. When such additions or deletions are made, the specification includes groups that are constructed to satisfy the recitation of all Markush groups set forth in the appended claims.

[0154] Specific embodiments of the present invention are described herein, including those embodiments known to the inventors to be the best mode for carrying out the invention. Of course, those skilled in the art will readily appreciate that the embodiments described herein may be modified in various ways upon review of the above detailed description. The inventors anticipate that such modifications may be adopted by those skilled in the art, and intend that the present invention may be practiced in other ways than as specifically described herein. Accordingly, the present invention includes all modifications of the subject matter recited in the appended claims and all equivalents of the subject matter of the present invention to the extent permitted within the scope of applicable law. Moreover, the present invention includes all combinations of the above-described elements in any and all variations thereof, unless otherwise indicated or the context clearly dictates otherwise.

[0155] Additionally, throughout this specification, various patents, publications, journal articles and other documents are cited (references herein). Each reference cited herein is individually incorporated herein by reference for the teachings thereof as if it were a part of this specification.

[0156] Finally, the embodiments of the invention disclosed herein are to be considered as illustrative of the principles of the invention. Other modifications may be adopted within the scope of the invention. Thus, by way of example, but not by way of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Thus, the invention is not to be limited to what has been precisely shown and described herein.

[0157] The details described herein are by way of example and are presented solely for the purpose of illustrating preferred embodiments of the present invention, to provide what is believed to be the most useful, and to facilitate an understanding of the principles and conceptual aspects of various embodiments of the present invention. In this regard, no structural details of the present invention are described in more detail than is necessary for a basic understanding of the present invention, and those skilled in the art will be able to easily understand how to actually embody some forms of the present invention by reading the description of the present invention in conjunction with the drawings and / or examples.

[0158] The definitions and explanations used in this disclosure are intended to control future interpretations, unless clearly and definitively changed in the following examples, or unless the interpretation is rendered meaningless or substantially meaningless by the meaning of the term. If the definition of a term is rendered meaningless or substantially meaningless by the interpretation of the term, please refer to the definition of the term from a dictionary known to those skilled in the art, such as Webster's Dictionary (3rd Edition) or Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

Claims

1. 1. An artificial expression construct comprising: (i) 3xcore3_eHGT_453m (SEQ ID NO: 104), core3_eHGT_453m (SEQ ID NO: 103), eHGT_023m (SEQ ID NO: 2), 3xCore-eHGT_023h v3 (SEQ ID NO: 26), Core-eHGT_023h v3 (SEQ ID NO: 25), eHGT_181h (SEQ ID NO: 8), eHGT_023h v2 (SEQ ID NO: 4), eHGT_356h (SEQ ID NO: 10), eHGT_467m (SEQ ID NO: 18), eHGT_483m (SEQ ID NO: 19), eHGT_796h (SEQ ID NO: 22), eHGT_588m (SEQ ID NO: 24), eHGT_007m (SEQ ID NO: 13), eHGT_589m (SEQ ID NO: 100), eHGT_963m (SEQ ID NO: 102), 3xcore2_eHGT_387m (SEQ ID NO: 106), core2_eHGT_387m (SEQ ID NO: 1 05), eHGT_882m (SEQ ID NO: 114), MGT_E122 (SEQ ID NO: 118), MGT_E146 (SEQ ID NO: 119), 3xcore_MGT_E116 (SEQ ID NO: 122), core_MGT_E116 (SEQ ID NO: 121), MGT_E150 (SEQ ID NO: 123), eHGT_1032h (SEQ ID NO: 124), eHGT_1027h (SEQ ID NO: 125) and eHGT_1027m (SEQ ID NO: 126), or A sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 104, 103, 2, 4, 8, 10, 13, 18, 19, 22, 24, 25, 26, 100, 102, 105, 106, 114, 118, 119, 121, 122, 123, 124, 125 or 126 and an enhancer having (ii) a promoter; and (iii) a coding sequence; An artificial expression construct comprising:

2. 2. The artificial expression construct of claim 1, wherein the coding sequence encodes a fluorescent protein or a neurotransmitter.

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

4. 4. The artificial expression construct of claim 3, wherein the capsid comprises PHP.eB, AAV-BR1, AAV-PHP.S, AAV-PHP.B, AAV-PPS, AAV9, AAVrh.10, AAV1R6 or AAV1R7.

5. 2. The artificial expression construct of claim 1, comprising or encoding a skipping element.

6. 6. The artificial expression construct of claim 5, wherein the skipping element comprises a T2A peptide, a P2A peptide, an E2A peptide, an F2A peptide, or an internal ribosome entry site (IRES).

7. An artificial expression construct as described in claim 1, contained within a viral vector.

8. A method for producing a nucleic acid sequence comprising the steps of: (a) a nucleic acid sequence selected from the group consisting of SEQ ID NO: 103, SEQ ID NO: 25, SEQ ID NO: 105, and SEQ ID NO: 121; or (b) a nucleic acid sequence selected from the group consisting of SEQ ID NO: 103, SEQ ID NO: 25, SEQ ID NO: 105, and SEQ ID NO: 121; comprising 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 103, SEQ ID NO: 25, SEQ ID NO: 105 or SEQ ID NO: 121; Artificial enhancers.

9. A method for producing a nucleic acid sequence comprising the steps of: (a) detecting a nucleic acid sequence comprising: a nucleic acid sequence selected from the group consisting of SEQ ID NO: 103, SEQ ID NO: 25, SEQ ID NO: 105, and SEQ ID NO: 121; or comprising three copies of a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 103, SEQ ID NO: 25, SEQ ID NO: 105 or SEQ ID NO: 121; The artificial enhancer of claim 8 .

10. A sequence selected from the group consisting of SEQ ID NO: 104, SEQ ID NO: 26, SEQ ID NO: 106 and SEQ ID NO: 122, or A sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 104, SEQ ID NO: 26, SEQ ID NO: 106 or SEQ ID NO: 122 The artificial enhancer of claim 8 , having the formula:

11. 1. A method for expressing a coding sequence in a population of cells of the cerebellum and a population of a second type of cell in vivo, comprising: providing the composition to a subject comprising a population of cells of the cerebellum and a population of a second type of cell at a sufficient dose and for a sufficient period of time, thereby expressing the coding sequence in said population of cells; The composition comprises an artificial expression construct, the artificial expression construct comprising: (i) eHGT_453m (SEQ ID NO: 14), 3xcore3_eHGT_453m (SEQ ID NO: 104), core3_eHGT_453m (SEQ ID NO: 103), eHGT_023m (SEQ ID NO: 2), 3xCore-eHGT_023h v3 (SEQ ID NO: 26), Core-eHGT_023h v3 (SEQ ID NO: 25), eHGT_181h (SEQ ID NO: 8), eHGT_023h v2 (SEQ ID NO: 4), eHGT_356h (SEQ ID NO: 10), eHGT_467m (SEQ ID NO: 18), eHGT_483m (SEQ ID NO: 19), eHGT_796h (SEQ ID NO: 22), eHGT_588m (SEQ ID NO: 24), eHGT_007m (SEQ ID NO: 13), eHGT_589m (SEQ ID NO: 100), eHGT_963m (SEQ ID NO: 102), 3xcore2_eHGT_387m (SEQ ID NO: 106), core2_eHGT_387m (SEQ ID NO: 1 05), eHGT_882m (SEQ ID NO: 114), MGT_E122 (SEQ ID NO: 118), MGT_E146 (SEQ ID NO: 119), 3xcore_MGT_E116 (SEQ ID NO: 122), core_MGT_E116 (SEQ ID NO: 121), MGT_E150 (SEQ ID NO: 123), eHGT_1032h (SEQ ID NO: 124), eHGT_1027h (SEQ ID NO: 125) and eHGT_1027m (SEQ ID NO: 126), or A sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 14, 104, 103, 2, 4, 8, 10, 13, 18, 19, 22, 24, 25, 26, 100, 102, 105, 106, 114, 118, 119, 121, 122, 123, 124, 125 or 126. and an enhancer having (ii) a promoter; and (iii) a coding sequence, The method, wherein the subject is a non-human subject.

12. A method for expressing a coding sequence in a population of cells of the cerebellum and a population of a second type of cell in vitro, comprising: providing the composition to a sample comprising a population of cells of the cerebellum and a population of a second type of cell at a sufficient dose and for a sufficient period of time to express the coding sequence in said cell population; The composition comprises an artificial expression construct, the artificial expression construct comprising: (i) eHGT_453m (SEQ ID NO: 14), 3xcore3_eHGT_453m (SEQ ID NO: 104), core3_eHGT_453m (SEQ ID NO: 103), eHGT_023m (SEQ ID NO: 2), 3xCore-eHGT_023h v3 (SEQ ID NO: 26), Core-eHGT_023h v3 (SEQ ID NO: 25), eHGT_181h (SEQ ID NO: 8), eHGT_023h v2 (SEQ ID NO: 4), eHGT_356h (SEQ ID NO: 10), eHGT_467m (SEQ ID NO: 18), eHGT_483m (SEQ ID NO: 19), eHGT_796h (SEQ ID NO: 22), eHGT_588m (SEQ ID NO: 24), eHGT_007m (SEQ ID NO: 13), eHGT_589m (SEQ ID NO: 100), eHGT_963m (SEQ ID NO: 102), 3xcore2_eHGT_387m (SEQ ID NO: 106), core2_eHGT_387m (SEQ ID NO: 1 05), eHGT_882m (SEQ ID NO: 114), MGT_E122 (SEQ ID NO: 118), MGT_E146 (SEQ ID NO: 119), 3xcore_MGT_E116 (SEQ ID NO: 122), core_MGT_E116 (SEQ ID NO: 121), MGT_E150 (SEQ ID NO: 123), eHGT_1032h (SEQ ID NO: 124), eHGT_1027h (SEQ ID NO: 125) and eHGT_1027m (SEQ ID NO: 126), or A sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 14, 104, 103, 2, 4, 8, 10, 13, 18, 19, 22, 24, 25, 26, 100, 102, 105, 106, 114, 118, 119, 121, 122, 123, 124, 125 or 126. and an enhancer having (ii) a promoter; and (iii) a coding sequence.

13. 13. The method of claim 11 or 12, wherein the artificial expression construct is associated with a capsid that crosses the blood-brain barrier.

14. The method of claim 13, wherein the capsid comprises PHP.eB, AAV-BR1, AAV-PHP.S, AAV-PHP.B, AAV-PPS, AAV9, AAVrh.10, AAV1R6 or AAV1R7.

15. 13. The method of claim 12, wherein said providing comprises pipetting performed on the brain slice.

16. 13. The method of claim 11 or 12, wherein the cells of the cerebellum comprise Purkinje cells, molecular layer interneuron (MLI) cells, deep cerebellar nuclei (DCN) cells, Bergmann glia, granule cells, mossy fiber (MF) cells, lobar white matter (WM) cells, or oligodendrocytes.

17. 13. The method of claim 11 or 12, wherein the second type of cell comprises a GABAergic neuron, a glutamatergic neuron, a non-neuronal cell, a cholinergic interneuron, a medium spiny neuron (MSN), or a spinal motor neuron.

18. The method described in claim 11 or 12, wherein the enhancer comprises the sequence shown in SEQ ID NO: 14, the cells of the cerebellum comprise deep cerebellar nuclei (DCN) cells of the cerebellum, and the second type of cells comprise layer 5 extratelencephalic projection glutamatergic (L5 ET) neurons of the neocortex.

19. 18. The method of claim 17, wherein the glutamatergic neurons include whole glutamatergic neurons, L5 ET neurons, L5 near-projecting (NP) neurons, L5 intratelencephalic (IT) neurons, or thalamic glutamatergic neurons.

20. The method of claim 11 , wherein said providing comprises administering to a living subject.

21. 21. The method of claim 20, wherein the administration to the living subject comprises intravenous injection, intraparenchymal injection into brain tissue, intracerebroventricular (ICV) injection, intracisternal (ICM) injection, or intrathecal injection.

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