Artificial expression constructs that selectively regulate gene expression in neocortical 5-glutamatergic neurons.

Abstract

To provide artificial expression constructs for selectively modulating gene expression in selected central nervous system cell types.SOLUTION: As target central nervous system cell populations, there are neocortical glutamatergic layer 5 neurons, for example, neocortical glutamatergic layer 5 extratelencephalic-projecting (L5 ET) neurons. In certain applications, aspects of the present disclosure can be used to deliver therapeutic payloads to upper motor neurons associated with, for example, Amyotrophic Lateral Sclerosis (ALS) or other neurodegenerative diseases in which L5 ET neurons are involved.SELECTED DRAWING: Figure 4B

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 013,342, filed on 21 April 2020, and its entire contents are incorporated herein by reference as if they were fully described herein.

[0002] Statements relating to research or development funded by the federal government This invention was completed with the support of the United States Government under grants MH114126 and MH121274 from the National Institute of Health. The United States Government has certain rights to this invention.

[0003] References regarding sequence listings The sequence listing relating to this application is provided in text format instead of on paper, and is incorporated herein by reference as constituting a part of this specification. The name of the text file containing the sequence listing is A166-0024PCT_ST25.txt. This text file is 211KB in size, was created on April 21, 2021, and was submitted electronically via EFS-Web.

[0004] Areas of disclosure This disclosure provides artificial expression constructs for selectively regulating gene expression in selected central nervous system cell types. These artificial expression constructs can be used to selectively express or modify synthetic genes in neocortical latitude 5 glutamatergic neurons, such as latitude 5 glutamatergic telencephalic projection (L5 ET) neurons. [Background technology]

[0005] Background of Disclosure To fully understand brain biology, it is necessary to distinguish and define different cell types, and to further study them, to identify artificial expression constructs that can selectively label and disrupt them. In mice, recombinase driver strains have proven highly effective in labeling cell populations that share marker gene expression. However, the creation, maintenance, and use of such strains that label cell types with high specificity are expensive and often require triple transgenic crosses, resulting in a low frequency of experimental animals. Furthermore, these tools require germline transgenic animals and are therefore not applicable to humans. [Overview of the Initiative]

[0006] Summary of Disclosure This disclosure provides an artificial expression construct that selectively drives gene expression in a target central nervous system cell population. The target central nervous system cell population includes: neocortical glutamatergic lamina 5 neurons, e.g., neocortical glutamatergic lamina 5 telencephalic projection (L5 ET) neurons. In specific applications, aspects of this disclosure can be used to deliver a therapeutic payload to upper motor neurons associated with, for example, amyotrophic lateral sclerosis (ALS) or other neurodegenerative diseases in which L5 ET neurons are involved.

[0007] Certain embodiments of the artificial expression construct selectively drive protein expression in L5 glutamatergic neurons using the following enhancers: eHGT_450m, eHGT_451m, eHGT_452m, eHGT_453m, eHGT_457m, eHGT_458m, eHGT_459m, eHGT_460m, eHGT_462m, eHGT_464m, eHGT_461m, eHGT_463m, eHGT_692h, eHGT_693h, eHGT_694h, eHGT_695h, eHGT_667m, eHGT_668m, eHGT_696m, eHGT_455m, eHGT_457h, eHGT_603m, eHGT_604m, eHGT_605m, eHGT_661m, eHGT_766m, eHGT_767m, eHGT_768m, eHGT_663m, eHGT_660m, eHGT_665m, eHGT_700m, eHGT_456m, eHGT_698m, eHGT_699m, eHGT_647m, eHGT_648m, eHGT_649m, eHGT_670m, eHGT_697m, eHGT_662m, and / or eHGT_583m.

[0008] In certain embodiments, the artificial enhancer element includes a linked enhancer core. Examples include linked cores of eHGT_459m, eHGT_453m, eHGT_462m, eHGT_461m, eHGT_464m, eHGT_452m, eHGT_451m, and / or eHGT_460m. These artificial enhancer elements can result in higher levels of transgene expression and faster initiation of expression compared to a single full-length original (native) enhancer.

[0009] In certain embodiments, the enhancer core includes the sequence described in any one of sequence numbers 45-52. In certain embodiments, these cores are concatenated and also have 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the core sequence. Sequence numbers 53-60 provide concatemers of three selected enhancer cores.

[0010] Certain embodiments of the artificial expression constructs utilize 3xcore2_eHGT_459m, 3xcore2_eHGT_453m, 3xcore2_eHGT_462m, 3xcore2_eHGT_461m, 3xcore2_eHGT_464m, 3xcore2_eHGT_452m, 3xcore2_eHGT_451m, and / or 3xcore2_eHGT_460m to selectively drive protein expression in L5 glutamatergic neurons.

[0011] Specific embodiments include vectors: CN2248, CN2249, CN2250, CN2251, CN2252, CN2253, CN2254, CN2255, CN2256, CN2315, CN2423, CN2424, CN2690, CN2695, CN2691, CN2692, CN2944, CN2886, CN2755, CN2938, CN2239, CN2410, CN2411, CN2412, CN2963, CN2623, CN262 4. Provides artificial expression constructs that include the characteristics of vectors described herein, such as CN2601, CN2843, CN2851, CN2853, CN2860, CN2828, CN2870, CN2871, CN2847, CN2686, CN2687, CN2878, CN2946, CN2943, CN3319, CN2918, CN3018, CN3030, CN3027, CN3032, CN3015, CN3012, and / or CN3024.

[0012] Many of the drawings submitted herein are better understood through color. The applicant considers the colored drawings to be part of the original application documents and reserves the right to present images of the colored drawings by following the procedures described below. [Brief explanation of the drawing]

[0013] [Figure 1]This outlines exemplary enhancer discovery for viral tools. To construct cell type-specific labeling tools, cells derived from adult mouse cortex can be isolated, and single-cell assays on transposase-accessible chromatin can be performed using sequencing (scATAC-seq). Samples can be clustered, and clusters can be identified by comparison with single-cell RNA sequencing (scRNA-seq) datasets. Single cells matching the same transcriptome type can then be pooled, and the genome can be searched for type-specific putative enhancers. Alternatively, some enhancers were selected using the human motor cortex SNARE-seq dataset (Bakken et al., Nature. 2021 doi:https: / / doi.org / 10.1101 / 2020.03.31.016972). The selected putative enhancer region can be cloned upstream of the minimal promoter in the AAV genome backbone, which can then be used to generate a self-complementary adeno-associated virus vector (scAAV) or a recombinant adeno-associated virus vector (rAAV). These viral tools were delivered posteriorly to the orbit to label specific neocortical populations. In cells with matching cell types, the enhancers recruit their homologous transcription factors to drive cell type-specific expression. In other cells, the viral genome is present, but the transcripts are not expressed. However, not all enhancers function as expected, so enhancer constructs need to be tested for specificity. [Figure 2A] This shows the mouse genome coordinates for enhancer eHGT_451m (shaded region), indicating the corresponding single-cell ATAC-seq peaks for each cell class and subclass. The arrows indicate the open chromatin peaks of the L5 ET neuron subclass. [Figure 2B]Epifluorescence micrograph showing native SYFP2 expression in the visual cortex two months after posterior delivery of the 6.00E11GC AAV vector #CN2249. This genomic coordinate is related to build Hg38, and the image was obtained from UCSC Genome Browser. Scale bar: 100 microns. [Figure 3A] This shows the mouse genome coordinates for enhancer eHGT_452m (shaded region), indicating the corresponding single-cell ATAC-seq peaks for each cell class and subclass. The arrows indicate the open chromatin peaks of the L5 ET neuron subclass. [Figure 3B] Epifluorescence micrograph showing native SYFP2 expression in the visual cortex two months after posterior delivery of the 1.45E12GC AAV vector #CN2250. These genomic coordinates are related to build Hg38, and the image was obtained from the UCSC Genome Browser. Scale bar: 100 microns. [Figure 4A] This shows the mouse genome coordinates for enhancer eHGT_453m (shaded region), indicating the corresponding single-cell ATAC-seq peaks for each cell class and subclass. The arrows indicate the open chromatin peaks of the L5 ET neuron subclass. [Figure 4B] Epifluorescence micrograph showing native SYFP2 expression in the visual cortex two months after posterior delivery of the 7.00E11GC AAV vector #CN2251. Scale bar: 100 microns. [Figure 4C]Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from the visual cortex region of mouse brains after retroorbital injection of CN2251 virus packaged in PHP.eB capsid. The number of cells mapped to the terminal end is shown in the bar graph below the dendrogram. Cell types of the transcriptome are shown below. This data demonstrates that eHGT_453m enhancer-driven reporter expression occurs selectively in all four types of L5 ET neurons in the visual cortex region. The bottom text is from left to right: 169 L2 / 3 IT VISp Rrad, 168 L2 / 3 IT VISp Adamts2, 167 L2 / 3 IT VISp Agmay, 164 L4 IT VISp Rspo1, 163 L5 IT VISp Hsd11b1 Endou, 162 L5 IT VISp Whrn Tox2, 160 L5 IT VISp Batf3, 158 L5 IT VISp Col6a1 Fezf1, 157 L5 IT VISp Col27a1, 154 L6 IT VISp Penk Col27a1, 153 L6 IT VISp Penk Fst, L6 IT VISp Col23a1 Adamts2, 149 L6 IT VISp Col18a1, 146 L6 IT VISp Car3, 144 L5 PT VISp Chrna6, 143 L5 PT VISp Lgr5, 142 L5 PT VISp C1ql2 Ptgfr, 141 L5 PT VISp C1qI2 Cdh13, 140 L5 PT VISp Krt80, 134 L5 NP VISp Trhr Cpne7, 133 L5 NP VISp Trhr Met, L5 CT Nxph2 Sla, 130 L5 CT VISp Krt80 Sla, L5 CT VISp Nxph2.Wls, 127 L5 CT VISp Ctxn3 Brinp3, 126 L5 CT VISp Ctxn3 Sla, 122 L5 CT VISp Gpr139, 120 L6b Col8a1 Rprm, 119 L6b VISp Mup5, 118 L6b VISp Col8a1 Rxfp1, 115 L6b P2ry12, L6b VISp Crh, 110 Lamp5 Krt73, Lamp5Fam19a1 Pax6、108 Lamp5 Fam19a1 Tmem182、106 Lamp5 Ntn1 Npy2r、105 Lamp5 Plch2 Dock5、101 Lamp5 Lsp1、100 Lamp5 Lhx6、Sncg Slc17a8、96 Sncg Vip Nptx2、95 Sncg Gpr50、93 Sncg Vip Itih5、90 Serpinf1 Clrn1、89 Serpinf1 Aqp5 Vip、85 Vip Igfbp6 Car10、84 Vip Igfbp6 Pltp、Vip Lmo1 Fam159b、Vip Lmo1 Myl1、79 Vip Igfbp4 Mab21l1、78 Vip Arhgap36 Hmcn1、77 Vip Gpc3 Slc18a3、74 Vip Ptprt Pkp2、73 Vip Rspo4 Rxfp1 Chat、71 Vip Lect1 Oxtr、70 Vip Rspo1 Itga4、67 Vip Chat Htr1f、66 Vip Pygm C1qI1、61 Vip CrispId2 Htr2c、60 Vip CrispId2 Kcne4、58 Vip Col15a1 Pde1a、54 Sst Chodl、53 Sst Mme Fam114a1、52 Sst Tac1 Htr1d、50 Sst Tac1 Tacr3、49 Sst Calb2 Necab1、48 Sst Calb2 Pdlim5、46 Sst Nr2f2 Necab1、45 Sst Myh8 Etv1、44 Sst Chrna1 Glra3、42 Sst Myh8 Fibin、40 Sst Chrna2 Ptgdr、39 Sst Tac2 Myn4、37 Sst Hpse Sema3c、36 Sst Hpse Cbln4、34 Sst Crh2 Efemp1、33 Sst Crh2 4930553C11Rik、31 Sst Esm1、29 Sst Tac2 Tacstd2、28 Sst Rxfp1 Eya1、27 Sst Rxfp1 Prdm8、23 Sst Nts、Pvalb Gabrg1、20 Pvalb Th Sst、18 Pvalb Calb1 Sst、17 Pvalb Akr1c18 Ntf3、16 Pvalb Sema3eKank4, 14 Pvalb Gpr149 IsIr, 11 Pvalb ReIn Itm2a, 10 Pvalb ReIn Tac1, 9 Pvalb Tpbg, 4 Pvalb Vlpr2, Meis2 Adamts19, 170 Astro Aqp4, 171 OPC Pdgfra Grm5, Oligo Serpinb1a, 174 Oligo Synpr, VLMC Osr1 Cd74, VLMC Osr1 Mc5r, VLMC Spp1 Col15a1, Peri Kcnj8, SMC Acta2, Endo Ctla2a, and 181 Microglia Siglech. [Figure 4D] Epi-fluorescence microscopy image of native SYFP2 fluorescence in fixed brain slices obtained from the primary motor cortex of a macaque 64 days after stereotaxic injection of the enhancer AAV vector CN2251 serotype PHP.eB. This genomic coordinate refers to build Hg38, and the image was obtained from the UCSC Genome Browser. Scale bar: 500 μm. [Figure 5A] Mouse genomic coordinates for the enhancer eHGT_459m (shaded region), showing the corresponding single-cell ATAC-seq peaks for each cell class and subclass. Arrows indicate the open chromatin peaks of the L5 ET neuron subclass. [Figure 5B] Epi-fluorescence micrograph image showing native SYFP2 expression in the visual cortex 2 months after retro-orbital delivery of 1.00E12 GC of AAV vector #CN2254. Scale bar: 100 microns. [Figure 5C] Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from the visual cortex region of the mouse brain after retro-orbital injection of CN2254 virus packaged in PHP.eB capsid. The number of cells mapped to the terminal end is shown in the bar graph below the dendrogram. The transcriptome cell types are shown at the bottom. This data indicates that eHGT_459m enhancer-driven reporter expression occurs primarily in only one of the four L5 ET neuron types in the visual cortex region. The characters at the bottom are the same as those described in Figure 4C from left to right. [Figure 5D] Epi-fluorescence microscopy image of native SYFP2 fluorescence in fixed brain slices obtained from the primary motor cortex of a macaque 113 days after stereotaxic injection of the enhancer AAV vector CN2254 serotype PHP.eB. This genomic coordinate refers to build Hg38, and the image was obtained from the UCSC Genome Browser. Scale bar: 500 μm. [Figure 6A] Mouse genomic coordinates for the enhancer eHGT_460m (shaded region), showing the corresponding single-cell ATAC-seq peaks for each cell class and subclass. Arrows indicate the open chromatin peaks of the L5 ET neuron subclass. [Figure 6B] Epi-fluorescence micrograph image showing native SYFP2 expression in the visual cortex 2 months after retro-orbital delivery of 6.50E11 GC of AAV vector #CN2255. This genomic coordinate refers to build Hg38, and the image was obtained from the UCSC Genome Browser. Scale bar: 100 microns. [Figure 7A] Mouse genomic coordinates for the enhancer eHGT_462m (shaded region), showing the corresponding single-cell ATAC-seq peaks for each cell class and subclass. Arrows indicate the open chromatin peaks of the L5 ET neuron subclass. [Figure 7B] Epi-fluorescence micrograph image showing native SYFP2 expression in the visual cortex 2 months after retro-orbital delivery of 6.50E11 GC of AAV vector #CN2256. This genomic coordinate refers to build Hg38, and the image was obtained from the UCSC Genome Browser. Scale bar: 100 microns. [Figure 8A] Mouse genomic coordinates for the enhancer eHGT_464m (shaded region), showing the corresponding single-cell ATAC-seq peaks for each cell class and subclass. Arrows indicate the open chromatin peaks of the L5 ET neuron subclass. [Figure 8B]Epifluorescence micrograph showing native SYFP2 expression in the visual cortex two months after posterior delivery of the 1.00E12GC AAV vector #CN2315. Scale bar: 100 microns. [Figure 8C] Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from the visual cortex region of mouse brains after retroorbital injection of CN2315 virus packaged in PHP.eB capsid. The number of cells mapped to the terminals is shown in the bar graph below the dendrogram. Cell types of the transcriptome are shown below. This data indicates that eHGT_464m enhancer-driven reporter expression occurs selectively in multiple types of L5 ET neurons within the visual cortex region. The letters at the bottom, from left to right, are the same as those in Figure 4C. [Figure 8D] Epifluorescence microscopy images of native SYFP2 fluorescence in fixed brain slices obtained from the primary motor cortex of a macaque 64 days after stereotactic injection of enhancer AAV vector CN2315 serotype PHP.eB. These genomic coordinates relate to build Hg38, and the images were obtained from UCSC Genome Browser. Scale bar: 500 μm. [Figure 9A] This shows the mouse genome coordinates for enhancer eHGT_461m (shaded region), indicating the corresponding single-cell ATAC-seq peaks for each cell class and subclass. The arrows indicate the open chromatin peaks of the L5 ET neuron subclass. [Figure 9B] Epifluorescence micrograph showing native SYFP2 expression in the visual cortex two months after posterior delivery of the 5.00E11GC AAV vector #CN2423. These genomic coordinates are related to build Hg38, and the image was obtained from the UCSC Genome Browser. Scale bar: 100 microns. [Figure 10A] This shows mouse genome coordinates for enhancer eHGT_463m (shaded region), indicating the corresponding single-cell ATAC-seq peaks for each cell class and subclass. The arrows indicate the open chromatin peaks of the L5 ET neuron subclass. [Figure 10B] Epifluorescence micrograph showing native SYFP2 expression in the visual cortex two months after posterior delivery of the 7.30E11GC AAV vector #CN2424. Scale bar: 100 microns. [Figure 10C] Mapping of single-cell transcriptome profiles of SYPF2+ cells sorted from the visual cortex region of mouse brains after post-orbital injection of CN2424 virus packaged in PHP.eB capsid. The number of cells mapped to the terminals is shown in the bar graph below the dendrogram. Cell types of the transcriptome are shown below. This data indicates that eHGT_463m enhancer-driven reporter expression occurs selectively in one of four types of L5 ET neurons within the visual cortex region. The letters at the bottom, from left to right, are the same as those shown in Figure 4C. These genomic coordinates are for build Hg38, and the image was obtained from UCSC Genome Browser. [Figure 11A] This shows mouse genome coordinates for enhancer eHGT_647m (shaded region), indicating the corresponding single-cell ATAC-seq peaks for each cell class and subclass. The arrows indicate the open chromatin peaks of the L5 ET neuron subclass. [Figure 11B] Epifluorescence micrograph showing native SYFP2 expression in the visual cortex 1.5 months after posterior delivery of the 7.00E11GC AAV vector #CN2847. These genomic coordinates are related to build Hg38, and the image was obtained from the UCSC Genome Browser. Scale bar: 100 microns. [Figure 12A] Sequences supporting this disclosure. Enhancer sequences disclosed herein. Each artificial expression construct described herein includes at least one of the enhancer sequences provided in Figure 12A. [Figure 12B] Sequences supporting this disclosure. Exemplary components that can be included (or encoded) within an artificial expression construct generated using an enhancer of (12A). [Figure 12C]Sequences supporting this disclosure. Representative artificial expression constructs generated using the enhancer of (12A) and the selected component of (12B). [Modes for carrying out the invention]

[0014] Detailed explanation To fully understand the biology of the brain, it is necessary to distinguish and define different cell types, and to identify artificial expression constructs that can selectively label and disrupt them in order to further study them. Tasic, Curr. Opin. Neurobiol. 50, 242-249 (2018), Zeng & Sanes, Nat. Rev. Neurosci. 18, 530-546 (2017). In mice, recombinase driver strains have been found to be highly effective in labeling cell populations that share marker gene expression. Daigle et al., Cell 174, 465-480. e22 (2018), Taniguchi et al., Neuron 71, 995-1013 (2011), Gong et al., J. Neurosci. 27, 9817-9823 (2007). However, the creation, maintenance, and use of such strains that label cell types with high specificity are expensive and often require triple transgenic crosses, resulting in a low frequency of experimental animals. Furthermore, these tools require germline transgenic animals and are therefore not applicable to humans.

[0015] This disclosure provides an artificial expression construct that selectively drives gene expression in a target central nervous system cell population. The target central nervous system cell population includes: neocortical glutamatergic lamina 5 neurons, e.g., neocortical glutamatergic telencephalic projection (L5 ET) neurons. In specific applications, aspects of this disclosure can be used to deliver a therapeutic payload to upper motor neurons associated with, for example, amyotrophic lateral sclerosis (ALS) or other neurodegenerative diseases in which L5 ET neurons are involved.

[0016] Specific embodiments of the artificial expression construct include the following enhancers: eHGT_450m, eHGT_451m, eHGT_452m, eHGT_453m, eHGT_457m, eHGT_458m, eHGT_459m, eHGT_460m, eHGT_462m, eHGT_464m, eHGT_461m, eHGT_463m, eHGT_692h, eHGT_693h, eHGT_694h, eHGT_695h, eHGT_667m, eHGT_668m, eHGT_696m, eHGT_455m, eHG T_457h, eHGT_603m, eHGT_604m, eHGT_605m, eHGT_661m, eHGT_766m, eHGT_767m, eHGT_768m, eHGT_663m, eHGT_660m, eHGT_665m, eHGT_700m, eHGT_456m, eHGT_698m, eHGT_699m, eHGT_647m, eHGT_648m, eHGT_649m, eHGT_670m, eHGT_697m, eHGT_662m, and / or eHGT_583m are used to selectively drive gene expression within L5 ET neurons.

[0017] In certain embodiments, the artificial enhancer element includes a linked core of the enhancers disclosed herein, for example, a linked core of eHGT_459m, eHGT_453m, eHGT_462m, eHGT_461m, eHGT_464m, eHGT_452m, eHGT_451m, and / or eHGT_460m. These artificial enhancer elements provide higher levels of transgene expression and faster initiation of expression compared to a single full-length original (native) enhancer.

[0018] In certain embodiments, the enhancer core may include the core array described in any one of sequence numbers 45-52. In certain embodiments, these cores are concatenated and include 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the core. Sequence numbers 53-60 provide concatemers of three selected enhancer cores.

[0019] Certain embodiments of the artificial expression construct utilize 3xcore2_eHGT_459m, 3xcore2_eHGT_453m, 3xcore2_eHGT_462m, 3xcore2_eHGT_461m, 3xcore2_eHGT_464m, 3xcore2_eHGT_452m, 3xcore2_eHGT_451m, and / or 3xcore2_eHGT_460m to selectively drive protein expression in L5 glutamatergic neurons.

[0020] Specific embodiments include vectors: CN2248, CN2249, CN2250, CN2251, CN2252, CN2253, CN2254, CN2255, CN2256, CN2315, CN2423, CN2424, CN2690, CN2695, CN2691, CN2692, CN2944, CN2886, CN2755, CN2938, CN2239, CN2410, CN2411, CN2412, CN2963, CN2623, CN262 4. Provides artificial expression constructs that include the characteristics of vectors described herein, such as CN2601, CN2843, CN2851, CN2853, CN2860, CN2828, CN2870, CN2871, CN2847, CN2686, CN2687, CN2878, CN2946, CN2943, CN3319, CN2918, CN3018, CN3030, CN3027, CN3032, CN3015, CN3012, and / or CN3024.

[0021] Aspects of this disclosure are described in the following further options and details: (i) artificial expression constructs and vectors for selective expression of genes in selected cell types, (ii) compositions for administration, (iii) cell lines containing artificial expression constructs, (iv) transgenic animals, (v) methods of use, (vi) kits and commercial products, (vii) exemplary embodiments, and (viii) concluding paragraphs.

[0022] (i) Expression constructs and vectors for the selective expression of genes in selected cell types. The expression constructs disclosed herein include (i) an enhancer sequence that induces the selective expression of a coding sequence in a target central nervous system cell type, (ii) the coding sequence to be expressed, and (iii) a promoter. The expression constructs may also include other regulatory elements if necessary or beneficial.

[0023] In certain embodiments, the “enhancer” or “enhancer element” is a cis-acting sequence that can function in either direction with respect to the promoter and coding sequence to enhance the level of transcription associated with the promoter, and can be located upstream or downstream of the promoter or coding sequence to be transcribed. Methods and techniques exist and are recognized in the art to measure the function(s) of enhancer element sequences. Specific examples of enhancer sequences used in the artificial expression constructs disclosed herein include: eHGT_450m, eHGT_451m, eHGT_452m, eHGT_453m, eHGT_457m, eHGT_458m, eHGT_459m, eHGT_460m, eHGT_462m, eHGT_464m, eHGT_461m, eHGT_ 463m, eHGT_692h, eHGT_693h, eHGT_694h, eHGT_695h, eHGT_667m, eHGT_668m, eHGT_696m, eH GT_455m, eHGT_457h, eHGT_603m, eHGT_604m, eHGT_605m, eHGT_661m, eHGT_766m, eHGT_767m, eHGT_768m, eHGT_663m, eHGT_660m, eHGT_665m, eHGT_700m, eHGT_456m, eHGT_698m, eHGT_699m, eHGT_647m, eHGT_648m, eHGT_649m, eHGT_670m, eHGT_697m, eHGT_662m, eHGT_583m, and linked co For example, there are 3xcore2_eHGT_459m, 3xcore2_eHGT_453m, 3xcore2_eHGT_462m, 3xcore2_eHGT_461m, 3xcore2_eHGT_464m, 3xcore2_eHGT_452m, 3xcore2_eHGT_451m, and 3xcore2_eHGT_460m.

[0024] In certain embodiments, the target central nervous system cell type enhancer is an enhancer that is uniquely or primarily utilized by the target central nervous system cell type. The target central nervous system cell type enhancer enhances gene expression in the target central nervous system cell type but does not substantially induce gene expression in other non-target cell types, and therefore has cell type-specific transcriptional activity.

[0025] If a coding sequence is selectively expressed in selected cells and substantially not expressed in other cell types, the product of the coding sequence is preferentially expressed in the selected cell types. In certain embodiments, preferential expression is greater than 50% compared to the reference cell type; greater than 60% compared to the reference cell type; greater than 70% compared to the reference cell type; greater than 80% compared to the reference cell type; or greater than 90% compared to the reference cell type. In certain embodiments, the reference cell type refers to untargeted cells. Untargeted cells are located within the same anatomical structure as the targeted cells and / or appear in a common anatomical region. In certain embodiments, the reference cell type is located within an anatomical structure adjacent to the anatomical structure containing the target cell type. In certain embodiments, the reference cell type is untargeted cells having a different gene expression profile than the target cells.

[0026] In certain embodiments, the coding sequence product may be expressed in non-selective cell types at levels less than 1% of the level at which the product is expressed in selected cells, or at low levels of 1%, 2%, 3%, 5%, 10%, 15%, or 20%. In certain embodiments, the target central nervous system cell type is the only cell type that expresses the appropriate combination of transcription factors that bind to the enhancers disclosed herein to drive gene expression. Thus, in certain embodiments, expression occurs only within the target cell type.

[0027] In certain embodiments, target cell types (e.g., neurons and / or non-neurons) can be identified based on transcriptional profiles, such as those described in Tasic et al., 2018 and Hodge et al., Nature, 573, 61-68 (2019). For reference, a description of cell types and identifying features is provided below:

[0028] Neocortic GABAergic subclass: ●All: Expresses the GABA synthesis genes Gad1 / GAD1 and Gad2 / GAD2. ● Lamp5, Sncg, Serpinf1, and Vip: Developingly originate from neuronal precursors derived from caudal ganglion luminescence (CGE) or preoptic area (POA). ● Sst and Pvalb: These are developmentally induced from neuronal precursors in medial ganglion luminescence (MGE). ● Lamp 5: Found in many cortical layers, especially the upper part (L1-L2 / 3), and mainly possesses glial cell morphology and single plexus morphology. ●Sncg: Found in many cortical layers, it has molecular overlap with Lamp5 and Vip cells, but Lamp5 or Vip expression is inconsistent, while Sncg expression is more consistent. ●Serpinf1: Found in many cortical layers, it has molecular overlap with Sncg and Vip cells, but Sncg or Vip expression is inconsistent, while Serpinf1 expression is more consistent. ●Vip: Found in many cortical layers, but particularly abundant in the upper layers (L1-L4), it highly expresses the neurotransmitter vasoactive intestinal peptide (Vip). ●Sst: These are found in many cortical layers, but are particularly frequent in the lower layers (L5-L6). They highly express the neurotransmitter somatostatin (Sst) and frequently block dendritic entry into postsynaptic neurons. This subclass includes sleep-active Sst Chodl neurons (which also express Nos1 and Tacr1) that are quite different from other Sst neurons but express several co-marker genes, including Sst. In humans, SST gene expression is often detected in layer 1 LAMP5+ cells. ● Pvalb: Found in many cortical layers, but particularly frequently in the lower layers (L5-L6). They highly express the calcium-binding protein parvalbumin (Pvalb), express the neuropeptide Tac1, and often attenuate postsynaptic neuronal output. Most GABAergic cells strongly express Pvalb. This subclass includes chandelier cells with a distinct chandelier-like morphology that express the markers Cpne5 and Vipr2 in mice, as well as NOG and UNC5B in humans. ●Meis2: Defined as a distinct subclass of neocortical GABAergic cytoplasm that expresses only the Meis2 gene, and which does not express some other genes expressed by all other neocortical GABAergic cytoplasms (e.g., Thy1 and Scn2b). This type is found in L6b and subcortical white matter.

[0029] Neocortic glutamatergic subclass: ● All: Express glutamate transmitters Slc17a6 and / or Slc17a7. They all express Snap25 and lack Gad1 / Gad2 expression. ●L2 / 3 IT: Primarily located in layers 2 and 3, and mainly possesses telencephalic (corticocortical) processes. ●L4 IT: Primarily located in layer 4, and mainly possesses either focal or telencephalonal (corticocortical) projections. ●L5 IT: Primarily located in layer 5, and mainly possesses telencephalic (corticocortical) processes. Also known as L5a. ● L5 ET: Primarily located in layer 5 and primarily possessing corticospinal processes. Also known as L5b, L5 CF (Corticofugal), or L5 PT (Pyramidal Tract Projection). This subclass includes cells located in the primary motor cortex and adjacent regions that are corticospinal process neurons associated with motor neuron / motor disorders such as ALS. This subclass includes thick, tufted pyramidal neurons, including characteristic subtypes found only in specialized regions, such as Betz cells, Meynart cells, and von Economo cells. ●L5 NP: Primarily resides in layer 5 and mainly has protrusions in its vicinity. ●L6 CT: Primarily located in layer 6 and mainly possesses the corticothalamic process. ● L6 IT: Primarily located in layer 6 and possessing mainly intracranial (corticocortical) processes. This subclass includes L6 IT Car3 cells, which are very similar to intraclavicular corticosteroid cells. ●L6b: Primarily located in the neocortical subplate (L6b), it consists of focal (near-cell body) processes, several corticocortical processes from the VISp to the anterior cyst, and subcortical processes to the thalamus. ●Cajal-Retzius cells, a distinct subclass defined as a single type within CR:L1, express distinct molecular markers Lhx5 and Trp73.

[0030] Cerebellar Purkinje cells are large GABAergic neurons, the only protruding neurons and their only product originating from the cerebellum. Their cell bodies form a monolayer known as the "Purkinje cell layer," and they express parvalbumin.

[0031] Deep cerebellar nucleus neurons: Neurons located within the deep cerebellar nucleus structure. These include glutamatergic and GABAergic cells that express the Pvalb gene.

[0032] Non-neuronal subclass: ● Astrocytes: Glial cells derived from neuroectoderm that express the marker Aqp4 and often GFAP, but do not express the neuronal marker SNAP25. They can have a distinct star-shaped morphology and are involved in supporting the metabolism of other cells in the brain. Multiple star cell morphologies have been observed in mice and humans. ● Oligodendrocytes: Glial cells derived from the neuroectoderm that express the marker Sox10. This category includes oligodendrocyte vesicle precursor cells (OPCs). Oligodendrocyte cells are a subclass primarily involved in neuronal myelination. ●VLMC: Vascular leptomeninge cells (VLMCs) are a part of the meninges that surround the outer layer of the cortex and express the marker genes Lum and Col1a1. ● Pericytes: Blood vessel-related cells that express the marker genes Kcnj8 and Abcc9. Pericytes surround endothelial cells and are important for regulating blood flow in capillaries, and are involved in blood-brain barrier permeability. ● SMC: A special type of smooth muscle cell that expresses the marker gene Acta2 and is associated with blood vessels. SMCs cover the arteries in the brain and are involved in blood-brain barrier permeability. ● Endothelial cells: Cells that reinforce the inner lining of blood vessels in the brain. Endothelial cells express the markers Tek and PDGF-B. ● Microglia: Hematopoietic-derived immune cells that are resident macrophages and perivascular macrophages (PVMs) of the brain, which may be transitionally associated with brain tissue or may be included as a byproduct of brain dissection. Microglia are known to express Cx3cr1, Tmem119, and PTPRC (CD45).

[0033] In certain embodiments, the coding sequence is a heterogeneous coding sequence that codes for an effector element. An effector element is a sequence that is expressed to achieve a desired effect, and that effect is actually achieved. Examples of effector elements include reporter genes / proteins and functional genes / proteins.

[0034] Exemplary reporter genes / proteins include those expressed by Addgene ID numbers 83894 (pAAV-hDlx-Flex-DTomato-Fishell_7), 83895 (pAAV-hDlx-Flex-GFP-Fishell_6), 83896 (pAAV-hDlx-GiDREADD-dTomato-Fishell-5), 83898 (pAAV-mDlx-ChR2-mCherry-Fishell-3), 83899 (pAAV-mDlx-GCaMP6f-Fishell-2), 83900 (pAAV-mDlx-GFP-Fishell-1), and 89897 (pcDNA3-FLAG-mTET2(N500)).Exemplary reporter genes include, in particular, those encoding 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, Amcyan, Midoriishi-Cyan, mTurquoise); green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green (mAzamigreen), CopGFP, AceGFP, avGFP, ZsGreen, Oregon Green (Trademark) (Thermo Fisher Scientific)); luciferase; orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric) Kusabira-Orange, mTangerine, tdTomato, dTomato); red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRuby, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred, Texas Red (Trademark) (Thermo Fisher Scientific)); far-red fluorescent proteins (e.g., mPlum and mNeptune); yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, SYFP2, Venus, YPet, PhiYFP, ZsYellowl); and tandem conjugates.

[0035] GFP is composed of 238 amino acids (26.9 kDa) and was originally isolated from the jellyfish Aequorea victoria / Aequorea aequorea / Aequorea forskalea, which fluoresce green when exposed to blue light. GFP from A. victoria has a large excitation peak at 395 nm and a smaller excitation peak at 475 nm. Its emission peak is at 509 nm, in the lower green part of the visible spectrum. GFP from the sea pansy (Renilla reniformis) has a single major excitation peak at 498 nm. Due to its potential for widespread use and the evolving needs of researchers, many different variants of GFP have been created through genetic engineering. The first major improvement was a single-point mutation (S65T) reported by Roger Tsien in Nature in 1995. This mutation dramatically improved the spectral characteristics of GFP, resulting in increased fluorescence, photostability, and a shift of the primary excitation peak to 488 nm, with peak emission maintained at 509 nm. Adding a 37°C folding efficiency (F64L) point mutant to this skeleton yielded enhanced GFP (EGFP). EGFP is also cited as 55,000 L / (mol●cm), with a density of 9.13 × 10⁻²¹ m 2 It possesses an extinction factor (denoted by ε), also known as the optical cross-section of the molecule. Superfolder GFP, a series of mutations that rapidly fold and mature GFP even when fused to an inadequately folded peptide, was reported in 2006.

[0036] Yellow fluorescent protein (YFP) is a genetic variant of green fluorescent protein derived from the jellyfish Aequorea victoria. Its excitation peak is at 514 nm and its emission peak is at 527 nm.

[0037] Exemplary functional molecules include functional ion transporters, cell transport proteins, enzymes, transcription factors, neurotransmitters, calcium reporters, channelrhodopsins, guide RNAs, nucleases, or designer receptors (DREADDs) that are exclusively activated by designer drugs.

[0038] Ion transporters are transmembrane proteins that mediate the transport of ions across the cell membrane. These ion transporters permeate most cell types and are important for regulating cellular excitability and homeostasis. Ion transporters are involved in numerous cellular processes, including action potentials, synaptic transmission, hormone secretion, and muscle contraction. Many important biological processes in living cells involve the movement of cations, such as calcium (Ca2+), potassium (K+), and sodium (Na+) ions, through such ion channels. In certain embodiments, ion transporters include voltage-gated sodium channels (e.g., SCN1A), potassium channels (e.g., KCNQ2), and calcium channels (e.g., CACNA1C).

[0039] Exemplary enzymes, transcription factors, receptors, membrane proteins, cell transport proteins, signaling molecules, and neurotransmitters include enzymes such as lactase, lipase, helicase, alpha-glucosidase, and amylase; transcription factors such as SP1, AP-1, heat shock factor protein 1, C / EBP (CCAA-T / enhancer-binding protein), and Oct-1; receptors such as transforming growth factor receptor beta-1, platelet-derived growth factor receptor, epidermal growth factor receptor, vascular endothelial growth factor receptor, and interleukin-8 receptor alpha; membrane proteins and cell transport proteins, such as clathrin, dynamin, caveolin, Rab-4A, and Rab-11A; signaling molecules such as nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGFβ), epidermal growth factor (EGF), GTPases, and HRas; and neurotransmitters such as cocaine and amphetamine regulatory transcripts, substance P, oxytocin, and somatostatin.

[0040] In certain embodiments, functional molecules include state-of-the-art cellular function reporters and calcium reporters. Intracellular calcium concentration is a key predictor of numerous cellular activities, including neuronal activation, muscle cell contraction, and second messenger signaling. A sensitive and convenient technique for monitoring intracellular calcium levels is via genetically encoded calcium indicators (GECIs). Among GECIs, a green fluorescent protein (GFP)-based calcium sensor called GCaMP is an efficient and widely used tool. GCaMP is formed by fusing the M13 and calmodulin proteins to the N-terminus and C-terminus of a cyclic mutant GFP. Some GCaMPs yield distinct fluorescence emission spectra (Zhao et al., Science, 2011, 333(6051):1888-1891). Exemplary GECIs exhibiting green fluorescence include GCaMP3, GCaMP5G, GCaMP6s, GCaMP6m, GCaMP6f, jGCaMP7s, jGCaMP7c, jGCaMP7b, and jGCaMP7f. Furthermore, GECIs exhibiting red fluorescence include jRGECO1a and jRGECO1b. AAV products containing GECIs are commercially available.For example, Vigene Biosciences offers AAV8-CAG-GCaMP3 (catalog number: BS4-CX3AAV8), AAV8-Syn-FLEX-GCaMP6s-WPRE (catalog number: BS1-NXSAAV8), AAV8-Syn-FLEX-GCaMP6s-WPRE (catalog number: BS1-NXSAAV8), AAV9-CAG-FLEX-GCaMP6m-WPRE (catalog number: BS2-CXMAAV9), AAV9-Syn-FLEX-jGCaMP7s-WPRE (catalog number: BS12-NXSAAV9), and AAV9-CAG-FLEX We provide AAV products including -jGCaMP7f-WPRE (catalog number: BS12-CXFAAV9), AAV9-Syn-FLEX-jGCaMP7b-WPRE (catalog number: BS12-NXBAAV9), AAV9-Syn-FLEX-jGCaMP7c-WPRE (catalog number: BS12-NXCAAV9), AAV9-Syn-FLEX-NES-jRGECO1a-WPRE (catalog number: BS8-NXAAAV9), and AAV8-Syn-FLEX-NES-jRCaMP1b-WPRE (catalog number: BS7-NXBAAV8).

[0041] In certain embodiments, calcium reporters include genetically encoded calcium indicators GECI, NTnC; myosin light chain kinase, GFP, calmodulin chimera; calcium indicator TN-XXL; BRET-type autoluminescent calcium indicator; and / or calcium indicator protein OeNL(Ca2+)-18u).

[0042] In certain embodiments, functional molecules include regulators of neuronal activity, such as channelrhodopsins (e.g., channelrhodopsin-1, channelrhodopsin-2, and their variants). Channelrhodopsins are a subfamily of retinilidene proteins (rhodopsins) that function as photosensitive 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 chimeras of the transmembrane domains of ChR1 and ChR2 in combination with site-directed mutagenesis. Zhang et al. (Nat Neurosci, 2008, 11(6):631-3) describe VChR1, a red-shifted channelrhodopsin variant. VChR1 exhibits low photosensitivity and poor membrane transport and expression. Other known channelrhodopsin variants include ChR2 variants described in Nagel et al., Proc Natl Acad Sci USA, 2003, 100(24):13940-5, ChR2 / H134R (Nagel, G. et al., Curr Biol, 2005, 15(24):2279-84), and ChD / ChEF / ChIEF (Lin, JY et al., Biophys J, 2009, 96(5):1803-14), which are activated by blue light (470 nm) but do not show sensitivity to orange / red light. Further variants are described in Lin, Experimental Physiology, 2010, 96.1:19-25, and Knopfel et al., The Journal of Neuroscience, 2010, 30(45):14998-15004.

[0043] In certain embodiments, the functional molecule may include DNA and RNA editing tools such as CRISPR / CAS (e.g., guide RNA and nucleases such as Cas, Cas9, or cpf1). The functional molecule may also include genetically modified Cpf1, e.g., as described in US2018 / 0030425, US2016 / 0208243, WO / 2017 / 184768, and Zetsche et al. (2015) Cell 163:759-771; a single gRNA (see, e.g., Jinek et al. (2012) Science 337:816-821, Jinek et al. (2013) eLife 2:e00471, Segal (2013) eLife 2:e00563), or an editase, guide RNA molecule, microRNA, or homologous recombination donor cassette.

[0044] The sequences are publicly available. Examples include lactase (e.g., GenBank:EAX11622.1), lipase (e.g., GenBank:AAA60129.1), helicase (e.g., GenBank:AMD82207.1), amylase (e.g., GenBank:AAA51724.1), alpha-glucosidase (e.g., GenBank:ABI53718.1), transcription factor SP1 (e.g., UniProtKB / Swiss-Prot:P08047.3), transcription factor AP-1 (e.g., NP_002219.1), and heat shock factor proteins. 1 (e.g., UniProtKB / Swiss-Prot:Q00613.1), CCAAT / enhancer-binding protein (C / EBP) beta isoform A (e.g., NP_005185.2), Oct-1 (e.g., UniProtKB / Swiss-Prot:P14859.2), TGFβ (e.g., GenBank:CAF02096.2), platelet-derived growth factor receptor (e.g., GenBank:AAA60049.1), epidermal growth factor receptor (e.g., GenBank:CAA25240.1), vascular endothelial growth factor receptor Clathrin body (e.g., GenBank: AAC16449.2), interleukin-8 receptor alpha (e.g., GenBank: AAB59436.1), caveolin (e.g., GenBank: CAA79476.1), dynamin (e.g., GenBank: AAA88025.1), clathrin heavy chain 1 isoform 1 (e.g., NP_004850.1), clathrin heavy chain 2 isoform 1 (e.g., NP_009029.3), clathrin light chain A isoform a (e.g., NP_001824.1), clathrin light chain B isoform Ras-related protein Rab-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 and amphetamine-modulating transcripts (chain A) (e.g., PDB:1HY9_A), protachykinin-1 (e.g., UniProtKB-P20366), oxytocin-neurophysin-1 (e.g., UniProtKB-P01178), somatostatin (e.g., GenBank:AAH32625.1), genetically encoded green calcium indicator NTnC (chain A) [synthetic construct] (e.g., PDB:5MWC_A), calcium indicator TN-XXL [synthetic construct] (e.g., GenBank:ACF93133.1), BRET-based self-luminescent calcium indicator [synthetic construct] (e.g., GenBank ADF42668.1), calcium indicator protein OeNL(Ca2+)-18u [synthetic construct] ((e.g., GenBank BBB18812.1), myosin light chain kinase, green fluorescent protein, calmodulin chimera (chain A) [synthetic construct] (e.g., PDB:3EKJ_A), 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-related protein (Cas) (e.g., GenBank:AKG27598.1), Cas9 [synthetic construct] (e.g., GenBank:AST09977.1), CRISPR-related end These include nuclease Cpf1 (e.g., UniProtKB / Swiss-Prot:U2UMQ6.1), ribonuclease 4 or ribonuclease L (e.g., UniProtKB / Swiss-Prot:Q05823.2), deoxyribonuclease II beta (e.g., GenBank:AAF76893.1), sodium channel protein type 1 subunit alpha (e.g., UniProtKB-P35498), potassium voltage-gated channel subfamily KQT member 2 (e.g., UniProtKB-O43526), ​​and voltage-gated L-type calcium channel subunit alpha-1C (e.g., UniProtKB-Q13936).

[0045] Further effector elements include Cre, iCre, dgCre, FlpO, and tTA2. iCre refers to codon-modified Cre. dgCre refers to an enhanced GFP / Cre recombinase fusion gene with an N-terminal fusion of the first 159 amino acids of the Escherichia coli K-12 strain chromosome dihydrofolate reductase gene (DHFR or folA) with the G67S mutation, and is modified to also include the R12Y / Y100I unstable domain mutation. FlpO refers to a codon-optimized form of FLPe that significantly increases protein expression and FRT recombination efficiency in mouse cells. Similar to the Cre / LoxP system, the FLP / FRT system is widely used for gene expression (and the generation of conditional knockout mice mediated by the FLP / FRT system). tTA2 refers to a tetracycline transactivator.

[0046] Exemplary expressible elements are expression products that do not contain effector elements, such as non-functional or defective proteins. In certain embodiments, expressible elements can provide a method for studying the effects of their functional counterparts. In certain embodiments, expressible elements are non-functional or defective based on genetically engineered mutations that render them non-functional. In these embodiments, non-expressible elements are as structurally similar as possible to their functional counterparts.

[0047] An exemplary self-cleaving peptide is the 2A peptide, which leads to the production of two proteins from a single mRNA. Because 2A sequences are short (e.g., 20 amino acids), they allow for greater use in size-restricted constructs. Specific examples include P2A, T2A, E2A, and F2A. In certain embodiments, the artificial expression construct includes an internal ribosome entry site (IRES) sequence. The IRES allows the ribosome to initiate translation at a second internal site on the mRNA molecule, leading to the production of two proteins from a single mRNA.

[0048] The coding sequences that encode the molecules described herein (e.g., RNA, proteins) can be obtained from publicly available databases and publications. The coding sequences may further include various sequence polymorphisms, mutations, and / or sequence variants, such modifications which do not affect the function of the encoded molecule. The term “encode” or “encoding” refers to the properties of a nucleic acid sequence, such as a vector, plasmid, gene, cDNA, or mRNA, that serves as a template for the synthesis of proteins and other molecules.

[0049] The term "gene" may include not only coding sequences but also regulatory regions such as promoters, enhancers, insulators, and / or terminal regions. The term may further include all introns and other DNA sequences spliced ​​from mRNA transcripts, as well as variants arising from alternative splice sites. Sequences may also include degenerate codons of a reference sequence, or sequences that can be introduced to provide codon preference in a particular organism or cell type.

[0050] Promoters can include general promoters, tissue-specific promoters, cell-specific promoters, and / or cytoplasm-specific promoters. Promoters can also be strong promoters, weak promoters, constitutive expression promoters, and / or inductive promoters. Inductive promoters induce expression in response to specific conditions, signals, or cellular events. For example, a promoter may be an inductive promoter that requires a specific ligand, small molecule, transcription factor, or hormone protein to produce transcription from the promoter. Specific examples of promoters include minBglobin, CMV, minCMV, and minCMV. * (minCMV * (minCMV is minCMV with the Sacl restriction site removed), minRho, minRho * (minRho *Examples include the minRho promoter (with the Sacl restriction site removed), the SV40 immediate early promoter, the Hsp68 minimal promoter (proHSP68), and the Rous sarcoma virus (RSV) long-term repeat (LTR) promoter. Minimal promoters do not have the activity to drive gene expression on their own, but can be activated to drive gene expression when ligated to a proximal enhancer element.

[0051] In certain embodiments, the expression construct is provided within a vector. The term vector refers to a nucleic acid molecule that can transfer or transport another nucleic acid molecule, such as an expression construct. The transferred nucleic acid is generally ligated, for example, into the vector nucleic acid molecule, or inserted. The vector may contain sequences that direct autonomous replication within a cell, or sequences that enable integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.

[0052] The term "viral vector" is widely used to refer to nucleic acid molecules containing virus-derived nucleic acid elements that facilitate the transfer and expression of non-native nucleic acid molecules into cells. The term "adeno-associated virus vector" primarily refers to viral vectors or plasmids containing structural and functional genetic factors, or parts thereof, derived from AAV. The term "retroviral vector" primarily refers to viral vectors or plasmids containing structural and functional genetic factors, or parts thereof, derived from retroviruses, etc. The term "lentiviral vector" primarily refers to viral vectors or plasmids containing structural and functional genetic factors, or parts thereof, derived from lentiviruses, etc. The term "hybrid vector" refers to a vector containing structural and / or functional genetic factors derived from two or more viral types.

[0053] An adenovirus vector is a construct that (a) supports the packaging of an artificial expression construct and (b) contains sufficient adenovirus sequences to express the cloned coding sequence in either sense or antisense direction. Recombinant adenovirus vectors contain genetically modified forms of adenoviruses. Knowledge of the genetic structure of adenoviruses, which are 36kb linear double-stranded DNA viruses, allows for the substitution of large elements of adenovirus DNA with foreign sequences of up to 7kb. In contrast to retroviruses, adenovirus DNA can replicate in an episomal manner without potential genotoxicity, so adenovirus infection of host cells does not lead to chromosomal integration. Furthermore, adenoviruses are structurally stable, and no genomic rearrangements have been detected after extensive amplification.

[0054] Adenoviruses are particularly well-suited for use as gene transfer vectors due to their medium genome size, ease of handling, high titer, broad target cell range, and high infectivity. The viral genome contains 100-200 base pair reverse repeats (ITRs) at both ends, which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are separated by the initiation of viral DNA replication. The E1 region (E1A and E1B) encodes proteins involved in regulating the transcription of the viral genome and several cellular genes. Expression of the E2 region (E2A and E2B) leads to the synthesis of proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell blockade. The late gene product, which contains most of the viral capsid proteins, is expressed only after significant treatment of a single primary transcript derived from the major late promoter (MLP). MLP is particularly efficient in the later stages of infection, and all mRNA derived from this promoter has a 5'-tripartite leader (TPL) sequence, which is preferred for translation.

[0055] Aside from the requirement that the adenovirus vector is replication-deficient, or at least conditionally defective, the properties of the adenovirus vector are not considered important for the successful practice of the particular embodiments disclosed herein. The adenovirus may be any of the 42 different known serotypes or subgroups A-F. In certain embodiments, adenovirus type 5 of subgroup C is a preferred starting material for obtaining a conditionally replication-deficient adenovirus vector for use in those embodiments, namely because adenovirus type 5 is a human adenovirus for which much of the biochemical and genetic information is publicly known, and because it has been historically used in most constructs that use adenovirus as a vector.

[0056] As explicitly stated, a typical vector is a replication defect and lacks the adenovirus E1 region. Therefore, it would be most convenient to introduce the polynucleotide encoding the target gene at the location where the E1 coding sequence is missing. However, the insertion site of the construct within the adenovirus sequence is not critical. The polynucleotide encoding the target gene can also be inserted in place of the deleted E3 region in an E3 substitution vector or helper cell line, or in the E4 region where a helper virus complements the E4 deficiency.

[0057] Adeno-associated virus (AAV) is a parvovirus discovered as a contaminant of adenovirus strains. It is a ubiquitous virus not associated with any disease (antibodies are present in 85% of the US human population). It is also classified as a helper virus because its replication depends on the presence of helper viruses such as adenoviruses. Various serotypes have been isolated, of which AAV-2 is the most characterized. AAV has single-stranded linear DNA that is encapsulated in capsid proteins VP1, VP2, and VP3 to form icosahedral virions with a diameter of 20-24 nm.

[0058] The AAV DNA is 4.7 kilobases long. It contains two open reading frames and two adjacent ITRs. The AAV genome has two main genes: rep and cap. The rep gene codes for proteins involved in viral replication, while cap codes for capsid proteins VP1-3. Each ITR forms a T-shaped hairpin structure. These terminal repeats are the only essential cis-component of AAV for chromosomal integration. Therefore, AAV can be used as a vector with all viral coding sequences removed and replaced with a cassette of genes for delivery. Three AAV viral promoters have been identified and named p5, p19, and p40 according to their mapped locations. Transcription from p5 and p19 results in the production of rep proteins, while transcription from p40 produces capsid proteins.

[0059] AAVs are noteworthy for use within this disclosure due to their superior safety profile, as well as the fact that their capsids and genomes can be modified to enable expression in selected cell populations. scAAV refers to self-complementary AAVs. pAAV refers to plasmid adeno-associated viruses. rAAV refers to recombinant adeno-associated viruses.

[0060] Other viral vectors can be used. For example, vectors derived from viruses such as vaccinia virus, poliovirus, and herpesvirus can be used. These offer several attractive properties to various mammalian cells.

[0061] Retroviruses are a common tool for gene delivery. A "retrovirus" is an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates that genomic DNA into the host genome. Once integrated into the host genome, the virus is called a "provirus." The provirus functions as a template for RNA polymerase II, inducing the expression of RNA molecules that encode structural proteins and enzymes necessary to produce new viral particles.

[0062] Exemplary retroviruses suitable for use in specific embodiments include: Moloney's mouse leukemia virus (M-MuLV), Moloney's mouse sarcoma virus (MoMSV), Harvey's mouse sarcoma virus (HaMuSV), mouse mammary cancer virus (MuMTV), gibbon leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, friend mouse leukemia virus, mouse stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentivirus.

[0063] "Lentivirus" refers to a group (or genus) of compound retroviruses. Exemplary lentiviruses include: HIV (human immunodeficiency virus, including HIV types 1 and HIV 2); bisnamedivirus (VMV); capriculoarthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In certain embodiments, an HIV-based vector skeleton (i.e., an HIV cis-action sequence element) may be used.

[0064] Improved safety for using certain vectors can be achieved by substituting the U3 region of the 5'LTR with a heterologous promoter to drive transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used for this purpose include, for example, the promoters for viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney's mouse leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase). Typical promoters can drive high levels of transcription in a Tat-independent manner. This substitution reduces the possibility of recombination to produce a reproducible virus because the complete U3 sequence is not present in the viral production system. In certain embodiments, heterologous promoters have further advantages in controlling how the viral genome is transcribed. For example, heterologous promoters can be inducible so that transcription of all or part of the viral genome occurs only when an inducer is present. Inducing factors include one or more chemical compounds, or physiological conditions for culturing host cells, such as temperature or pH.

[0065] In certain embodiments, the viral vector includes a TAR element. The term "TAR" refers to the "transactivation response" gene element located in the R region of the lentiviral LTR. This element interacts with the lentiviral transactivator (tat) gene element to enhance viral replication. However, this element is not required in embodiments where the U3 region of the 5' LTR is replaced with a heterologous promoter.

[0066] The "R region" refers to a region within the retroviral LTR that begins at the start of the capping group (i.e., at the start of transcription) and ends immediately before the start of the poly(A) region. The R region is also defined as being adjacent to the U3 and U5 regions. The R region plays a role in enabling the transfer of nascent DNA from one end of the genome to the other during reverse transcription.

[0067] In certain embodiments, the expression of heterologous sequences in viral vectors is enhanced by incorporating post-transcriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vector. Various post-transcriptional regulatory elements can enhance the expression of heterologous nucleic acids. Examples include the woodchuck virus post-transcriptional regulatory element (WPRE, Zufferey et al., 1999, J. Virol., 73:2886); the post-transcriptional regulatory element present in hepatitis B virus (HPRE) (Smith et al., Nucleic Acids Res. 26(21):4818-4827, 1998); and similar elements (Liu et al., 1995, Genes Dev., 9:1766). In certain embodiments, the vector includes a post-transcriptional regulatory element such as WPRE, WPRE3, or HPRE. In certain embodiments, the vector lacks or does not include a post-transcriptional regulatory element such as WPRE, WPRE3, or HPRE.

[0068] Elements that direct the efficient termination and polyadenylation of heterologous nucleic acid transcripts can enhance heterologous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. In certain embodiments, the vector contains a 3' polyadenylation sequence of the polynucleotide encoding the molecule to be expressed (e.g., a protein). The term "poly(A) site" or "poly(A) sequence" refers to a DNA sequence that directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by adding a poly(A) tail to the 3' end of the coding sequence, thus contributing to improved translation efficiency. Certain embodiments may utilize BGHpA or SV40pA. In certain embodiments, a preferred embodiment of the expression construct includes a terminator element. These elements can help increase transcription levels and minimize reading from the construct to other plasmid sequences.

[0069] In certain embodiments, the viral vector further comprises one or more insulator elements. The insulator elements may contribute to protecting viral vector expression sequences, such as effector elements or expressible elements, from integrated site effects (i.e., positional effects, see, e.g., Burgess-Beusse et al., PNAS., USA, 99:16433, 2002, and Zhan et al., Hum. Genet., 109:471, 2001), which may result in uncontrolled expression of the import sequence, mediated by cis-acting elements present in the genomic DNA. In certain embodiments, the viral import vector comprises one or more insulator elements in the 3'LTR, and upon integration of the provirus into the host genome, the provirus replicates the 3'LTR, thereby comprising one or more insulators in both the 5'LTR and 3'LTR. Suitable insulators for use in specific embodiments include chicken β-globin insulators (Chung et al. Cell 74:505, 1993, Chung et al., PNAS USA 94:575, 1997, and Bell et al. Cell 98:387, 1999), SP10 insulators (Abhyankar et al., JBC 282:36143, 2007), or other small CTCF-recognizing sequences that function as enhancer-blocking insulators (Liu et al. Nature, Biotechnology, 33:198, 2015).

[0070] Beyond the foregoing description, a wide range of suitable expression vector types are known to those skilled in the art. These may include commercially available expression vectors designed for common recombination procedures, such as plasmids containing one or more reporter genes and regulatory elements necessary for the expression of a reporter gene in cells. Numerous vectors are commercially available from, for example, Invitrogen, Stratagene, Clontech, and others, and are described in many relevant guides. In certain embodiments, suitable expression vectors may include any plasmid, cosmid, or phage construct capable of supporting the expression of an encoding gene in mammalian cells, such as the pUC or Bluescript plasmid series.

[0071] Specific embodiments of the vectors disclosed herein include: [Table 1-1] [Table 1-2]

[0072] Those skilled in the art can readily identify the sequences of subcomponents within a large vector sequence, and can readily identify them based on the content of this disclosure (see Figure 12C). The identifiable and enumerated nucleotides between the subcomponents reflect restriction enzyme recognition sites used in construct assembly (cloning), and in some cases, further nucleotides do not convey identifiable function. These segments of the complete vector sequence can be adjusted based on different cloning strategies and / or the use of the vector. Generally, short six-nucleotide palindromic sequences reflect vector construction artifacts that are not important to the vector function.

[0073] In certain embodiments, a vector having a capsid that crosses the blood-brain barrier (BBB) ​​(e.g., AAV) is selected. In certain embodiments, the vector is modified to include a capsid that crosses the BBB. 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. These include 2016;34(2): 204), AAV-PPS (Chen et al., Nat Med. 2009;15: 1215), and PHP.eB. In certain embodiments, the PHP.eB capsid, unlike AAV9, references AAV9, and the amino acid S-AQ-A (SEQ ID NO: 144), which begins at residue 586, is changed to S-DGTLAVPFK-A (SEQ ID NO: 145). In certain embodiments, PHP.eB references SEQ ID NO: 85.

[0074] AAV9 is a naturally occurring AAV serotype that, unlike many other naturally occurring serotypes, can cross the blood-brain barrier (BBB) ​​after intravenous injection. It transforms a large portion of the central nervous system (CNS) and thus enables minimally invasive treatments, as described in relation to clinical trials for the treatment of spinal muscular atrophy (SMA) syndrome with AveXis (AVXS-101, NCT03505099) and CLN3 gene-associated neuronal ceroid-lipofuse syndrome (NCT03770572) (Naso et al., BioDrugs. 2017;31(4):317).

[0075] AAVrh.10 was originally isolated from rhesus monkeys and, compared to other common serotypes used for gene delivery, exhibits low seropositivity in humans (see Selot et al., Front Pharmacol. 2017;8:441). It has been evaluated in clinical trials for LYS-SAF302, LYSOGENE, and NCT03612869.

[0076] Two mutants isolated from a library of chimeric AAV vectors, AAV1R6 and AAV1R7 (in which the AAV1 capsid domain was replaced with AAVrh.10), retained the ability to cross the blood-brain barrier and transduce the central nervous system (CNS), while showing a significant reduction in hepatic and vascular endothelial transduction.

[0077] Similarly, rAAVrh.8 isolated from rhesus macaques exhibits global transduction of glial and neuronal cell types in clinically important areas after peripheral administration, and also shows reduced peripheral tissue tropism compared to other vectors.

[0078] AAV-BR1 is an AAV2 variant that displays the NRGTEWD ​​(SEQ ID NO: 146) epitope, isolated during in vivo screening of a random AAV display peptide library. It exhibits high specificity with high transgene expression in the brain and minimal off-target affinity (including in the liver) (Korbelin et al., EMBO Mol Med. 2016;8(6):609).

[0079] AAV-PHP.S (Addgene, Watertown, MA) is a variant of AAV9 created using the CREATE method, encoding the 7-mer sequence QAVRTSL (sequence number 147), and transducing neurons in the enteric nervous system, thereby strongly transducing peripheral sensory afference into the spinal cord and brainstem.

[0080] AAV-PHP.B (Addgene, Watertown, MA) is a variant of AAV9 generated by the CREATE method, encoding the 7-mer sequence TLAVPFK (SEQ ID NO: 148). It translocates genes across the entire CNS with higher efficiency than AAV9, transducing a large portion of astrocytes and neurons across multiple CNS regions.

[0081] AAV-PPS was created by inserting the DSPAHPS (sequence number 149) epitope into the capsid of AAV2, and it shows dramatically improved brain tropism compared to AAV2.

[0082] For further information on capsids that cross the blood-brain barrier, see Chan et al., Nat. Neurosci, 2017 Aug:20(8):1172-1179.

[0083] (ii) Compositions for administration. The artificial expression constructs and vectors of this disclosure (hereinafter referred to as physiologically active components) can be formulated with carriers suitable for administration to cells, tissue slices, animals (e.g., mice, non-human primates), or humans. The physiologically active components in the compositions described herein can be prepared in a neutral form, as free bases or as pharmacokinetically acceptable salts.

[0084] As pharmaceutically acceptable salts, there are acid addition salts (formed from free amino groups of proteins), which are formed from inorganic acids, such as hydrochloric acid or phosphoric acid, or from organic acids, such as acetic acid, oxalic acid, tartaric acid, and mandelic acid. Salts formed from free carboxyl groups can be obtained from inorganic bases, such as sodium, potassium, ammonium, calcium, or ferric hydroxide, as well as from organic bases, such as isopropylamine, trimethylamine, histidine, and procaine.

[0085] Carriers for physiologically active components can include solvents, dispersion media, vehicles, coatings, diluents, isotonic agents, and absorption retarders, buffers, solutions, suspensions, colloids, etc. The use of such carriers for physiologically active components is well known in the art. Any conventional culture medium or agent can be used with the compositions described herein, except when the conventional medium or agent is incompatible with the physiologically active component.

[0086] The term "pharmaceutically acceptable carrier" refers to a carrier that does not cause allergic or similar adverse reactions when administered to humans, and in certain embodiments, when administered intravenously (for example, into the posterior orbital plexus).

[0087] In certain embodiments, the composition may be formulated for intravenous, intraparenchymal, intraocular, intravitreous, parenteral, subcutaneous, intraventricular, intramuscular, subarachnoid, intraspinal, intraperitoneal, oral or intranasal inhalation, or for direct injection or application to one or more cells, tissues, or organs.

[0088] The composition may include liposomes, lipids, lipid complexes, microspheres, fine particles, nanospheres, and / or nanoparticles.

[0089] The formation and use of liposomes are generally known to those skilled in the art. Liposomes with improved serum stability and circulating half-lives have been developed (see, for example, U.S. Patent No. 5,741,516). Furthermore, various methods for liposomes and liposome-like preparations as potential drug carriers have been described (see, for example, U.S. Patents No. 5,567,434; No. 5,552,157; ​​No. 5,565,213; No. 5,738,868; and No. 5,795,587).

[0090] Furthermore, this disclosure provides pharmaceutically acceptable nanocapsule formulations of physiologically active components. Nanocapsules can generally encapsulate compounds in a stable and reproducible manner (Quintanar-Guerrero et al. Drug Dev Ind Pharm 24(12):1113-1128,1998; Quintanar-Guerrero et al. Pharm Res. 15(7):1056-1062,1998; Quintanar-Guerrero et al., J. Microencapsul. 15(1):107-119,1998; Douglas et al. Crit, Rev Ther Drug Carrier Syst 3(3):233-261,1987). To avoid side effects due to intracellular polymer overload, such ultrafine particles can be designed using polymers that can be degraded in vivo. Biodegradable polyalkylcyanoacrylate nanoparticles that meet these requirements are planned for use in this disclosure. Such particles can be readily produced as described in Couvreuret alo., J Pharm Sci 69(2):199-202, 1980; Couvreuret al., Crit Rev Ther Drug Carrier Syst. 5(1)1-20, 1988; zur Muhlenet al., Eur J Pharm Biopharm, 45(2):149-155, 1998; Zambaux et al., J Control Release 50(1-3):31-40, 1998, and U.S. Patent No. 5,145,684.

[0091] The injectable composition may include a sterile aqueous solution, or a dispersion and sterile powder for preparing a sterile injectable solution or dispersion at the time of use (U.S. Patent No. 5,466,468). For delivery by injection, the form is sterile and fluid enough to be delivered by syringe. In certain embodiments, the composition is stable under manufacturing and storage conditions and optionally contains one or more preservative compounds to protect against microbial contamination, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. Adequate fluidity can be maintained, for example, by the use of a coating such as lecithin, maintaining the required particle size in the case of a dispersion, and / or by using a surfactant. Prevention of microbial action can be achieved with various antimicrobial and / or antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In various embodiments, the preparation may include isotonic agents, such as sugars or sodium chloride. Sustained absorption of the injectable composition can be achieved by including agents that delay absorption into the composition, such as aluminum monostearate and gelatin. The injectable composition may be appropriately buffered as needed, and the liquid diluent may first be made isotonic with sufficient saline or glucose.

[0092] The dispersion may be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. As shown, under normal storage and use conditions, these preparations may contain preservatives to prevent microbial growth.

[0093] Sterile compositions can be prepared by incorporating physiologically active components into an appropriate amount of a solvent containing other optional components (e.g., those described above), followed by filtration sterilization. Generally, dispersions are prepared by incorporating various sterilized physiologically active components into a sterile vehicle containing a basic dispersion medium and other necessary components (e.g., those described above). In the case of sterile powders for preparing sterile injection solutions, preferred preparation methods may be vacuum drying and freeze-drying techniques, from which powders of physiologically active components and any further desired components are obtained from a pre-sterilized filtered solution.

[0094] Oral compositions may be in liquid form, for example, as a solution, syrup, or suspension, or they may be presented as drug products to be reconstituted with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifiers (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoic acid or sorbic acid). The composition may take the form of tablets or capsules prepared with pharmaceutically acceptable excipients such as, for example, binders (e.g., pre-gelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well known in the art.

[0095] The inhalable composition may be delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer, using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dose unit may be determined by providing a valve for delivering the measured amount. Capsules and cartridges, for example, gelatin capsules and cartridges for use in inhalers or fillers, may be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

[0096] The composition also includes a microchip device (U.S. Patent No. 5,797,898), an ophthalmic formulation (Bourlais et al., Prog Retin Eye Res, 17(1):33-58, 1998), a transdermal matrix (U.S. Patents No. 5,770,219 and 5,783,208), and feedback-controlled delivery (U.S. Patent No. 5,697,899).

[0097] Furthermore, auxiliary active ingredients can also be incorporated into the composition.

[0098] Typically, a composition may contain at least 0.1% of a physiologically active component, although the proportion of the physiologically active component can certainly vary, and conveniently, this could be 1 or 2% to 70% or 80% or more, or 0.5% to 99%, of the total weight or volume of the composition. Naturally, the amount of physiologically active component in each physiologically useful composition can be prepared in such a way that a suitable dose is obtained for any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, and other pharmacological considerations are planned by those skilled in the art who prepare such pharmaceutical formulations, and therefore, various compositions and dosages may be desirable.

[0099] In certain embodiments, for administration to humans, the composition should meet sterility, pyrogenicity, and general safety and purity standards required by the U.S. Food and Drug Administration (FDA) or other applicable regulatory bodies in the country.

[0100] (iii) Cell lines containing the artificial expression construct. This disclosure includes cells containing the artificial expression construct described herein. Cells transformed with the artificial expression construct can be used for a number of purposes, including neuroanatomical studies, evaluation of functional and / or non-functional proteins, and drug screening to evaluate the regulatory properties of enhancers.

[0101] Various host cell lines can be used, but in certain embodiments, the cells are mammalian cells.In a particular embodiment, the artificial expression constructs are eHGT_450m, eHGT_451m, eHGT_452m, eHGT_453m, eHGT_457m, eHGT_458m, eHGT_459m, eHGT_460m, eHGT_462m, eHGT_464m, eHGT_461m, eHGT_463m, eHGT_692h, eHGT_693h, eHGT_694h, eHGT_695h, eHGT_667m, eHGT_668m, eHGT_696m, eHGT_455m, eHGT_457h, eHGT_603m, eHGT_604m, e HGT_605m, eHGT_661m, eHGT_766m, eHGT_767m, eHGT_768m, eHGT_663m, eHGT_660m, eHGT_665m, eHGT_700m, eHGT_456m, eHGT_698m, eHGT_699m, eHGT_6 47m, eHGT_648m, eHGT_649m, eHGT_670m, eHGT_697m, eHGT_662m, eHGT_583m, 3xcore2_eHGT_459m, 3xcore2_eHGT_453m, 3xcore2_eHGT_462m, 3xcore2 _eHGT_461m, 3xcore2_eHGT_464m, 3xcore2_eHGT_452m, 3xcore2_eHGT_451m, and / or 3xcore2_eHGT_460m and / or CN2248, CN2249, CN2250, CN2251, CN2252, CN2253, CN2254, CN2255, CN2256, CN2315, CN2423, CN2424, CN2690, CN2695, CN2691, CN2692, CN2944, CN2886, CN2755, CN2938, CN2239, CN2410, CN The cell line comprises enhancer and / or vector sequences of 2411, CN2412, CN2963, CN2623, CN2624, CN2601, CN2843, CN2851, CN2853, CN2860, CN2828, CN2870, CN2871, CN2847, CN2686, CN2687, CN2878, CN2946, CN2943, CN3319, CN2918, CN3018, CN3030, CN3027, CN3032, CN3015, CN3012, and / or CN3024, and the cell line is human, primate, or mouse cell.Concatemers of 1, 2, 4, 5, 6, 7, 8, 9, or 10 copies of the disclosed enhancer core can also be used. Furthermore, cell lines that can be used for transformation in this disclosure include primary cell lines derived from living tissues such as rat or mouse brains, and organ-type cell cultures containing brain slices derived from animals such as rats or mice. The PC12 cell line (available from the American Type Culture Collection, ATCC, Manassas, VA) has been shown to express numerous neuronal marker proteins in response to neuronal growth factor (NGF). The PC12 cell line is considered a neuronal cell line and can be used in this disclosure. JAR cells (available from ATCC) are platelet-derived cell lines that express certain neuronal genes, such as the serotonin transporter gene, and can be used in conjunction with the embodiments described herein.

[0102] WO91 / 13150 describes various cell lines, including neuronal cell lines, and methods for producing them. Similarly, WO97 / 39117 describes neuronal cell lines and methods for producing such cell lines. The neuronal cell lines disclosed in these patent applications are applicable to use in this disclosure.

[0103] In certain embodiments, “neuronal” means a nerve cell, related to a nerve cell, or containing a nerve cell. A nerve cell is defined by the presence of an axon and dendrites. The term “neuronal-specific” means an activity found or occurring in nerve cells or cells derived from nerve cells that is not found, does not occur, or substantially not found or substantially not occurring in non-nerve cells or cells not derived from nerve cells, such as glial cells like astrocytes or oligodendrocytes.

[0104] In certain embodiments, non-neuronal cell lines, including mouse embryonic stem cells, may be used. Using cultured mouse embryonic stem cells, gene construct expression can be analyzed using transient transfection with plasmid constructs. Mouse embryonic stem cells are pluripotent and undifferentiated. These cells can be maintained in this undifferentiated state by leukemia inhibitors (LIFs). LIF withdrawal induces differentiation of embryonic stem cells. In culture, stem cells form various differentiated cell types. Differentiation is triggered by the expression of tissue-specific transcription factors, and the function of enhancer sequences can be evaluated. (See, for example, Fiskerstrand et al., FEBS Lett 458:171-174, 1999.)

[0105] Methods for differentiating stem cells into different cell types include replacing the stem cell culture medium with a medium containing basic fibroblast growth factor (bFGF) heparin, N2 supplements (e.g., transferrin, insulin, progesterone, putrescine, and selenate), laminin, and polyornithine. The process for producing myelinated oligodendrocyte cells 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, while Chatzi, et al., 2009, Exp. Neurol. 217:407-16 describes a procedure for producing GABAergic neurons. This procedure involves exposing stem cells to total trans RA for 3 days. Subsequently, 95% of the GABA neurons grew after being cultured in serum-free neuronal induction medium containing Neurobasal medium supplemented with B27, bFGF, and EGF.

[0106] U.S. Publication No. 2012 / 0329714 describes increasing the number of neural stem cells using prolactin, and U.S. Publication No. 2012 / 0308530 describes a culture surface with amino groups that promotes neuronal differentiation into neurons, astrocytes, and oligodendrocytes. Therefore, the fate of neural stem cells can be controlled by various extracellular factors. Commonly used factors include brain-derived growth factor (BDNF; Shetty and Turner, 1998, J. Neurobiol. 35:395-425); fibroblast growth factor (bFGF; U.S. Patent No. 5,766,948; FGF-1, FGF-2); neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4); Caldwell, et al., 2001, Nat. Biotechnol. 1; 19:475-9); ciliary neurotrophic factor (CNTF); BMP-2 (U.S. Patent Nos. 5,948,428 and 6,001,654); and isobutyl These include 3-methylxanthine; leukemia-suppressing growth factor (LIF; U.S. Patent No. 6,103,530); somatostatin; amphiregulin; neurotrophins (e.g., cyclic adenosine monophosphate; epidermal growth factor (EGF); dexamethasone (glucocorticoid hormone); forskolin; GDNF family receptor ligands; potassium; retinoic acid (U.S. Patent No. 6,395,546); tetanus toxin; and transformation of growth factor-α and TGF-β (U.S. Patents No. 5,851,832 and 5,753,506).

[0107] In certain embodiments, a yeast one-hybrid system is used to produce eHGT_450m, eHGT_451m, eHGT_452m, eHGT_453m, eHGT_457m, eHGT_458m, eHGT_459m, eHGT_460m, eHGT_462m, eHGT_464m, eHGT_461m, eHGT_463m, eHGT_692h, eHGT_693h, eHGT_694h, eHGT_695h, eHGT_667m, eHGT_668m, eHGT_696m, eHGT_455m, eHGT_457h, eHGT_603m Compounds that inhibit specific protein / DNA interactions, such as eHGT_604m, eHGT_605m, eHGT_661m, eHGT_766m, eHGT_767m, eHGT_768m, eHGT_663m, eHGT_660m, eHGT_665m, eHGT_700m, eHGT_456m, eHGT_698m, eHGT_699m, eHGT_647m, eHGT_648m, eHGT_649m, eHGT_670m, eHGT_697m, eHGT_662m, eHGT_583m, and their core transcription factors, can also be identified.

[0108] Transgenic animals are described below. Cell lines may also be derived from such transgenic animals. For example, primary tissue cultures derived from transgenic mice (as described below, for example) can provide cell lines with artificial expression constructs already incorporated into the genome (see, for example, MacKenzie and Quinn, Proc Natl Acad Sci USA 96:15251-15255, 1999).

[0109] (iv) Transgenic animals. Another aspect of the present disclosure includes transgenic animals whose genomes are operably linked to heterologous coding sequences eHGT_450m, eHGT_451m, eHGT_452m, eHGT_453m, eHGT_457m, eHGT_458m, eHGT_459m, eHGT_460m, eHGT_462m, eHGT_464m, eHGT_461m , eHGT_463m, eHGT_692h, eHGT_693h, eHGT_694h, eHGT_695h, eHGT_667m, eHGT_668m, eHGT_696 m, eHGT_455m, eHGT_457h, eHGT_603m, eHGT_604m, eHGT_605m, eHGT_661m, eHGT_766m, eHGT_76 7m, eHGT_768m, eHGT_663m, eHGT_660m, eHGT_665m, eHGT_700m, eHGT_456m, eHGT_698m, eHGT_6 99m, eHGT_647m, eHGT_648m, eHGT_649m, eHGT_670m, eHGT_697m, eHGT_662m, eHGT_583m, 3xcor Includes artificial expression constructs containing e2_eHGT_459m, 3xcore2_eHGT_453m, 3xcore2_eHGT_462m, 3xcore2_eHGT_461m, 3xcore2_eHGT_464m, 3xcore2_eHGT_452m, 3xcore2_eHGT_451m, and / or 3xcore2_eHGT_460m. Concatemers of 1, 2, 4, 5, 6, 7, 8, 9, or 10 copies of the disclosed enhancer core may also be used.In a particular embodiment, the genome of the transgenic animal is CN2248, CN2249, CN2250, CN2251, CN2252, CN2253, CN2254, CN2255, CN2256, CN2315, CN2423, CN2424, CN2690, CN2695, CN2691, CN2692, CN2944, CN2886, CN2755, CN2938, CN2239, CN2410, CN2411, CN241 2, including CN2963, CN2623, CN2624, CN2601, CN2843, CN2851, CN2853, CN2860, CN2828, CN2870, CN2871, CN2847, CN2686, CN2687, CN2878, CN2946, CN2943, CN3319, CN2918, CN3018, CN3030, CN3027, CN3032, CN3015, CN3012, and / or CN3024.In certain embodiments, when non-embedded vectors are used, the transgenic animal expresses artificial constructs eHGT_450m, eHGT_451m, eHGT_452m, eHGT_453m, eHGT_457m, eHGT_458m, eHGT_459m, eHGT_460m, eHGT_462m, eHGT_464 in one or more of its cells. m, eHGT_461m, eHGT_463m, eHGT_692h, eHGT_693h, eHGT_694h, eHGT_695h, eHGT_667m, eHGT_6 68m, eHGT_696m, eHGT_455m, eHGT_457h, eHGT_603m, eHGT_604m, eHGT_605m, eHGT_661m, eHGT_ 766m, eHGT_767m, eHGT_768m, eHGT_663m, eHGT_660m, eHGT_665m, eHGT_700m, eHGT_456m, eHG T_698m, eHGT_699m, eHGT_647m, eHGT_648m, eHGT_649m, eHGT_670m, eHGT_697m, eHGT_662m, eH GT_583m, 3xcore2_eHGT_459m, 3xcore2_eHGT_453m, 3xcore2_eHGT_462m, 3xcore2_eHGT_461m, 3xcore2_eHGT_464m, 3xcore2_eHGT_452m, 3xcore2_eHGT_451m, and / or 3xcore2_eHGT_460m and / or CN2248, CN2249, CN2250, CN2251, CN2252, CN2253, CN2254, CN2255, CN2256, CN2315, CN2423, CN2424, CN2690, CN2695, CN2691, CN2692, CN2944, CN2886, CN2755, CN2938, CN2239, CN2410, CN2411, CN2412, CN2963, C Includes N2623, CN2624, CN2601, CN2843, CN2851, CN2853, CN2860, CN2828, CN2870, CN2871, CN2847, CN2686, CN2687, CN2878, CN2946, CN2943, CN3319, CN2918, CN3018, CN3030, CN3027, CN3032, CN3015, CN3012, and / or CN3024.Concatemers of 1, 2, 4, 5, 6, 7, 8, 9, or 10 copies of the disclosed enhancer core can also be used.

[0110] Detailed methods for producing transgenic animals are described in U.S. Patent No. 4,736,866. Transgenic animals may be any non-human species, but preferably include non-human primates (NHPs), sheep, horses, cattle, pigs, goats, dogs, cats, rabbits, chickens, and rodents, such as guinea pigs, hamsters, gerbils, rats, mice, and ferrets.

[0111] In certain embodiments, the construction of a transgenic animal results in an organism having a genetically modified construct present in all cells within the same genomic integration site. Therefore, cell lines derived from such transgenic animals will be as consistent in all cells as if the genetically modified construct were located in the same genomic integration site, and thus will undergo the same position-effect mutations. In contrast, introducing a gene into a cell line or primary cell culture can result in heterologous expression of the construct. A drawback of this approach is that the expression of the introduced DNA may be influenced by the specific genetic background of the host animal.

[0112] As shown above in relation to cell lines, the artificial expression constructs of this disclosure may be used to genetically modify mouse embryonic stem cells using techniques known in the art. Typically, the artificial expression construct is introduced into cultured mouse embryonic stem cells. The transformed ES cells are then injected into a blastocyst from a host mother, and the host embryo is re-transplanted into the mother. This results in a chimeric mouse whose tissues are 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, the mice from which the cultured ES cells used for introduction originate are selected to have a different coat color from the host mouse into which the transformed cells are injected into its embryo. The chimeric mice then have a variety of coat colors. At least some of these can be crossed with a suitable strain to produce offspring that carry the transgene, as long as the germline tissues originate from the genetically modified cells.

[0113] In addition to the delivery methods described above, the following techniques are also intended as alternative methods for delivering artificial expression constructs to target cells or selected tissues and organs of animals, particularly cells, organs, or tissues of vertebrates: ultrasound (e.g., ultrasound as described in U.S. Patent No. 5,656,016); intraosseous injection (U.S. Patent No. 5,779,708); microchip devices (U.S. Patent No. 5,797,898); ophthalmic formulations (Bourlais et al., Prog Retin Eye Res, 17(1):33-58, 1998); transdermal matrices (U.S. Patents No. 5,770,219 and U.S. Patent No. 5,783,208); feedback-controlled delivery (U.S. Patent No. 5,697,899); and other delivery methods available and / or described elsewhere in this disclosure.

[0114] (v) Method of use. In certain embodiments, compositions containing the physiologically active ingredients described herein are administered to a subject to produce a physiological effect.

[0115] In certain embodiments, this disclosure includes the use of the artificial expression constructs described herein to modulate the expression of heterologous genes partially or completely encoded at a downstream position of an enhancer in a genetically engineered sequence. Thus, methods of using the disclosed artificial expression constructs in the research, clinical trials, and potential development of pharmaceuticals for preventing, treating, or mitigating symptoms of disease, dysfunction, or impairment are provided herein.

[0116] Specific embodiments are those described herein as eHGT_450m, eHGT_451m, eHGT_452m, eHGT_453m, eHGT_457m, eHGT_458m, eHGT_459m, eHGT_460m, eHGT_462m, eHGT_464m, eHGT_461m, eHGT_463m, eHGT_692h, eHGT_693h, eHGT_694h, eHGT_695h, eHGT_667m, eHGT_668m, eHGT_696m, eHGT_455m, eHGT_457h, eHGT_603m, eHGT_604m, e HGT_605m, eHGT_661m, eHGT_766m, eHGT_767m, eHGT_768m, eHGT_663m, eHGT_660m, eHGT_665m, eHGT_700m, eHGT_456m, eHGT_698m, eHGT_699m, eHGT_ 647m, eHGT_648m, eHGT_649m, eHGT_670m, eHGT_697m, eHGT_662m, eHGT_583m, 3xcore2_eHGT_459m, 3xcore2_eHGT_453m, 3xcore2_eHGT_462m, 3xcore 2_eHGT_461m, 3xcore2_eHGT_464m, 3xcore2_eHGT_452m, 3xcore2_eHGT_451m, and / or 3xcore2_eHGT_460m, and / or CN2248, CN2249, CN2250, CN2251, CN2252, CN2253, CN2254, CN2255, CN2256, CN2315, CN2423, CN2424, CN2690, CN2695, CN2691, CN2692, CN2944, CN2886, CN2755, CN2938, CN2239, CN2410 The method includes administering an artificial expression construct containing CN2411, CN2412, CN2963, CN2623, CN2624, CN2601, CN2843, CN2851, CN2853, CN2860, CN2828, CN2870, CN2871, CN2847, CN2686, CN2687, CN2878, CN2946, CN2943, CN3319, CN2918, CN3018, CN3030, CN3027, CN3032, CN3015, CN3012, and / or CN3024 to a target to drive selective expression of the gene in a selected cell type.Concatemers of 1, 2, 4, 5, 6, 7, 8, 9, or 10 copies of the disclosed enhancer core can also be used. The subject can be an isolated cell, a cell network, a tissue slice, a laboratory animal, a veterinary animal, or a human.

[0117] As is well known in the medical technology field, the dosage for any one subject depends on many factors, including the subject's size, surface area, age, the particular compound to be administered, sex, administration time and administration route, general health, and other drugs being administered concurrently. While the dose of the compound of the present disclosure varies, in certain embodiments, the dose is 10 of the artificial expression construct of the present disclosure 5 ~10 100 copies. In certain embodiments, a patient receiving intravenous administration, intraparenchymal administration, intraspinal administration, retro-orbital administration, or intrathecal administration can be injected with 10 6 ~10 22 copies of the artificial expression construct.

[0118] An "effective amount" is the amount of the composition necessary to produce a desired physiological change in a subject. Effective amounts are often administered for research purposes. The effective amounts disclosed herein can cause a statistically significant effect in an animal model or an in vitro assay.

[0119] The amount of the expression construct of such a composition and the administration time will be within the scope of those skilled in the art having the benefit of the present teachings. However, administration of an effective amount of the disclosed composition will most likely be achievable by a single administration, such as a single injection of a sufficient number of infectious particles to produce an effect in the subject. Alternatively, in some circumstances, it may be desirable to provide multiple or sequential administrations of the artificial expression construct composition or other genetic constructs, over either a relatively short or a relatively long period of time, as may be determined by the individual supervising the administration of such compositions. For example, the number of infectious particles administered to a mammal is 10, which can be given either as a single dose or divided into two or more administrations, as may be required to achieve the intended effect 7 , 10 8, 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or even higher infectivity particles / ml. In fact, in certain embodiments, it may be desirable to administer two or more different expression constructs in combination to achieve the desired effect.

[0120] In certain circumstances, it may be desirable to deliver the artificial expression construct in a suitably formulated composition disclosed herein by any of the following means: pipetting, posterior orbital injection, subcutaneous, intraocular, intravitreous, parenteral, subcutaneous, intravenous, intraparenchymal, intrathecal, intraspinal, intraperitoneal, oral or nasal inhalation, or direct application or injection into one or more cells, tissues, or organs. The methods of administration may also include those forms as described in U.S. Patents 5,543,158, 5,641,515, and 5,399,363.

[0121] (vi) Kits and commercially available packages. Kits and commercially available packages contain the artificial expression constructs described herein. The artificial expression constructs can be isolated. In certain embodiments, the components of the expression product can be isolated from each other. In certain embodiments, the expression product may be in a vector, in a viral vector, in an intracellular, in a tissue slice or sample, and / or in a transgenic animal. Such kits may further include one or more reagents, restriction enzymes, peptides, therapeutic agents, pharmaceutical compounds, or means of delivering compositions such as syringes or injectable preparations.

[0122] Embodiments of the kit or commercially available package also include, for example, a description of the use of the components contained herein in basic research, electrophysiological research, neuroanatomical research, and / or research and / or treatment of disorders, diseases or conditions.

[0123] The following exemplary embodiments are included to demonstrate specific embodiments of the Disclosure. Those skilled in the art will understand that many modifications can be made to the specific embodiments disclosed herein in light of the Disclosure, and that similar or comparable results can be obtained without departing from the spirit and scope of the Disclosure.

[0124] (vii) Exemplary embodiments. 1. The connecting core of the enhancer disclosed herein. 2. A linked core of eHGT_459m, eHGT_453m, eHGT_462m, eHGT_461m, eHGT_464m, eHGT_452m, eHGT_451m, or eHGT_460m enhancers. 3. The linked core of Embodiment 1 or 2, wherein the linked core includes the sequence described in Sequence ID No. 45, Sequence ID No. 46, Sequence ID No. 47, Sequence ID No. 48, Sequence ID No. 49, Sequence ID No. 50, Sequence ID No. 51, and / or Sequence ID No. 52. 4. The linked core of Embodiment 1 or 2, wherein the linked core includes the sequence described in Sequence ID No. 45, Sequence ID No. 46, Sequence ID No. 47, Sequence ID No. 48, Sequence ID No. 49, Sequence ID No. 50, Sequence ID No. 51, or Sequence ID No. 52. 5. A connecting core according to any of embodiments 1 to 4, wherein the connecting core includes 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the enhancer core. A linked core of Embodiment 5, including 6.2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of SEQ ID NOs. 45, 46, 47, 48, 49, 50, 51, and / or 52. A linked core of Embodiment 5, including 7.2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 52. 8.2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of sequence number 45; Sequence ID 46 in copies 2, 3, 4, 5, 6, 7, 8, 9, or 10; Array 47 in 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies; Sequence ID 48 in copies 2, 3, 4, 5, 6, 7, 8, 9, or 10; 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of sequence number 49; Array 50 in copies 2, 3, 4, 5, 6, 7, 8, 9, or 10; 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of sequence number 51; or 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of sequence number 52, A connecting core of embodiment 5, including the following. 9.3 A linked core of Embodiment 5, including SEQ ID NO: 45; SEQ ID NO: 46; SEQ ID NO: 47; SEQ ID NO: 48; SEQ ID NO: 49; SEQ ID NO: 50; SEQ ID NO: 51; or SEQ ID NO: 52. 10. The connecting core of Embodiment 5, wherein the connecting core includes SEQ ID NO: 53; SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; or SEQ ID NO: 60. 11.eHGT_450m, eHGT_451m, eHGT_452m, eHGT_453m, eHGT_457m, eHGT_458m, eHGT_459m, eHGT_460m, eHGT_462m, eHGT_464m, eHGT_46 1m, eHGT_463m, eHGT_692h, eHGT_693h, eHGT_694h, eHGT_695h, eHGT_667m, eHGT_668m, eHGT_696m, eHGT_455m, eHGT_457h, eHGT_603 Isolated enhancers selected from m, eHGT_604m, eHGT_605m, eHGT_661m, eHGT_766m, eHGT_767m, eHGT_768m, eHGT_663m, eHGT_660m, eHGT_665m, eHGT_700m, eHGT_456m, eHGT_698m, eHGT_699m, eHGT_647m, eHGT_648m, eHGT_649m, eHGT_670m, eHGT_697m, eHGT_662m, and eHGT_583m. 12. (i) an enhancer or its linked core according to any of Embodiments 1 to 11; (ii) a promoter; and (iii) an artificial expression construct comprising a heterogeneous coding sequence. 13. The artificial expression construct according to Embodiment 12, wherein the heterogeneous code sequence codes for an effector element or an expressible element. 14. The artificial expression construct according to Embodiment 13, wherein the effector element comprises a reporter protein or a functional molecule. 15. The artificial expression construct according to Embodiment 14, wherein the reporter protein comprises a fluorescent protein. 16. An artificial expression construct according to Embodiment 13 or 14, wherein the functional molecule comprises a functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cell transport protein, signaling molecule, neurotransmitter, calcium reporter, channelrhodopsin, CRISPR / CAS molecule, editase, guide RNA molecule, homologous recombination donor cassette, designer receptor (DREADD) exclusively activated by a designer drug, growth factor, or survival-promoting gene. 17. An artificial expression construct according to any one of embodiments 13 to 16, wherein the expressible element includes a non-functional molecule. 18. An artificial expression construct according to Embodiment 17, wherein the non-functional molecule comprises a non-functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cell transport protein, signaling molecule, neurotransmitter, calcium reporter, channelrhodopsin, CRISPR / CAS molecule, editase, guide RNA molecule, homologous recombination donor cassette, DREADD, growth factor, or survival-promoting gene. 19. The artificial expression construct according to any one of embodiments 12 to 18, wherein the artificial expression construct is associated with a capsid that crosses the blood-brain barrier. 20. The artificial expression construct according to Embodiment 19, wherein the capsid comprises PHP.eB, AAV-BR1, AAV-PHP.S, AAV-PHP.B, or AAV-PPS. 21. An artificial expression construct according to any one of embodiments 12 to 20, wherein the artificial expression construct includes or encodes a skipping element. 22. The artificial expression construct according to Embodiment 21, wherein the skipping element comprises a 2A peptide and / or an internal ribosome entry site (IRES). 23. The artificial expression construct according to Embodiment 22, wherein the 2A peptide comprises T2A, P2A, E2A, or F2A. 24. The artificial expression constructs are: eHGT_450m, eHGT_451m, eHGT_452m, eHGT_453m, eHGT_457m, eHGT_458m, eHGT_459m, eHGT_460m, eHGT_462m, eHGT_464m, eHGT_461m, eHGT_463m, eHGT_692h, eHGT_693h, eHG T_694h, eHGT_695h, eHGT_667m, eHGT_668m, eHGT_696m, eHGT_455m, eHGT_457h, eHGT_603m, e HGT_604m, eHGT_605m, eHGT_661m, eHGT_766m, eHGT_767m, eHGT_768m, eHGT_663m, eHGT_660m, eHGT_665m, eHGT_700m, eHGT_456m, eHGT_698m, eHGT_699m, eHGT_647m, eHGT_648m, eHGT_649 m, eHGT_670m, eHGT_697m, eHGT_662m, eHGT_583m, 3xcore2_eHGT_459m, 3xcore2_eHGT_453m, 3 xcore2_eHGT_462m, 3xcore2_eHGT_461m, 3xcore2_eHGT_464m, 3xcore2_eHGT_452m, 3xcore2_eHGT_451m, 3xcore2_eHGT_460m, AAV, scAAV, rAAv, minBglobin, CMV, minCMV, minRho, minRho * An artificial expression construct according to any of Embodiments 12 to 23, comprising or encoding a set of features selected from fluorescent proteins (e.g., EGFP, SYFP, SYFP2, GFP), Cre, iCre, dgCre, FlpO, tTA2, SP10, WPRE, WPRE3, and / or BGHpA. 25. The artificial expression construct is: eHGT_450m-minBglobin-heterogeneous coding sequence-WPRE3-BGHpA; eHGT_451m-minBglobin-heterogeneous coding sequence-WPRE3-BGHpA; eHGT_452m-minBglobin-heterogeneous coding sequence-WPRE3-BGHpA; eHGT_453m-minBglobin-heterogeneous coding sequence-WPRE3-BGHpA; eHGT_457m-minBglobin-heterogeneous coding sequence-WPRE3-BGHpA; eHGT_458m-minBglobin-heterogeneous coding sequence-W PRE3-BGHpA;eHGT_459m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_460m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_462m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_464m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_461m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_463m-minBglobin-heterogeneous code array-WPRE3 -BGHpA;eHGT_692h-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_693h-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_694h-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_695h-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_667m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_668m-minBglobin-heterogeneous code array-WPRE3-BG HpA;eHGT_696m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_455m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_457h-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_603m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_604m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_605m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_661m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_766m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_767m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_768m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_663m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_660m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_665m-minB globin-heterogeneous code array-WPRE3-BGHpA;eHGT_700m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_456m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_698m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_699m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_647m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_648m-minBglobin-heterogeneous code array WPRE3-BGHpA;eHGT_649m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_670m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_697m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_662m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_583m-minBglobin-heterogeneous code array-WPRE3-BGHpA;3xcore2_eHGT_4 59m-minBglobin-heterogeneous code array-WPRE3-BGHpA;3xcore2_eHGT_453m-minBglobin-heterogeneous code array-WPRE3-BGHpA;3xcore2_eHGT_462m-minBglobin-heterogeneous code array-WPRE3-BGHpA;3xcore2_eHGT_461m-minBglobin-heterogeneous code array-WPRE3-BGHpA;3xcore2_eHGT_464m-minBglobin-heterogeneous code array-WPRE3-BGHpA;3xcore2_eHGT_452m-minBglobin-heterogeneous code array-WPRE3-BGHpA;3xcore2_eHGT_451m-minBglobin-heterogeneous code array-WPRE3-BGHpA;3xcore2_eHGT_460m-minBglobin-heterogeneous code array-WPRE3-BGHpA;eHGT_450m-minimum promoter-heterogeneous code array-WPRE3-BGHpA;eHGT_451m-minimum promoter-heterogeneous code array-WPRE3-BGHpA;eHGT_452m-minimum promo Promoter-Heterogeneous code sequence-WPRE3-BGHpA;eHGT_453m-Minimum promoter-Heterogeneous code sequence-WPRE3-BGHpA;eHGT_457m-Minimum promoter-Heterogeneous code sequence-WPRE3-BGHpA;eHGT_458m-Minimum promoter-Heterogeneous code sequence-WPRE3-BGHpA;eHGT_459m-Minimum promoter-Heterogeneous code sequence-WPRE3-BGHpA;eHGT_460m-Minimum promoter-Heterogeneous code sequence-WPRE3-BGHpA;eHGT_462m-Minimum promoter-Heterogeneous code Sequence-WPRE3-BGHpA;eHGT_464m-Minimum Promoter-Heterogeneous Code Sequence-WPRE3-BGHpA;eHGT_461m-Minimum Promoter-Heterogeneous Code Sequence-WPRE3-BGHpA;eHGT_463m-Minimum Promoter-Heterogeneous Code Sequence-WPRE3-BGHpA;eHGT_692h-Minimum Promoter-Heterogeneous Code Sequence-WPRE3-BGHpA;eHGT_693h-Minimum Promoter-Heterogeneous Code Sequence-WPRE3-BGHpA;eHGT_694h-Minimum Promoter-Heterogeneous Code Sequence-WPRE 3-BGHpA;eHGT_695h-minimum promoter-heterogeneous code sequence-WPRE3-BGHpA;eHGT_667m-minimum promoter-heterogeneous code sequence-WPRE3-BGHpA;eHGT_668m-minimum promoter-heterogeneous code sequence-WPRE3-BGHpA;eHGT_696m-minimum promoter-heterogeneous code sequence-WPRE3-BGHpA;eHGT_455m-minimum promoter-heterogeneous code sequence-WPRE3-BGHpA;eHGT_457h-minimum promoter-heterogeneous code sequence-WPRE3-BGHpA;eHGT_603m-minimum promoter-heterogeneous code sequence-WPRE3-BGHpA;eHGT_604m-minimum promoter-heterogeneous code sequence-WPRE3-BGHpA;eHGT_605m-minimum promoter-heterogeneous code sequence-WPRE3-BGHpA;eHGT_661m-minimum promoter-heterogeneous code sequence-WPRE3-BGHpA;eHGT_766m-minimum promoter-heterogeneous code sequence-WPRE3-BGHpA;eHGT_767m-minimum promoter-heterogeneous code sequence-WPRE3-BGHpA;eHGT_ 768m-Minimum Promoter-Heterogeneous Code Sequence-WPRE3-BGHpA;eHGT_663m-Minimum Promoter-Heterogeneous Code Sequence-WPRE3-BGHpA;eHGT_660m-Minimum Promoter-Heterogeneous Code Sequence-WPRE3-BGHpA;eHGT_665m-Minimum Promoter-Heterogeneous Code Sequence-WPRE3-BGHpA;eHGT_700m-Minimum Promoter-Heterogeneous Code Sequence-WPRE3-BGHpA;eHGT_456m-Minimum Promoter-Heterogeneous Code Sequence-WPRE3-BGHpA;eHGT_698m-Minimum Small promoter - heterogeneous code sequence - WPRE3 - BGHpA; eHGT_699m - minimum promoter - heterogeneous code sequence - WPRE3 - BGHpA; eHGT_647m - minimum promoter - heterogeneous code sequence - WPRE3 - BGHpA; eHGT_648m - minimum promoter - heterogeneous code sequence WPRE3 - BGHpA; eHGT_649m - minimum promoter - heterogeneous code sequence - WPRE3 - BGHpA; eHGT_670m - minimum promoter - heterogeneous code sequence - WPRE3 - BGHpA; eHGT_697m - minimum promoter -Heterogeneous code sequence-WPRE3-BGHpA;eHGT_662m-Minimum promoter-Heterogeneous code sequence-WPRE3-BGHpA;eHGT_583m-Minimum promoter-Heterogeneous code sequence-WPRE3-BGHpA;3xcore2_eHGT_459m-Minimum promoter-Heterogeneous code sequence-WPRE3-BGHpA;3xcore2_eHGT_453m-Minimum promoter-Heterogeneous code sequence-WPRE3-BGHpA;3xcore2_eHGT_462m-Minimum promoter-Heterogeneous code sequence-WPRE3-BGHpA;An artificial expression construct according to any of Embodiments 12 to 24, comprising or encoding a set of features selected from 3xcore2_eHGT_461m-minimum promoter-heterogeneous coding sequence-WPRE3-BGHpA; 3xcore2_eHGT_464m-minimum promoter-heterogeneous coding sequence-WPRE3-BGHpA; 3xcore2_eHGT_452m-minimum promoter-heterogeneous coding sequence-WPRE3-BGHpA; 3xcore2_eHGT_451m-minimum promoter-heterogeneous coding sequence-WPRE3-BGHpA; or 3xcore2_eHGT_460m-minimum promoter-heterogeneous coding sequence-WPRE3-BGHpA. 26. A vector comprising an artificial expression construct according to any of Embodiments 12 to 25. 27. The vector according to Embodiment 26, wherein the vector includes a viral vector. 28. The vector according to Embodiment 26 or 27, wherein the viral vector comprises a recombinant adeno-associated virus (AAV) vector. 29. An adeno-associated virus (AAV) vector comprising at least one heterologous coding sequence, wherein the heterologous coding sequence is a promoter and eHGT_450m, eHGT_451m, eHGT_452m, eHGT_453m, eHGT_457m, eHGT_458m, eHGT_459m, eHGT_460m, eHGT_462m, eHGT_464m, eHGT _461m, eHGT_463m, eHGT_692h, eHGT_693h, eHGT_694h, eHGT_695h, eHGT_667m, eHGT_668m, eHGT_696 m, eHGT_455m, eHGT_457h, eHGT_603m, eHGT_604m, eHGT_605m, eHGT_661m, eHGT_766m, eHGT_767m, eHG T_768m, eHGT_663m, eHGT_660m, eHGT_665m, eHGT_700m, eHGT_456m, eHGT_698m, eHGT_699m, eHGT_64 7m, eHGT_648m, eHGT_649m, eHGT_670m, eHGT_697m, eHGT_662m, eHGT_583m, 3xcore2_eHGT_459m, 3xco The adeno-associated virus (AAV) vector under the control of an enhancer selected from re2_eHGT_453m, 3xcore2_eHGT_462m, 3xcore2_eHGT_461m, 3xcore2_eHGT_464m, 3xcore2_eHGT_452m, 3xcore2_eHGT_451m, and / or 3xcore2_eHGT_460m. 30. Transgenic cells comprising an expression construct or vector as described in any of the prior embodiments. 31. Transgenic cells of Embodiment 30, wherein the transgenic cells are L5 glutamatergic neurons. 32. The transgenic cell of Embodiment 31 in which the L5 glutamatergic neuron is an L5 ET neuron. 33. A non-human transgenic animal comprising an artificial expression construct, vector, or transgenic cell as described in any of the prior embodiments. 34. The non-human transgenic animal according to Embodiment 33, wherein the non-human transgenic animal is a mouse or a non-human primate. 35. An administerable composition comprising an expression construct, vector, or transgenic cell as described in any of the prior embodiments. 36. A kit comprising an artificial expression construct, vector, transgenic cells, transgenic animals, and / or an administerable composition as described in any of the prior embodiments. 37. A method for selectively expressing a heterologous gene in a cell population in vivo or in vitro, comprising providing a sample or subject containing a cell population with an administerable composition according to Embodiment 35 in a sufficient dose and over a sufficient period of time, thereby selectively expressing the gene in the cell population. 38. The method according to Embodiment 37, wherein the heterogene codes for an effector element or an expressible element. 39. The method according to Embodiment 38, wherein the effector element comprises a reporter protein or a functional molecule. 40. The method according to Embodiment 39, wherein the reporter protein comprises a fluorescent protein. 41. The method according to Embodiment 39 or 40, wherein the functional molecule comprises a functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cell transport protein, signaling molecule, neurotransmitter, calcium reporter, channelrhodopsin, CRISPR / CAS molecule, editase, guide RNA molecule, homologous recombination donor cassette, DREADD, growth factor, or survival-promoting gene. 42. The method according to any one of embodiments 37 to 41, wherein the expressible element includes a non-functional molecule. 43. The method according to Embodiment 42, wherein the non-functional molecule includes a non-functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cell transport protein, signaling molecule, neurotransmitter, calcium reporter, channelrhodopsin, CRISPR / CAS molecule, editase, guide RNA molecule, homologous recombination donor cassette, DREADD, growth factor, or survival-promoting gene. 44. The method according to any one of embodiments 37 to 43, wherein the provision includes pipetting. 45. The method according to Embodiment 44, wherein the pipetting is performed on brain slices. 46. ​​The method according to embodiment 45, wherein the brain slice includes an L5 ET neuron. 47. The method according to embodiment 45 or 46, wherein the brain slice is from a mouse, human, or non-human primate. 48. The method according to any one of Embodiments 37 to 43, wherein the provision includes administration to a living organism. 49. The method according to Embodiment 48, wherein the organism is a human, a non-human primate, or a mouse. 50. The method according to Embodiment 48 or 49, wherein the administration to the living organism is performed by injection. 51. The method according to Embodiment 50, wherein the injection includes intravenous injection, intraparenchymal injection into brain tissue, intraventricular (ICV) injection, intracisional (ICM) injection, or subarachnoid injection. 52. The method according to any one of embodiments 48 to 51, wherein the organism has a neurodegenerative disease. 53. The method according to any one of embodiments 48 to 52, wherein the organism has ALS or multiple sclerosis. 54.CN2248, CN2249, CN2250, CN2251, CN2252, CN2253, CN2254, CN2255, CN2256, CN2315, CN2423, CN2424, CN269 0, CN2695, CN2691, CN2692, CN2944, CN2886, CN2755, CN2938, CN2239, CN2410, CN2411, CN2412, CN2963, CN2623 Artificial expression constructs including CN2624, CN2601, CN2843, CN2851, CN2853, CN2860, CN2828, CN2870, CN2871, CN2847, CN2686, CN2687, CN2878, CN2946, CN2943, CN3319, CN2918, CN3018, CN3030, CN3027, CN3032, CN3015, CN3012, and / or CN3024.

[0125] (viii) Closing paragraph This includes variants of the sequences disclosed and referenced herein. Guidance is available using computer programs well known in the art, such as DNASTAR® (Madison, Wisconsin) software, to determine which amino acid residues can be substituted, inserted, or deleted without impairing biological activity. Preferably, the amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve the substitution of one amino acid family associated with their side chains.

[0126] Suitable conservative amino acid substitutions in peptides or proteins are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art generally recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its 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 the following conserved substitution families: 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 (Major aliphatic, nonpolar residues): Isoleucine (I Group 10: (Polyliphatic, nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (Sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, WH Freeman and Company.

[0127] When making such modifications, the hydrophilicity index of amino acids may be considered. The importance of hydrothermal amino acid indexes in conferring biological functions that interact with proteins is generally understood in the art (Kyte and Doolittle, 1982, J.Mol.Biol.157(1),105-32). Each amino acid has been assigned a hydrophobic index based on its hydrophobic and charge properties (Kyte and Doolittle, 1982). These values ​​are as follows: Ile (+4.5), Val (+4.2), Leu (+3.8), Phe (+2.8), Cys (+2.5), Met (+1.9), Ala (+1.8), Gly (-0.4), Thr (-0.7), Ser (-0.8), Trp (-0.9), Tyr (-1.3), Pro (-1.6), His (-3.2), Glutamate (-3.5), Gln (-3.5), Aspartate (-3.5), Asn (-3.5), Lys (-3.9), and Arg (-4.5).

[0128] In the art, it is known that certain amino acids may be substituted with other amino acids having similar hydrophilicity indices or scores, resulting in proteins with similar biological activity, i.e., proteins that are still biologically functionally equivalent. When making such changes, substitutions of amino acids with hydrophilicity indices within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more preferred. It is also understood in the art that substitutions of similar amino acids can be effectively carried out based on hydrophilicity.

[0129] As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to amino acid residues: Arg (+3.0), Lys (+3.0), aspartic acid (+3.0±1), glutamic acid (+3.0±1), Ser (+0.3), Asn (+0.2), Gln (+0.2), Gly (0), Thr (-0.4), Pro (-0.5±1), Ala (-0.5), His (-0.5), Cys (-1.0), Met (-1.3), Val (-1.5), Leu (-1.8), Ile (-1.8), Tyr (-2.3), Phe (-2.5), Trp (-3.4). It is understood that amino acids can be substituted with other amino acids having similar hydrophilicity values, and that a biologically, and especially immunologically, equivalent protein can still be obtained. In such changes, substitutions of amino acids with hydrophilicity values ​​of ±2 are preferred, those of ±1 are particularly preferred, and those of ±0.5 are even more preferred.

[0130] As described above, amino acid substitutions may be based on the relative similarity of amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc.

[0131] As shown elsewhere, gene sequence variants may include codon-optimizing variants, sequence polymorphisms, splice variants, and / or mutations that do not statistically significantly affect the function of the encoded product.

[0132] The variants of proteins, nucleic acids, and gene sequences disclosed herein also include sequences having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the proteins, nucleic acids, or gene sequences disclosed herein.

[0133] "% sequence identity" refers to the relationship between two or more sequences, as determined by comparing them. In the art, "identity" also means the degree of sequence correlation between protein, nucleic acid, or gene sequences, as determined by the matching of such sequence strings. "Identity" (often referred to as "similarity") can be readily calculated using known methods; see 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 ed.) Humana Press, NJ (1994), Sequence Analysis in Molecular Biology (Von Heijne, G. ed.) Academic Press (1987), and Sequence Analysis Primer (Gribskov, M. and Devereux, J. ed.) Oxford University Press, NY (1992). Preferred methods for determining identity are designed to give the best match between the sequences tested. Methods for determining identity and similarity are coded into publicly available computer programs. Sequence alignment and identity percentage calculation may be performed using the Megalign program in the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple sequence alignments can also be performed using the Clustal alignment method (with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10, Higgins and Sharp CABIOS, 5, 151-153 (1989))).Related programs also include the GCG suite 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. Editors: Suhai, Sandor. Publisher: Plenum, New Including York, NY. In the context of this disclosure, if sequence analysis software is used for analysis, it will be understood that the results of the analysis are based on the “default values” of the referenced program. As used herein, “default values” means any set of values ​​or parameters that are initially loaded by the software when it is first initialized.

[0134] The variants also include nucleic acid molecules that hybridize to the sequences disclosed herein under stringent hybridization conditions and provide the same functionality as the reference sequences. Exemplary stringent hybridization conditions may involve overnight incubation at 43°C in a solution containing 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhard solution, 10% dextran sulfate, and 20 μg / ml denatured shear salmon sperm DNA, followed by washing the filter in 0.1×SSC at 50°C. Changes in the stringency of hybridization and signal detection are primarily achieved by formamide concentration (lower percentages of formamide result in lower stringency), salt conditions, or temperature manipulation. For example, moderately high stringency conditions include overnight incubation at 37°C in a solution containing 6×SSPE (20×SSPE = 3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, and 100 μg / ml salmon sperm blocking DNA, followed by washing at 50°C with 1×SSPE and 0.1% SDS. In addition, to achieve even lower stringency, washing performed after stringent hybridization can be done with a higher salt concentration (e.g., 5×SSC). Modifications in the above conditions can be achieved through the inclusion and / or substitution of alternative blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may necessitate modifications to the hybridization conditions described above due to compatibility issues.

[0135] The term "concatamerize" is widely used to describe joining together elements to form a chain or a series. It is used to describe joining nucleotide or amino acid sequences together to form individual nucleotide or amino acid sequences. The terms "concatamerize" and "concatenate" are interchangeable.

[0136] As will be understood by those skilled in the art, each embodiment disclosed herein includes, essentially consists of, or may consist of, the specific elements, steps, ingredients, or components described herein. Therefore, the terms “include” or “including” should be interpreted as “include, consist of, or essentially consist of.” The transitional terms “comprise” or “comprises” mean that the embodiment anticipates the inclusion of an unspecified element, step, ingredient, or component, but not limited to a major quantity. The transitional phrase “consists of” excludes any unspecified element, step, ingredient, or component. The transitional phrase “essentially consists of” limits the scope of the embodiment to a specific element, step, ingredient, or component, and those that do not substantially affect the embodiment. Substantial effects would result in a statistically significant reduction in selective expression in L5 glutamatergic neurons, such as L5 ET neurons.

[0137] In certain embodiments, artificial means mean that they do not exist in nature.

[0138] Unless otherwise indicated, all numbers expressing properties such as the amount, molecular weight, and reaction conditions of components used herein and in the claims should be understood in all cases to be modified by the term "approximately." Therefore, unless otherwise indicated, the numerical parameters described herein and in the appended claims are approximations that may vary depending on the desired properties obtained by the invention. At the very least, and not as an attempt to limit the application of the principle of value to the claims, each numerical parameter should be interpreted in light of the number of significant digits reported and by applying common rounding techniques. If further clarification is needed, the term “approximately” has the meaning reasonably assigned by a person skilled in the art to be within the range of the stated number or range, i.e., ±20%, ±19%, ±18%, ±17%, ±16%, ±15%, ±14%, ±13%, ±12%, ±11%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the stated value.

[0139] Although the numerical ranges and parameters describing the broad scope of the present invention are approximations, the numerical values ​​described in specific examples are reported as accurately as possible. However, each numerical value inherently contains a certain error that inevitably arises from the standard deviation found in each test measurement.

[0140] In the context describing the present invention (particularly in the context of the following claims), “a,” “an,” “the,” and similar references should be construed to encompass both singular and plural unless otherwise indicated herein or unless explicitly stated otherwise or the context expressly contradicts it. The enumeration of value ranges herein is intended solely as a shorthand for individually referring to each distinct value within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually listed herein. All methods described herein may be carried out in any preferred order unless otherwise indicated herein or the context expressly contradicts it. Any and all examples provided herein, or the use of exemplary language (e.g., “etc.”), are intended merely to better illuminate the present invention and do not impose any limitation on the scope of the present invention as separately claimed. No language herein should be construed to indicate any element not described in any of the claims that is essential to the practice of the present invention.

[0141] The grouping of alternative elements or embodiments of the Invention disclosed herein should not be construed as limiting. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. For convenience and / or patentability reasons, it is anticipated that one or more members of a group may be included in or removed from a group. In the event of such inclusion or removal, this specification shall be deemed to include the modified group, thereby satisfying all descriptions of the Markush groups used in the appended claims.

[0142] Specific embodiments of the present invention are described herein, including the best modes known to the inventors for carrying out the invention. Of course, variations relating to these described embodiments will be obvious to those skilled in the art by reading the preceding description. The inventors expect that those skilled in the art will adopt such modifications as needed, and the inventors intend that the invention may be carried out in ways other than those specifically described herein. Accordingly, the invention includes all modifications and equalities of the subject matter enumerated in the claims appended herein, as permitted by applicable law. Furthermore, unless otherwise indicated herein, or unless clearly inconsistent with the context, any combination of the elements described above in all possible variations thereof is incorporated into the invention.

[0143] Furthermore, numerous references are made throughout this specification to patents, printed publications, journal articles, and other written texts (materials referenced herein). Each of these referenced materials is individually incorporated herein by reference in its entirety for reference teaching purposes.

[0144] Finally, it should be understood that the embodiments of the present invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be adopted are within the scope of the invention. Therefore, alternative configurations of the invention may be used in accordance with the teachings herein, but not limiting them, for example. Thus, the invention is not limited to those precisely shown and described.

[0145] The details provided herein are presented, for example, solely for illustrative purposes of a preferred embodiment of the present invention and are intended to provide what is considered to be the most useful and readily understandable description of the principles and conceptual aspects of various embodiments of the present invention. In this regard, no attempt has been made to show the structural details of the present invention; rather, an attempt is needed to understand the present invention fundamentally, and an attempt is needed to make it clear to those skilled in the art, through the description taken together with the drawings and / or examples, how some forms of the present invention can actually be embodied.

[0146] The definitions and descriptions used in this disclosure are intended to be controlled in any future interpretation unless expressly and expressly modified in the following examples, or unless the application of the meaning would render any interpretation meaningless or essentially meaningless. Where the interpretation of a term would render it meaningless or essentially meaningless, the definition should be taken from dictionaries 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. (i) (a) Sequence ID No. 10 or 49 or (b) an enhancer comprising a sequence having at least 95% sequence identity with the sequence of sequence number 10 or 49 and having enhancer activity; (ii) promoter; and (iii) An artificial expression construct containing a coding sequence.

2. The artificial expression construct according to claim 1, wherein the coding sequence codes for a fluorescent protein.

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

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

5. The artificial expression construct according to claim 1, wherein the artificial expression construct includes or encodes a skipping element.

6. The artificial expression construct according to claim 5, wherein the skipping element comprises a 2A peptide and / or an internal ribosome entry site (IRES).

7. The artificial expression construct according to claim 1, wherein the artificial expression construct is present in a viral vector.

8. The artificial expression construct according to claim 7, wherein the viral vector comprises a recombinant adeno-associated virus (AAV) vector.

9. A transgenic cell comprising the artificial expression construct described in claim 1.

10. The transgenic cell according to claim 9, wherein the transgenic cell is an L5 glutamatergic neuron.

11. The transgenic cell according to claim 10, wherein the L5 glutamatergic neuron is an L5 ET neuron.

12. The sequence of sequence number 49, or A sequence having at least 95% sequence identity with the sequence of Sequence ID No. 49 and possessing enhancer activity. An artificial enhancer containing a core having the following properties.

13. Sequence ID 49, copies 2, 3, 4, 5, 6, 7, 8, 9, or 10, or A sequence having at least 95% sequence identity with the sequence shown in Sequence ID No. 49, and possessing enhancer activity, in copies 2, 3, 4, 5, 6, 7, 8, 9, or 10. The artificial enhancer according to claim 12, having the following characteristics.

14. Three copies of sequence number 49, or A sequence having at least 95% sequence identity with the sequence shown in Sequence ID No. 49, and possessing enhancer activity, comprising three copies. The artificial enhancer according to claim 13, having the following characteristics.

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